Integrated device with external light source for molecular detection, detection and analysis

Abstract

The present invention relates to a system and method for analyzing single molecules and performing nucleic acid sequencing. An integrated device includes a plurality of pixels having a sample well configured to receive a sample that emits radiation when excited. The integrated device includes at least one waveguide configured to propagate excitation energy from an area of ​​the integrated device for coupling with an excitation energy source to the sample well. A pixel may also include at least one element for directing emission energy toward a sensor within the pixel. The system also includes an instrument connected to the integrated device. The instrument may include an excitation energy source for providing excitation energy to the integrated device by coupling to an excitation energy coupling region of the integrated device. The sample may be labeled with a marker of a plurality of markers distinguishable by a temporal parameter of the emission energy, and the construction of the sensor within a pixel may allow detection of a temporal parameter associated with the marker of the labeled sample.

Description

[0001] This application is a divisional application of the invention patent application with Chinese application number 201580054785.3 (corresponding to PCT international application number PCT / US2015 / 044379), application date August 7, 2015, and invention name “Integrated device with external light source for detecting, detecting and analyzing molecules”.

[0002] Related Applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 035,258, filed on August 8, 2014, entitled “Integrated Device with External Light Source for Detection, Detection and Analysis of Molecules” and U.S. Provisional Patent Application No. 62 / 164,464, filed on May 20, 2015, entitled “Integrated Device with External Light Source for Detection, Detection and Analysis of Molecules”, the entire contents of each of the above patent applications are incorporated herein by reference.

[0004] This application is related to the following U.S. patent applications:

[0005] U.S. Provisional Patent Application No. 62 / 164,506, filed May 20, 2015, entitled “INTEGRATED APPARATUS FOR TIME DIVISION OF RECEIVED PHOTONS”;

[0006] U.S. Provisional Patent Application No. 62 / 164,485, entitled “Pulsed Laser,” filed on May 20, 2015;

[0007] U.S. Provisional Patent Application No. 62 / 164,482, entitled “Methods for Nucleic Acid Sequencing,” filed on May 20, 2015;

[0008] U.S. Provisional Patent Application No. 62 / 035,242, filed on August 8, 2014, entitled “Optical System and Detection Chip for Detection, Detection and Analysis of Molecules,” which is hereby incorporated by reference in its entirety;

[0009] U.S. Non-Provisional Patent Application No. R0708.70002US02, filed on the same date and entitled “INTEGRATED APPARATUS FOR TIME DIVISION OF RECEIVED PHOTONS”;

[0010] U.S. non-provisional patent application No. R0708.70003US01, entitled "Optical system and detection chip for detecting, detecting and analyzing molecules," filed on the same date.

[0011] The entire contents of each of the related applications listed above are incorporated herein by reference. Technical Field

[0012] The present application generally relates to devices, methods and techniques for performing rapid, massively parallel quantitative analysis of biological and / or chemical samples, and methods of making the devices. Background Art

[0013] Detection and analysis of biological samples can be performed using biological assays ("bioassays"). Bioassays typically involve large and expensive laboratory equipment, which requires training of research assistants in order to operate the equipment and perform the bioassays. In addition, bioassays are typically performed in batches, so large quantities of samples of a particular type are necessary for detection and quantification.

[0014] Some bioassays are performed by labeling samples with fluorescent markers that emit light of a specific wavelength. These markers are illuminated with a light source, resulting in luminescence, and the luminescence is detected with a light detector to quantify the amount of luminescence emitted by the marker. Bioassays using fluorescent markers typically involve expensive laser light sources for illuminating the sample, and complex, bulky detection optics and electronics to collect the luminescence from the illuminated sample. Summary of the invention

[0015] The technology described herein relates to an apparatus and method for rapidly analyzing a sample using an active pixel integrated device that can interact with a mobile computing instrument. The integrated device can take the form of a disposable or reusable chip laboratory or packaging module that is configured to receive a small amount of sample and perform a large number of analyses on the sample inside the sample in parallel. The integrated device can be used in some embodiments to detect the presence of a specific chemical or biological analyte, in some embodiments to evaluate a chemical or biological reaction, and in some embodiments to determine a gene sequence. According to some embodiments, the integrated device can be used for single molecule gene sequencing.

[0016] According to some embodiments, the user places the sample in a chamber on the integrated device and inserts the integrated device into a receiving instrument. The receiving instrument interacts with the integrated device alone or automatically in connection with a computer, receives data from the integrated device, processes the received data, and provides the results of the analysis to the user. As can be appreciated, the integration and computational intelligence on a chip, receiving instrument, and / or computer reduces the skill level required of the user.

[0017] According to some embodiments of the present application, an integrated device including a plurality of pixels is provided, wherein one pixel of the plurality of pixels includes: a sample well for receiving excitation energy from an excitation source outside the integrated device, and at least one sensor for receiving luminescence from a sample located in the sample well and generating a signal providing sample identification information based on the received luminescence.

[0018] In some embodiments, the signal is an indicator of the time parameter of the received luminescence. In some embodiments, the time parameter is the lifetime associated with the luminescence from the sample. In some embodiments, the signal is an indicator of the luminescence spectrum. In some embodiments, the signal is an indicator of the characteristic wavelength of the luminescence. In some embodiments, the signal and the excitation energy represent the absorption spectrum of the sample. In some embodiments, the signal and the excitation energy represent the characteristic wavelength absorbed by the sample.

[0019] According to some embodiments of the present application, an integrated device including a pixel region is provided, the pixel region including a plurality of pixels. One of the plurality of pixels has: a sample well on a surface of the integrated device, wherein the sample well is configured to receive a sample; at least one sensor for receiving luminescence from the sample well; and at least one waveguide for delivering excitation energy to the vicinity of the sample well. The integrated device includes an excitation source coupling region, the excitation source coupling region including a coupling component for receiving excitation energy from an external excitation energy source and coupling the excitation energy into the waveguide.

[0020] According to some embodiments of the present application, a system including an excitation source module is provided, the excitation source module including an excitation source for emitting a pulse of excitation energy having a first time period and an integrated device. The integrated device includes: a sample well for receiving a sample, the sample emitting cold light when coupled to the pulse of excitation energy; a sensor for detecting the luminescence in a second time period, wherein the second time period occurs after the first time period; a first energy path along which the pulse of excitation energy moves from the excitation source to an energy source coupling component; a second energy path along which the pulse of excitation energy moves from the energy source coupling component to the sample well; and a third energy path along which the luminescence moves from the sample well to the sensor.

[0021] According to some embodiments of the present application, a method for detecting the presence of a molecule in a sample is provided. The method includes introducing a sample labeled with a plurality of luminescent markers into a sample well, wherein at least a portion of the plurality of luminescent markers have different luminescence lifetime values. The method also includes: irradiating the sample well with a pulse of light, measuring the arrival time of photons emitted from the sample well, and determining a characteristic of the marker based on the time of arrival of the photons.

[0022] According to some embodiments of the present application, an integrated device including a sample well and a sensor is provided. The sample well is used to receive a sample labeled with a plurality of luminescent markers, each of the plurality of luminescent markers having a different luminescence lifetime value. The sensor is used to detect luminescence from one of the plurality of luminescent markers in a plurality of time periods, wherein the plurality of time periods are selected so as to distinguish among the plurality of luminescent markers.

[0023] According to some embodiments of the present application, an integrated device including a sample well and a plurality of sensors is provided. The sample well is used to receive a sample marked with one of a plurality of luminescent markers. Each of the plurality of luminescent markers emits luminescence in one of a plurality of spectral ranges, and a portion of the plurality of luminescent markers that emit luminescence in one of a plurality of spectral ranges each has a different luminescence lifetime value. Each of the plurality of sensors is used to detect a spectral range of the plurality of spectral ranges in a plurality of time periods, and the plurality of time periods are selected to distinguish between a portion of the plurality of luminescent markers.

[0024] According to some embodiments, a system including a plurality of excitation sources and an integrated device is provided. The plurality of excitation sources are used to emit a plurality of excitation energies, wherein each of the plurality of excitation sources emits a pulse of an excitation energy of the plurality of excitation energies. The integrated device includes a sample well for receiving a sample labeled with one of a plurality of luminescent markers. A portion of the plurality of luminescent markers emits cold light after being irradiated with one of a plurality of excitation energies each having a different lifetime value. The integrated device also includes a sensor for detecting luminescence from one of the plurality of luminescent markers in a plurality of time periods after a pulse of an excitation energy of the plurality of excitation energies, wherein the timing of the pulse of the one of the plurality of excitation energies and the plurality of time periods are distinguished among the plurality of luminescent markers.

[0025] According to some embodiments of the present application, a method for forming an integrated device is provided, the method comprising forming a plurality of sensor regions, wherein one of the plurality of sensor regions comprises a plurality of sensors, thereby forming a plurality of sample wells, wherein one of the plurality of sample wells is aligned with a corresponding one of the plurality of sensor regions, and forming at least one waveguide for coupling excitation energy separated from the plurality of sample wells and guiding the excitation energy to at least one sample well.

[0026] According to some embodiments of the present application, an apparatus is provided, comprising: at least one excitation source for providing at least one excitation energy, an excitation source positioning system for aligning at least one excitation energy emitted by the at least one excitation source with a coupling region of an integrated device, and a readout circuit for receiving at least one readout signal representative of the emission energy detected by a sensor on the integrated device.

[0027] According to some embodiments of the present application, a method for sequencing a target nucleic acid molecule is provided. The method includes providing an integrated device, which includes a sample well for accommodating a target nucleic acid molecule, a polymerase, and multiple types of nucleotides or nucleotide analogs. Each type of multiple types of nucleotides or nucleotide analogs is labeled with one or more markers. The method also includes performing an extension reaction at the priming position of the target nucleic acid molecule in the presence of a polymerase, thereby sequentially incorporating at least a portion of the nucleotides or nucleotide analogs into a growing chain that is complementary to the target nucleic acid molecule, wherein the markers that label the nucleotides or nucleotide analogs incorporated into the growing chain when excited with excitation energy produce emission from the sample well, and the emission lifetimes are distinguishable for multiple types of nucleotides or nucleotide analogs. The method also includes identifying nucleotides or nucleotide analogs based on a signal received from a sensor that is an indicator of the emission lifetime, thereby sequencing the target nucleic acid molecule.

[0028] According to some embodiments of the present application, a method for nucleic acid sequencing is provided. The method includes providing an integrated device, which includes multiple sample wells and an excitation energy source operably coupled to the multiple sample wells. Individual sample wells of the multiple sample wells contain target nucleic acid molecules, polymerases, and nucleotides or nucleotide analogs. One of the multiple markers labels each nucleotide or nucleotide analog. The method also includes: subjecting the target nucleic acid molecule to a polymerization reaction in the presence of nucleotides or nucleotide analogs and a polymerase, thereby obtaining a growing chain that is complementary to the target nucleic acid molecule. In the case of incorporating nucleotides or nucleotide analogs into the growing chain, multiple markers emit emission light when excited by excitation energy from an excitation source. The method also includes: detecting the lifetime of the luminescence while performing an extension reaction, wherein the luminescence lifetime is distinguishable for multiple markers; and identifying the sequence of the target nucleic acid molecule based on the luminescence lifetime.

[0029] According to some embodiments of the present application, a method for analyzing a sample is provided. The method includes: depositing the sample on a surface of an integrated device having a plurality of pixels, wherein each pixel has a sample well for receiving a sample labeled with a first marker of a plurality of markers and a sensor region having at least one sensor; aligning the integrated device with an instrument having at least one excitation energy source for coupling excitation energy to the sample well of the first pixel and a readout circuit for receiving a readout signal of at least one sensor of the sensor region of the first pixel; irradiating the first marker with the excitation energy; and detecting the lifetime of emission energy generated by emission of the first marker based on the readout signal of at least one sensor of the sensor region of the first pixel.

[0030] The term "pixel" used in the present disclosure may refer to a unit cell of an integrated device. The unit cell may include a sample well and a sensor. The unit cell may also include at least one excitation coupling optical structure (which may be referred to as a "first structure"), which is used to enhance the coupling of excitation energy from the excitation source to the sample well. The unit cell may also include at least one emission coupling structure, which is used to enhance the coupling of emission from the sample well to the sensor. The unit cell may also include integrated electronic devices (e.g., CMOS devices). There may be multiple pixels arranged in an array on the integrated device.

[0031] The term "optical" as used in this disclosure may refer to the visible, near infrared, and short wavelength infrared spectral bands.

[0032] The term "label" used in the present disclosure may refer to a tag, a probe, or a reporter, and includes a marker attached to a sample to be analyzed, or a marker attached to a reagent that can bind to a sample.

[0033] As used in this disclosure, the phrase "excitation energy" may refer to any form of energy (eg, radiative or non-radiative) that is delivered to a sample and / or a marker within a sample well. Radiative excitation energy may include optical radiation at one or more characteristic wavelengths.

[0034] The phrase "characteristic wavelength" as used in this disclosure may refer to a central wavelength or dominant wavelength within a limited bandwidth of radiation. In some cases, "characteristic wavelength" may refer to a peak wavelength of the radiation bandwidth. Examples of characteristic wavelengths of fluorophores are 563 nm, 595 nm, 662 nm, and 687 nm.

[0035] The phrase "characteristic energy" as used in this disclosure may refer to energy associated with a characteristic wavelength.

[0036] The term "emission" as used in this disclosure may refer to emission from a tag and / or a sample. This may include radiative emission (e.g., light emission) or non-radiative energy transfer (e.g., Dexter energy transfer or Resonance Energy Transfer). Emission is caused by excitation of the sample and / or marker within the sample well.

[0037] As used in this disclosure, the phrases "emission from a sample well" or "emission from a sample" may refer to emission from a flag and / or a sample within a sample well.

[0038] The term "self-aligned" as used in the present disclosure may refer to a microfabrication process in which at least two different elements (e.g., a sample well and an emission coupling structure, a sample well and an excitation source) may be fabricated and aligned to one another without employing two separate lithographic patterning steps, wherein a first lithographic patterning step (e.g., photolithography, ion beam lithography, EUV lithography) prints a pattern for the first element and a second lithographic patterning step coincides with the first lithographic patterning step and prints a pattern for the second element. The self-aligned process may include including the patterns for both the first and second elements in a single lithographic patterning step, or may include utilizing features of a fabricated structure of the first element to form the second element.

[0039] The term "sensor" as used in this disclosure may refer to one or more integrated circuit devices for sensing emission from a sample well and generating at least one electrical signal representative of the sensed emission.

[0040] The term "nanoscale" as used in the present disclosure may refer to structures having at least one dimension or minimum feature size of about 150 nanometers (nm) or less but not greater than about 500 nm.

[0041] The term "micrometer-scale" as used in this disclosure may refer to structures having at least one dimension or smallest characteristic dimension between about 500 nm and about 100 micrometers.

[0042] The phrase "enhancing excitation energy" as used in the present disclosure may refer to increasing the intensity of the excitation energy at the excitation region of the sample well. For example, the intensity may be increased by concentrating and / or resonating the excitation energy incident on the sample well. In some cases, the intensity may be increased using an anti-reflection coating or lossy layer that allows the excitation energy to penetrate further into the excitation region of the sample well. The enhancement of the excitation energy may be a comparative reference to an embodiment that does not include a structure for enhancing the excitation energy in the excitation region of the sample well.

[0043] The terms "about," "approximately," and "substantially" as used in this disclosure may refer to a value and are intended to include the referenced value plus and minus acceptable variations. In some embodiments, the amount of variation may be less than 5%, in some embodiments less than 10%, and in some embodiments less than 20%. In embodiments where the device may function properly over a wide range of values ​​(e.g., including a range of one or more orders of magnitude), the amount of variation may be a factor of 2. For example, if the device may function properly for values ​​within the range of 20 to 350, then "about 80" may include values ​​between 40 and 160.

[0044] The term "adjacent" as used in this disclosure may refer to two elements that are arranged very close to each other (e.g., within a distance of less than about one-fifth of the lateral or vertical dimension of a pixel). In some cases, there may be intervening structures or layers between adjacent elements. In some cases, adjacent elements may be in close proximity to each other and there may be no intervening structures or elements.

[0045] The term "detection" as used in this disclosure may refer to receiving emissions from a sample well at a sensor and generating at least one electrical signal representative of or associated with the emissions. The term "detection" as used in this disclosure may also refer to determining the presence of a particular sample or marker in a sample well, or identifying its characteristics, based on the emissions from the sample well.

[0046] The foregoing and other aspects, embodiments and features of the present disclosure may be more fully understood based on the following description in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] It will be appreciated by those skilled in the art that the drawings described herein are for illustrative purposes only. It should be understood that in some cases various aspects of the present invention may be magnified or enlarged to facilitate understanding of the present invention. In the accompanying drawings, similar reference numerals generally refer to similar features, functionally similar and / or structurally similar elements in all the various drawings. These drawings may not be drawn to scale, but rather emphasis is placed on illustrating the principles of the present disclosure. These drawings are not intended to limit the scope of the present disclosure in any way.

[0048] The features and advantages of the present invention will become apparent from the detailed description set forth below in conjunction with the accompanying drawings.

[0049] When describing embodiments with reference to the accompanying drawings, directional words ("above", "below", "top", "bottom", "left", "right", "horizontal", "vertical", etc.) may be used. Such directional words are merely used as an aid to the reader when viewing the drawings in a normal orientation. These directional words are not intended to describe the preferred or only orientations of the embodied device. The device may adopt other orientations.

[0050] Figure 1-1 A graph showing the probability of a photon being emitted from a marker as a function of time.

[0051] Figure 1-2A Shown are emission timing spectra according to some embodiments.

[0052] Figure 1-2B Absorption wavelength spectra according to some embodiments are shown.

[0053] Figure 1-2C Emission wavelength spectra according to some embodiments are shown.

[0054] Figure 1-3A The phase space for emission wavelength and emission lifetime is shown.

[0055] Figure 1-3B The phase space for absorption wavelength and emission lifetime is shown.

[0056] Figure 1-4 The phase space for emission wavelength, absorption wavelength, and emission lifetime is shown.

[0057] Figure 2-1A is a block diagram of an apparatus that can be used for rapid mobile analysis of biological and chemical samples, according to some embodiments.

[0058] Figure 2-1B is a block diagram of an integrated device and apparatus according to some embodiments.

[0059] Figure 2-2 An integrated device according to some embodiments is shown.

[0060] Figure 3-1A A row of pixels of an integrated device according to some embodiments is shown.

[0061] Figure 3-1B Excitation energy coupling to sample wells in a row of pixels and emission energy directed from each sample well toward a sensor are shown in accordance with some embodiments.

[0062] Figure 3-2 An integrated device and excitation source according to some embodiments are shown.

[0063] Figure 4-1A Edge coupling of an excitation source to a waveguide is shown according to some embodiments.

[0064] Figure 4-1B A grating coupler for coupling an integrated device to an excitation source is shown in accordance with some embodiments.

[0065] Figure 4-2 An integrated device and excitation source according to some embodiments are shown.

[0066] Figure 4-3A An exemplary excitation-coupling region is shown according to some embodiments.

[0067] Figure 4-3B Shows Figure 4-3A Simulation of light intensity in the excitation coupling region is shown in .

[0068] Figure 4-3C A grating coupler and a waveguide are shown according to some embodiments.

[0069] Figure 4-4The losses of different waveguide structures are plotted as a function of the bending radius.

[0070] Figure 4-5 A star coupler is shown according to some embodiments.

[0071] Figure 4-6 A star coupler for coupling input light from two grating couplers is shown in accordance with some embodiments.

[0072] Figure 4-7 The configuration of an MMI beam splitter according to some embodiments is shown.

[0073] Figure 4-8 A simulation of the light intensity passing through an MMI beam splitter is shown.

[0074] Figure 4-9A A grating coupler according to some embodiments is shown.

[0075] Figure 4-9B and Figure 4-9C A grating coupler according to some embodiments is shown.

[0076] Figure 5-1 A sample well formed in a pixel region of an integrated device is shown according to one embodiment.

[0077] Figure 5-2 Excitation energy incident on a sample well is shown according to some embodiments.

[0078] Figure 5-3 Shown is the decay of excitation energy along a sample well formed as a zero-mode waveguide, according to some embodiments.

[0079] Figure 5-4 A sample well is shown including a dimple that, in some embodiments, increases excitation energy at an excitation region associated with the sample well.

[0080] Figure 5-5 The excitation intensity of a sample well with a dimple and without a dimple according to one embodiment is compared.

[0081] Figure 5-6A A sample well and a pit formed at a protrusion are shown according to some embodiments.

[0082] Figure 5-6B A sample well and a pit are shown according to some embodiments.

[0083] Figure 5-7A A sample well having tapered sidewalls is shown in accordance with some embodiments.

[0084] Figure 5-7BA sample well including curved sidewalls and a recess having a small lateral dimension is shown according to some embodiments.

[0085] Figure 5-7C and Figure 5-7D A sample well formed by a surface plasmon structure is shown.

[0086] Figure 5-7E A sample well including excitation energy enhancement structures formed along sidewalls of the sample well is shown in accordance with some embodiments.

[0087] Figure 5-7F A sample well formed in a multi-layer stack is shown in accordance with some embodiments.

[0088] Figure 5-8 A surface coating formed on a sample well surface is shown according to some embodiments.

[0089] Figure 5-9A to Figure 5-9E Structures associated with a lift-off process to form a sample well are shown according to some embodiments.

[0090] Figure 5-9F Structures associated with an alternative lift-off process for forming a sample well are shown according to some embodiments.

[0091] Figure 5-10A to Figure 5-10D Structures associated with a direct etch process to form a sample well are shown according to some embodiments.

[0092] Figure 5-11 A sample well is shown that may be formed in multiple layers using a lift-off process or a direct etching process according to some embodiments.

[0093] Figure 5-12 Structures associated with an etching process that may be used to form pits are shown according to some embodiments.

[0094] Figure 5-13A to Figure 5-13C Structures associated with alternative processes for forming pits are shown according to some embodiments.

[0095] Figure 5-14A to Figure 5-14D Structures associated with processes for depositing adhesion and passivation layers are shown according to some embodiments.

[0096] Figure 5-15 Structures associated with a process for centrally depositing an adhesive within a sample well are shown, according to some embodiments.

[0097] Figure 5-16 A dimpled sample well is shown in accordance with some embodiments.

[0098] Figure 6-1AA simulation of excitation radiation coupled from a waveguide to a sample well is shown, according to some embodiments.

[0099] Figure 6-1B A simulation of excitation radiation coupled to a sample well is shown, according to some embodiments.

[0100] Figure 6-2A , Figure 6-2B and Figure 6-2C An integrated device with a microcavity according to some embodiments is shown.

[0101] Figure 6-3A An integrated device with a microcavity according to some embodiments is shown.

[0102] Figure 6-3B An integrated device with a microcavity according to some embodiments is shown.

[0103] Figure 6-3C An integrated device with a microcavity according to some embodiments is shown.

[0104] Figure 6-3D An integrated device with a microcavity according to some embodiments is shown.

[0105] Figure 6-4 An integrated device with a microcavity according to some embodiments is shown.

[0106] Figure 6-5 A simulation of excitation radiation propagating in an integrated device with a microcavity is shown, according to some embodiments.

[0107] Figure 6-6A , Figure 6-6B and Figure 6-6C A simulation of excitation radiation propagating in an integrated device with a microcavity is shown, according to some embodiments.

[0108] Figure 6-6D A simulation of excitation radiation propagating in an integrated device with a microcavity is shown, according to some embodiments.

[0109] Figure 6-7A An integrated device with a microcavity according to some embodiments is shown.

[0110] Figure 6-7B A simulation of excitation radiation propagating in an integrated device with a microcavity is shown, according to some embodiments.

[0111] Figure 6-7C A simulation of excitation radiation propagating in an integrated device with a microcavity is shown, according to some embodiments.

[0112] Figure 6-7DA cross-sectional view of an integrated device according to some embodiments is shown.

[0113] Figure 6-8A and Figure 6-8B An integrated device with a tapered waveguide is shown according to some embodiments.

[0114] Figure 6-9A and Figure 6-9B An integrated device with a tapered waveguide is shown according to some embodiments.

[0115] Figure 6-10 A graph showing the losses as a function of the taper length.

[0116] Figure 6-11A An integrated device with a sample well sinker is shown according to some embodiments.

[0117] Figure 6-11B and Figure 6-11C An integrated device with a sample well sinker is shown according to some embodiments.

[0118] Figure 6-12 An array of sample wells of an integrated device is shown according to some embodiments.

[0119] Figure 6-13 An integrated device including a waveguide with variable dimensions is shown according to some embodiments.

[0120] Figure 6-14 An integrated device including a waveguide with variable dimensions is shown according to some embodiments.

[0121] Figure 7-1A , Figure 7-1B and Figure 7-1C Components for coupling emission energy from a sample well of an integrated device are shown in accordance with some embodiments.

[0122] Figure 7-2A A simulation of the emission energy from a sample well is shown.

[0123] Figure 7-2B A graph showing the emission energy emitted from a sample well at an angle.

[0124] Figure 7-3 Graphs showing absorbance and reflectance as a function of wavelength are shown.

[0125] Figure 7-4A and Figure 7-4B A polarizing filter is shown according to some embodiments.

[0126] Figure 7-5 A wavelength filter according to some embodiments is shown.

[0127] Figure 7-6 A graph showing light transmittance as a function of wavelength is shown.

[0128] Figure 7-7 A multi-wavelength filter according to some embodiments is shown.

[0129] Figure 7-8 A graph showing light transmittance as a function of wavelength is shown.

[0130] Figure 7-9A and Figure 7-9B A sensor with time bins is shown according to some embodiments.

[0131] Figure 8-0A An exemplary system for providing light pulses according to some embodiments is shown.

[0132] Figure 8-0B A graph showing light intensity as a function of time is shown.

[0133] Figure 8-1 A graph showing carrier density as a function of time is shown.

[0134] Figure 8-2 Shown is a shaped electrical signal used to form a light output according to some embodiments.

[0135] Figure 8-3 Light output from an excitation source is shown according to some embodiments.

[0136] Figure 8-4 Light output from an excitation source is shown according to some embodiments.

[0137] Figure 8-5 The performance of a laser diode according to some embodiments is shown.

[0138] Figure 8-6A A transmission line pulsar is shown in accordance with some embodiments.

[0139] Figure 8-6B Shown are light pulses obtained using a transmission-line pulsar, according to some embodiments.

[0140] Figure 8-7 A circuit for obtaining light pulses according to some embodiments is shown.

[0141] Figure 8-8 A circuit for obtaining light pulses according to some embodiments is shown.

[0142] Figure 8-9A A circuit for obtaining light pulses according to some embodiments is shown.

[0143] Figure 8-9B Shown from Figure 8-9A The electrical signals of the circuit shown in .

[0144] Figure 8-10A A circuit for obtaining light pulses according to some embodiments is shown.

[0145] Figure 8-10B Shown from Figure 8-10A The electrical signals of the circuit shown in .

[0146] Figure 8-11A An arrangement for combining light sources according to some embodiments is shown.

[0147] Figure 8-11B Shows Figure 8-10A A graph of the performance of the circuit shown in FIG.

[0148] Figure 9-1 An excitation source module and basic instrumentation according to some embodiments are shown.

[0149] Figure 9-2 An excitation source module and basic instrumentation according to some embodiments are shown.

[0150] Figure 9-3 Optical components for aligning an excitation source with an integrated device are shown according to some embodiments.

[0151] Figure 9-4 An excitation source module and basic instrumentation according to some embodiments are shown.

[0152] Figure 9-5 to Figure 9-11 An excitation source module and basic instrumentation according to some embodiments are shown.

[0153] Figure 9-12 to Figure 9-19 Components for passive alignment of an excitation source with an integrated device are shown in accordance with some embodiments.

[0154] Figure 9-20 A monitoring sensor is shown according to some embodiments.

[0155] Figure 9-21 An integrated device with monitoring sensors according to some embodiments is shown.

[0156] Figure 9-22 Arrangements of waveguides and monitoring sensors of an integrated device are shown according to some embodiments.

[0157] Figure 9-23 A monitoring sensor of an integrated device is shown according to some embodiments.

[0158] Figure 9-24Optical components for coupling excitation energy to an integrated device are shown according to some embodiments.

[0159] Figure 9-25A Components for coupling excitation energy to an integrated device are shown in accordance with some embodiments.

[0160] Figure 9-25B Components for coupling excitation energy to an integrated device are shown in accordance with some embodiments.

[0161] Figure 9-25C Components for coupling excitation energy to an integrated device are shown in accordance with some embodiments.

[0162] Figure 10-1 A schematic diagram of a sample well containing various components for nucleic acid sequencing is shown, showing a target space, a polymerase complex, a target nucleic acid, a complementary strand and primers, and an adapter for immobilization.

[0163] Figure 10-2 An exemplary experiment for nucleic acid sequencing showing four stages of a sequencing reaction; (A) prior to incorporation of fluorescently labeled nucleotides; (B) the first incorporation event; (C) the period between the first and second incorporation events; and (D) the second incorporation event; and corresponding examples of raw data and processed data during stages (A)-(D).

[0164] Figure 10-3 An exemplary process of surface pretreatment is shown, which includes the following steps: (a) Al 2 O 3 Deposition, (b) PEG-phosphonate passivation, (c) biotin / PEG-silanization, (d) complex loading, and (e) sequencing reaction initiation.

[0165] Figure 10-4 A schematic diagram for performing measurements according to some embodiments is shown.

[0166] Figure 10-5 A Fresnel lens according to some embodiments is shown.

[0167] Figure 10-6 Shown is a graph of an optical signal as a function of time according to some embodiments.

[0168] Figure 10-7 Signal curves for markers in time bins are shown according to some embodiments.

[0169] Figure 10-8 Shown is a graph of an optical signal as a function of time according to some embodiments.

[0170] Figure 10-9Signal curves for markers in time bins are shown according to some embodiments.

[0171] Figure 10-10 A schematic diagram for performing measurements according to some embodiments is shown.

[0172] Figure 10-11 A graph showing lifetime as a function of emission wavelength according to some embodiments.

[0173] Figure 10-12 A graph showing an optical signal as a function of wavelength according to some embodiments.

[0174] Figure 10-13 Shown is a graph of an optical signal as a function of time according to some embodiments.

[0175] Figure 10-14 Signal curves for a marker in time bins for multiple sensors are shown in accordance with some embodiments.

[0176] Figure 10-15 Shown is a graph of an optical signal as a function of time according to some embodiments.

[0177] Figure 10-16 Shown are signal curves of markers in time bins for multiple sensors according to some embodiments.

[0178] Figure 10-17 A schematic diagram for performing measurements according to some embodiments is shown.

[0179] Figure 10-18 A graph showing an optical signal as a function of wavelength according to some embodiments.

[0180] Figure 10-19 Shown is a graph of an optical signal as a function of time according to some embodiments.

[0181] Figure 10-20 Shown are signal curves of markers in time bins for multiple sensors according to some embodiments.

[0182] Figure 11-1 A method for fabricating a sample well is shown according to some embodiments.

[0183] Figure 11-2 A method for fabricating a sample well is shown according to some embodiments.

[0184] Figure 11-3 A method for fabricating a sample well is shown according to some embodiments.

[0185] Figure 11-4AA method for fabricating a sample well is shown according to some embodiments.

[0186] Figure 11-4B A method for fabricating a sample well is shown according to some embodiments.

[0187] Figure 11-5 A method for fabricating a sample well layer having a sinker is shown according to some embodiments.

[0188] Figure 11-6 A method for fabricating a sample well layer having a sinker is shown according to some embodiments.

[0189] Figure 11-7 A method for making concentric gratings according to some embodiments is shown.

[0190] Figure 11-8 A method for making concentric gratings according to some embodiments is shown.

[0191] Figure 11-9 A method for making concentric gratings according to some embodiments is shown.

[0192] Figure 11-10 An exemplary microcavity design according to some embodiments is shown.

[0193] Figure 11-11 A method for making a refractive optical component according to some embodiments is shown.

[0194] Figure 11-12 Images showing different steps in making a refractive optic according to some embodiments.

[0195] Figure 11-13 A refractive optic is shown according to some embodiments.

[0196] Figure 11-14 A method for making a refractive optical component according to some embodiments is shown.

[0197] Figure 11-15 A method for making a refractive optical component according to some embodiments is shown.

[0198] Figure 11-16 A Fresnel lens according to some embodiments is shown.

[0199] Figure 11-17 A Fresnel lens according to some embodiments is shown.

[0200] Figure 11-18 A Fresnel lens according to some embodiments is shown.

[0201] Figure 11-19 A method for making a Fresnel lens according to some embodiments is shown.

[0202] Figure 11-20 A method for making a Fresnel lens according to some embodiments is shown.

[0203] Figure 11-21 A method for making a Fresnel lens according to some embodiments is shown.

[0204] Figure 11-22 A method for making a Fresnel lens according to some embodiments is shown. DETAILED DESCRIPTION

[0205] The inventor has recognized and realized that the detection and quantitative compact high-speed device for performing single molecule or particle can reduce the cost of performing the complex quantitative measurement of biological and / or chemical samples, and rapidly promote the speed of biochemical technology discovery. In addition, the cost-effective device that can be easily transported can not only transform the mode of the bioassay performed in developed countries, but also provide the rapid acquisition of the basic diagnostic test that can significantly improve their health and welfare for the first time for the people in developing countries. For example, in some embodiments, the diagnostic test for performing the bioassay device is for performing the diagnostic test to biological samples (such as blood, urine and / or saliva). The device can be used by individuals in their homes, by doctors in remote clinics or any other locations (such as village doctors' offices) in developing countries. This diagnostic test can include the detection of biomolecules (such as nucleic acid molecules or proteins) in the biological sample of the subject. In some examples, the diagnostic test includes sequencing of the nucleic acid molecules in the biological sample of the subject, such as sequencing of the cell-free deoxyribonucleic acid molecules or expression products in the biological sample of the subject. For example, embodiments described herein may be used for diagnostic testing of blood, urine, and / or saliva that can be used by individuals in their homes, by physicians in remote clinics in developing countries, or any other location (e.g., a rural doctor's office).

[0206] Pixelated sensor devices with a large number of pixels (e.g., hundreds, thousands, millions or more) allow for the parallel detection of multiple individual molecules or particles. For example, but not limited to, these molecules can be proteins and / or DNA. In addition, high-speed devices that can acquire data at rates greater than 100 frames per second allow for the detection and analysis of dynamic processes or changes that occur over time within the sample being analyzed.

[0207] The inventors have recognized and appreciated that one obstacle that prevents bioassay devices from being made more compact is the need to filter the excitation light to avoid causing adverse detection events at the sensor. Optical filters used to transmit the desired signal light (luminescence) and fully block the excitation light can be thick, bulky, and expensive and do not allow for changes in the angle of incidence of the light, thereby preventing miniaturization. However, the inventors have recognized and appreciated that the use of a pulsed excitation source can reduce the need for such power, or in some cases eliminate the need for such filters. By using a sensor that can determine the time when a photon is detected relative to the excitation light pulse, the signal light can be separated from the excitation light based on the time when the photon is received rather than based on the spectrum of the received light. Therefore, in some embodiments, the need for bulky optical filters is reduced and / or eliminated.

[0208] The inventors have recognized and appreciated that luminescence lifetime measurements can also be used to identify molecules present in a sample. An optical sensor capable of detecting when a photon is detected can measure the luminescence lifetime of a molecule excited by the excitation light using statistical data collected from many events. In some embodiments, luminescence lifetime measurements can also be performed in addition to spectral measurements of luminescence. Alternatively, spectral measurements of luminescence can be completely omitted in the identification of sample molecules. Luminescence lifetime measurements can be performed using a pulsed excitation source. In addition, luminescence lifetime measurements can be performed using an integrated device including a sensor, or a device in which the light source is located in a system separate from the integrated device.

[0209] The inventors have also recognized and appreciated that incorporating a sample well (which may include a nanopore) and a sensor into a single integrated device capable of measuring luminescence emitted from a biological sample can reduce the cost of manufacturing such a device, thereby forming a disposable bioanalysis integrated device. A disposable, single-use integrated device that interacts with a base instrument can be used in parts of the world without the constraints of requiring a high-cost biological laboratory for sample analysis. Thus, automated bioanalysis can be brought to areas of the world where quantitative analysis of biological samples could not previously be performed. For example, a blood test for an infant can be performed by placing a blood sample on a disposable integrated device, placing the disposable integrated device in a small, portable base instrument for analysis, and processing the results using a computer for immediate viewing by the user. The data can also be transmitted to a remote location for analysis over a data network, and / or archived for later clinical analysis.

[0210] The inventors have also recognized and appreciated that disposable devices that are disposed of after use can be manufactured more simply and at a lower cost by not including a light source on the integrated device. Instead, the light source can include reusable components incorporated into the system that interact with the disposable integrated device to analyze the sample.

[0211] The inventors have also recognized and appreciated that when a sample is labeled with multiple different types of luminescent markers, any suitable luminescent marker signature may be used to identify the type of marker present in a particular pixel of an integrated device. For example, a luminescent signature emitted by a marker and / or signature of excitation absorption may be used to identify the marker. In some embodiments, the emission energy of the luminescence (which is directly related to the wavelength of the light) may be used to distinguish a first type of marker from a second type of marker. Additionally or alternatively, luminescence lifetime measurements may also be used to identify the type of marker present at a particular pixel. In some embodiments, luminescence lifetime measurements may be performed using a pulsed excitation source using a time sensor that can distinguish when photons are detected with sufficient resolution to obtain lifetime information. Additionally or alternatively, the energy of the excitation light absorbed by different types of markers may be used to identify the type of marker present at a particular pixel. For example, a first marker may absorb light of a first wavelength but not equally absorb light of a second wavelength, while a second marker may absorb light of a second wavelength but not equally absorb light of the first wavelength. Thus, when more than one excitation light source, each having a different excitation energy, is used to illuminate a sample in an interleaved manner, the absorbed energy of the marker may be used to identify the type of marker present in the sample. Different marks may also have different luminescence intensities. Therefore, the detected intensity of the luminescence may also be used to identify the type of mark present at a particular pixel.

[0212] A non-limiting example of an application of the device contemplated by the inventors is a device capable of performing sequencing of biomolecules, such as nucleic acids or polypeptides (e.g., proteins) having multiple amino acids. Diagnostic tests that can be performed using such a device include sequencing nucleic acid molecules in a subject's biological sample, such as sequencing cell-free DNA molecules or expression products in a subject's biological sample.

[0213] The application provides a device, system and method for detecting biomolecules or their subunits (e.g., nucleic acid molecules). This detection may include sequencing. Biomolecules can be extracted from the biological sample obtained from the study subject. The biological sample can be extracted from the body fluid or tissue of the study subject, such as respiratory fluid, saliva, urine or blood (e.g., whole blood or plasma). The study subject may suspect that there is a healthy state, such as a disease (e.g., cancer). In some instances, one or more nucleic acid molecules can be extracted from the body fluid or tissue of the study subject. One or more nucleic acids can be extracted from one or more cells obtained from the study subject, such as the part of the tissue of the study subject, or obtained from the acellular body fluid (e.g., whole blood) of the study subject.

[0214] Sequencing can include determining individual subunits of a template biomolecule (e.g., a nucleic acid molecule) by synthesizing another biomolecule that is complementary or similar to the template, such as by synthesizing a nucleic acid molecule that is complementary to the template nucleic acid molecule and identifying the incorporation of nucleotides over time (e.g., by sequencing by synthesis). As an alternative, sequencing can include direct identification of individual subunits of a biomolecule.

[0215] During sequencing, signals that are indicators of individual subunits of a biomolecule can be collected in a memory and processed in real time or at a later point in time to determine the sequence of the biomolecule. Such processing can include comparison of the signal to a reference signal that enables identification of the individual subunits, which in some cases results in a read. A read can be a sequence of sufficient length (e.g., at least about 30, 50, 100 base pairs (bp) or more) that can be used to identify a larger sequence or region, such as a location that can be aligned to a chromosome or genomic region or gene.

[0216] Markers can be used to identify individual subunits of biomolecules. In some instances, luminescent markers are used to identify individual subunits of biomolecules. Luminescent markers (also referred to herein as "markers") can be exogenous or endogenous markers. Exogenous markers can be external luminescent markers used in reporter genes and / or tags for luminescent labeling. Examples of exogenous markers may include, but are not limited to: fluorescent molecules, fluorophores, fluorescent dyes, fluorescent stains, organic dyes, fluorescent proteins, enzymes, atomic groups involved in fluorescence resonance energy transfer (FRET), enzymes, and / or quantum dots. Such exogenous markers can be coupled to probes or specifically bound to specific targets or component functional groups (e.g., molecules, ions, and / or ligands). Attaching exogenous markers to probes allows identification of targets by detecting the presence of exogenous markers. Examples of probes may include protein, nucleic acid (e.g., DNA, RNA) molecules, lipids, and antibody probes. The combination of exogenous markers and functional groups can form any suitable probe, tag, and / or label for detection, including molecular probes, labeled probes, hybridization probes, antibody probes, protein probes (e.g., biotin-binding probes), enzyme labels, fluorescent probes, fluorescent tags, and / or enzyme reporters.

[0217] Although the present disclosure refers to luminous labels, other types of labels can be used with the devices, systems and methods provided herein. Such labels can include mass labels or electrostatic labels.

[0218] While exogenous markers may be added to the sample, endogenous markers may already be part of the sample. Endogenous markers may include any luminescent marker present that can luminesce or "autofluoresce" in the presence of excitation energy. Autofluorescence of endogenous fluorophores may provide label-free and non-invasive labeling without the need to introduce exogenous fluorophores. Examples of such endogenous fluorophores may include, for example, but are not limited to: hemoglobin, oxyhemoglobin, lipids, collagen and elastin crosslinks, reduced nicotinamide adenine dinucleotide (NADH), oxidized flavins (FAD and FMN), lipofuscin, keratins, and / or porphyrins.

[0219] Although some embodiments may involve diagnostic testing by detecting single molecules in a sample, the inventors have also recognized that some embodiments may utilize single molecule detection capabilities to perform nucleic acid (e.g., DNA, RNA) sequencing of one or more nucleic acid fragments, such as genes or polypeptides. Nucleic acid sequencing allows the determination of the order and position of nucleotides in a target nucleic acid molecule. Nucleic acid sequencing techniques may vary in the methods used to determine nucleic acid sequences and in the rates, read lengths, and error rates of sequencing processes. For example, some nucleic acid sequencing methods are based on sequencing by synthesis, in which the characteristics of nucleotides are determined as nucleotides incorporated into a newly synthesized nucleic acid chain that is complementary to the target nucleic acid molecule. Some sequencing by synthesis methods require the presence of a group of target nucleic acid molecules (e.g., copies of the target nucleic acid) or an amplification step of the target nucleic acid to obtain a population of target nucleic acids.

[0220] Since the need for simple and less complex devices for performing single molecule detection and / or nucleic acid sequencing has been recognized, the inventors have conceived a technique for detecting single molecules using a group of fluorescent labels, thereby labeling different molecules using a group of labels, such as optical (e.g., luminescent) labels, thereby labeling different molecules. Such single molecules can be nucleotides or amino acids with labels. The labels can be detected when bound to a single molecule, when released from a single molecule, or when bound to a single molecule and released from a single molecule. In some examples, the labels are fluorescent labels. Each fluorescent label in the selected group is associated with each molecule. For example, a group of four labels can be used to "label" the nucleobases present in DNA, and each label of the group is associated with a different nucleobase, for example, a first label is associated with adenine (A), a second label is associated with cytosine (C), a third label is associated with guanine (G), and a fourth label is associated with thymine (T). In addition, each fluorescent label in the label of the group has different characteristics, which can be used to distinguish the first label of the group from other labels in the group. Thus, each tag may be uniquely identified using one or more of these distinguishable characteristics. For example, but not limited to, characteristics of a tag that may be used to distinguish one tag from another tag may include: the emission energy and / or wavelength of light emitted by the tag in response to an excitation energy, and / or the wavelength and / or energy of the excitation light that excites a particular tag. Distinguishing a tag in a set of four markers uniquely identifies the nucleobase associated with that tag.

[0221] Luminescent signs may vary in the wavelength of the light they emit, the temporal characteristics of the light they emit (e.g., their emission delay period), and their response to excitation energy (e.g., the probability that they absorb excitation photons). Thus, luminescent signs may be identified or distinguished from other luminescent signs based on the detection of these characteristics. Such identification or distinction techniques may be used alone or in any suitable combination.

[0222] In some embodiments, the integrated photodetector described in the present application can measure or distinguish the luminescence lifetime, such as the fluorescence lifetime. The lifetime measurement is based on the excitation of one or more markers (e.g., fluorescent molecules) and the measurement of the time change in the emitted luminescence. The probability of the marker emitting a photon decreases exponentially over time after the marker reaches an excited state. The rate of probability reduction can be a characteristic of the marker and can be different for different markers. Detection of the temporal characteristics of the light emitted by the marker can allow identification of markers relative to each other and / or differentiation of markers. The decrease in the probability of emitting a photon over time can be represented by an exponential decay function p(t)=e^(-t / τ), where p(t) is the probability of photon emission at time t, and τ is a time parameter of the marker. The time parameter τ represents the time after excitation when the probability of the marker emitting a photon is a certain value. The time parameter τ is a characteristic of the marker that can be different from its absorption and emission spectral characteristics. This time parameter τ is called the luminescence lifetime, fluorescence lifetime, or simply the "lifetime" of the marker.

[0223] Figure 1-1 The probability of emitting a photon as a function of time is plotted for two markers with different lifetimes. The marker represented by probability curve B has a probability of emission that decays more rapidly than the probability of emission of the marker represented by probability curve A. The marker represented by probability curve B has a shorter time parameter τ or lifetime than the marker represented by probability curve A. In some embodiments, the marker may have a lifetime in the range of 0.1-20 ns. However, the techniques described herein are not limited to the lifetime of the marker used.

[0224] The lifetime of a marker can be used to distinguish among more than one marker, and / or can be used to identify a marker. In some embodiments, lifetime measurements can be performed where multiple markers with different lifetimes are excited by an excitation source. As an example, four markers with lifetimes of 0.5, 1, 2, and 3 nanoseconds, respectively, can be excited by a light source that emits light with a selected wavelength (e.g., 635 nm, by way of example). These markers can be identified or distinguished from each other based on measuring the lifetime of the light emitted by the markers.

[0225] Lifetime measurements may employ relative intensity measurements by comparing how the intensity changes over time, as opposed to absolute intensity values. Thus, lifetime measurements may avoid some of the difficulties of absolute intensity measurements. Absolute intensity measurements may depend on the concentration of the marker present, and may require calibration steps for varying marker concentrations. In contrast, lifetime measurements may be insensitive to the concentration of the marker.

[0226] Embodiments may utilize any suitable combination of marker features to distinguish a first marker in a group of markers from other markers in the same group. For example, some embodiments may utilize only timing information of emitted light from a marker to identify the marker. In such embodiments, each marker in a selected marker group has an emission lifetime that is different from other markers in the group, and the luminescent markers are all excited by light from a single excitation source. Figure 1-2A Emission timings from four light-emitting signs are shown according to one embodiment, wherein the four signs exhibit different average emission lifetimes (τ). The probability that a sign is measured to have a lifetime of a particular value is referred to herein as the sign's "emission timing." A first emission timing 1-101 from a first light-emitting sign has a peak probability of having a lifetime at τ1, a second emission timing 1-102 from a second light-emitting sign has a peak probability of having a lifetime at τ2, a third emission timing 1-103 from a third light-emitting sign has a peak probability of having a lifetime at τ3, and a fourth emission timing 1-104 from a fourth light-emitting sign has a peak probability of having a lifetime at τ4. In this embodiment, the lifetime probability peaks of the four light-emitting signs may have any suitable values ​​that satisfy the relationship τ1<τ2<τ3<τ4. Due to slight variations in the lifetime of a particular light-emitting sign, the four timing emission patterns may or may not overlap, as shown in FIG. Figure 1-2A In this embodiment, the excitation wavelengths at which the four markers each maximally absorb light from the excitation source are approximately the same, but this need not be the case. Using the marker set described above, four different molecules can be labeled with individual markers from the marker set, these markers can be excited using a single excitation source, and these markers can be distinguished from each other by detecting the emission lifetime of the marker using an optical system and sensor. Although Figure 1-2A Four different markers are shown, but it will be appreciated that any suitable number of markers may be used.

[0227] Other embodiments may utilize any suitable combination of signature features to determine the features of a signature in a set of signatures. Examples of signature features that may be used include, but are not limited to, excitation wavelength, emission wavelength, and emission lifetime. The combination of signature features from phase space and each signature may be represented as a point inside this phase space. The signatures within a set of signatures should be selected so that the "distance" between the signatures within the set is sufficiently large so that the detection mechanism can distinguish each signature from the other signatures in the set. For example, in some embodiments, a set of signatures may be selected in which a subset of the signatures have the same emission wavelength, but different emission lifetimes and / or different excitation wavelengths. In other embodiments, a set of signatures may be selected in which a subset of the signatures have the same emission lifetimes, but different emission wavelengths and / or different excitation wavelengths. In other embodiments, a set of signatures may be selected in which a subset of the signatures have the same excitation wavelength, but different emission wavelengths and / or different emission lifetimes.

[0228] For example but not limited to, Figure 1-2B Emission spectra from four light-emitting signs are shown according to one embodiment, wherein two signs have a first peak emission wavelength and the other two signs have a second peak emission wavelength. The first emission spectrum 1-105 from the first light-emitting sign has a peak emission wavelength at λ1, the second emission spectrum 1-106 from the second light-emitting sign also has a peak emission wavelength at λ1, the third emission spectrum 1-107 from the third light-emitting sign has a peak emission wavelength at λ2, and the fourth emission spectrum 1-108 from the fourth light-emitting sign also has a peak emission wavelength at λ2. In this embodiment, the emission peaks of the four light-emitting signs may have any suitable values ​​that satisfy the relationship λ1<λ2. In embodiments, for example, where the peak emission wavelengths of more than one light-emitting sign are the same, the individual characteristics of each sign having the same emission wavelength must be different. For example, two signs that emit light at λ1 may have different emission lifetimes. Figure 1-3A This is schematically shown in a phase space partitioned by emission wavelength and emission lifetime. The first marker has an emission wavelength λ1 and an emission lifetime τ1, the second marker has an emission wavelength λ1 and an emission lifetime τ4, the third marker has an emission wavelength λ2 and an emission lifetime τ1, and the fourth marker has an emission wavelength λ2 and an emission lifetime τ4. Therefore, in Figure 1-3A All four markers in the marker set shown in are distinguishable from each other. Using such a marker set allows differentiation between the four markers even when the absorption wavelength of the four markers is the same. A sensor can be used that can detect the time of emission of photoluminescence as well as the emission wavelength.

[0229] For example but not limited to, Figure 1-2CAbsorption spectra from four light-emitting signs according to another embodiment are shown. In this embodiment, two signs have a first peak absorption wavelength and the other two signs have a second peak absorption wavelength. The first absorption spectrum 1-109 of the first light-emitting sign has a peak absorption wavelength at λ3, the second absorption spectrum 1-110 of the second light-emitting sign has a peak absorption wavelength at λ4, the third absorption spectrum 1-111 of the third light-emitting sign has a peak absorption wavelength at λ3, and the fourth absorption spectrum 1-112 of the fourth light-emitting sign has a peak absorption wavelength at λ4. It should be noted that in Figure 1-2C Markers that share a common absorption peak wavelength can be distinguished using another other marker characteristic, such as emission lifetime. Figure 1-3B This is schematically shown in a phase space divided by absorption wavelength and emission lifetime. The first marker has an absorption wavelength λ3 and an emission lifetime τ1, the second marker has an absorption wavelength λ3 and an emission lifetime τ4, the third marker has an absorption wavelength λ4 and an emission lifetime τ1, and the fourth marker has an absorption wavelength λ4 and an emission lifetime τ4. Therefore, in Figure 1-3A All four flags in the flag group shown in are distinguishable from each other.

[0230] The use of such a marker set allows differentiation between four markers, even when the emission wavelengths of the four markers are indistinguishable. It is possible to use two excitation sources emitting at different wavelengths or a single excitation source capable of emitting at multiple wavelengths, together with a sensor that can detect the emission time of the photoluminescence. If the wavelength of the luminescence excited for each detected emission event is known, then it can be determined which marker is present. The excitation source can be switched between a first excitation wavelength and a second excitation wavelength, which is called interleaving. Alternatively, two or more pulses of a first excitation wavelength can be used, followed by two or more pulses of a second excitation wavelength.

[0231] The number of excitation sources or excitation wavelengths used to distinguish between markers is not limited to two, and in some embodiments more than two excitation wavelengths or energies may be used to distinguish between markers. In such embodiments, the intensity or number of photons emitted in response to multiple excitation wavelengths may be used to distinguish between markers. By detecting the number of photons emitted in response to exposing the marker to a certain excitation wavelength, a marker may be distinguished from multiple markers. In some embodiments, the marker is identified by irradiating the marker with one of multiple excitation energies at a time and identifying the excitation energy from the multiple excitation energies in which the marker emits the highest number of photons. In other embodiments, the number of photons emitted from the marker in response to different excitation energies may be used to identify the marker. A first marker having a probability of emitting photons in response to a first excitation energy higher than a second excitation energy may be distinguished from a second marker having a probability of emitting photons in response to a second excitation energy higher than the first excitation energy. In this way, a marker having a distinguishable probability of emitting a specific amount of photons in response to different excitation energies may be identified by measuring the emitted photons while exposing an unknown marker to different excitation energies. In such embodiments, the marker may be exposed to multiple excitation energies and the identification of the marker may be achieved by determining whether the marker emits any light and / or a specific number of emitted photons. Any suitable number of excitation energy sources may be used. In some embodiments, four different excitation energies may be used to distinguish among different markers (e.g., four different markers). In some embodiments, three different excitation energies may be used to distinguish among different markers. The presence of a marker may be distinguished by combining the amount of photons emitted in response to different excitation energies (including emission lifetime and emission spectrum) and other characteristics of the marker.

[0232] In other embodiments, more than two characteristics of a marker in a marker set may be used to distinguish which marker is present. Figure 1-4 An illustrative phase space partitioned by the absorption wavelength, emission wavelength, and emission lifetime of each marker is shown. Figure 1-4 In the embodiment of the present invention, eight different markers are distributed in phase space. Four of the eight markers have the same emission wavelength, the different four markers have the same absorption wavelength and the different four markers have the same emission lifetime. However, when all three characteristics of the marker are considered, each marker can be distinguished from each other marker. The embodiment is not limited to any number of markers. This concept can be extended to include any number of markers that can be distinguished from each other using at least these three marker characteristics.

[0233] Although not shown in the figures, other embodiments may determine the characteristics of the illuminated markers based solely on absorption frequency. Such an embodiment is possible if the excitation light can be tuned to a specific wavelength that matches the absorption spectrum of each marker in the marker group. In such an embodiment, the optical system and sensor used to direct and detect the light emitted from each marker does not need to be able to detect the wavelength of the emitted light. This can be advantageous in some embodiments because it reduces the complexity of the optical system and sensor because detection of the emission wavelength is not required in such embodiments.

[0234] As described above, the inventors have recognized and appreciated the need to be able to distinguish different luminescent signatures from one another using various characteristics of each signature. The type of characteristics used to determine signature characteristics affects the physical devices used to perform this analysis. The present application discloses several embodiments of apparatus, devices, instruments, and methods for performing these various experiments.

[0235] The inventors have recognized and appreciated that a low-cost, disposable, integrated device including optical components and sensors can be used in conjunction with an instrument including an excitation source to measure different characteristics of light emitted from one or more markers used to tag a biological sample, thereby analyzing the sample. Using a low-cost integrated device can reduce the cost of performing a given biological assay. The biological sample is placed on the integrated device and can be discarded when the biological assay is completed. The integrated device interacts with a more expensive multiple-use instrument that can be reused with many different disposable integrated devices. The low-cost integrated device that interacts with a compact portable instrument can be used anywhere in the world without the constraints of a high-cost biological laboratory that requires laboratory expertise to analyze the sample. Therefore, automated biological analysis can be brought to areas where quantitative analysis of biological samples could not be performed before. For example, a blood test for an infant can be performed by placing a blood sample on a disposable integrated device, analyzing the disposable integrated device in a small portable instrument, and processing the results using a computer connected to the instrument for immediate viewing by the user. The data can also be transmitted to a remote location over a data network for analysis and / or archived for later clinical analysis. Alternatively, the instrument may include one or more processors for analyzing data acquired from sensors of the integrated device.

[0236] Various implementations are described in more detail below.

[0237] I. System overview

[0238] The system includes an integrated device and an instrument for interacting with the integrated device. The integrated device includes an array of pixels, wherein one pixel includes a sample well and at least one sensor. The surface of the integrated device has a plurality of sample wells, wherein the sample well is used to receive a sample from a sample placed on the surface of the integrated device. A sample may contain a plurality of samples, and in some embodiments, different types of samples. A plurality of sample wells may be designed so that at least a portion of the sample wells are used to receive a sample from a sample. In some embodiments, these numbers of samples inside the sample wells may be distributed among the sample wells so that some sample wells accommodate one sample and other sample wells accommodate zero, two or more samples. For example, the sample may contain a plurality of single-stranded DNA templates, and the sample wells on the surface of the integrated device may receive the single-stranded DNA templates. The sample wells of at least a portion of the integrated device may accommodate the single-stranded DNA templates. The sample may also accommodate a labeled dNTP, which then enters the sample well and allows identification of nucleotides when it is incorporated into the complementary strand of DNA. In this example, "sample" may refer to both the single-stranded DNA and the labeled dNTPs that are now incorporated using a polymerase. In some embodiments, the sample may include a single-stranded DNA template, and then the tagged dNTPs may be introduced into the sample well as the nucleotides are incorporated into the complementary strand of the DNA inside the sample well. In this way, the timing of nucleotide incorporation may be controlled when the tagged dNTPs are introduced into the sample well of the integrated device.

[0239] Excitation energy is provided from an excitation source located at a location separate from the pixel array of the integrated device. The excitation energy is directed at least partially toward one or more pixels using components of the integrated device to illuminate an illumination region within the sample well. Then, when located within the illumination region and in response to being illuminated by the excitation energy, the marker or tag can emit emission energy. In some embodiments, the one or more excitation sources are part of an instrument of the system, wherein the components of the instrument and the integrated device are used to direct the excitation energy toward the one or more pixels. In other embodiments, the one or more excitation sources are located on the integrated device but in a separate region from the array of pixels, and the components in the integrated device are used to direct the excitation energy from the excitation source region to the one or more pixels.

[0240] Then, one or more sensors within a pixel of the integrated device can be used to detect the emission energy emitted by the sample. In some embodiments, the sizes of multiple sensors are designed and arranged to capture the spatial distribution of emission energy. In some embodiments, one or more sensors can be used to detect timing characteristics (e.g., fluorescence lifetime) related to the emission energy of the sample. Then, the output signal from one or more sensors can be used to distinguish a mark from multiple marks, wherein the multiple marks can be used to identify the sample inside the sample. In some embodiments, multiple excitation energies can be used to excite the sample, and the emission energy and / or timing characteristics emitted by the sample in response to the multiple excitation energies can distinguish a mark from multiple marks.

[0241] A schematic diagram of the system 2-100 is shown in Figure 2-1A and Figure 2-1B . The system includes two integrated devices 2-102 that interact with an instrument 2-104. In some embodiments, the instrument 2-104 may include one or more excitation sources 2-106. In some embodiments, the excitation sources may be external to both the instrument 2-104 and the integrated device 2-102, and the instrument 2-104 may be configured to receive excitation energy from the excitation sources and direct the excitation energy to the integrated device. The integrated device is interconnected with the instrument using any suitable socket that accepts the integrated device and holds it in precise optical alignment with the excitation source. The excitation source 2-106 can be used to provide excitation energy to the integrated device 2-102. Although in Figure 2-1B The excitation source is shown as being located on the instrument, but in some cases the excitation source may also be located on the integrated device in an area separate from the pixel. Figure 2-1B As schematically shown, the integrated device 2-102 has a plurality of pixels, wherein each pixel 2-112 is capable of independently analyzing a sample. Such a pixel 2-112 may be referred to as a "passive pixel" because the pixel receives excitation energy from an excitation source 2-106 that is separate from the pixel, wherein the excitation source excites the plurality of pixels. Each pixel 2-112 has a sample well 2-108 for holding and analyzing the sample, and a sensor 2-110 for detecting emission energy emitted by the sample in response to irradiating the sample with the excitation energy provided by the excitation source 2-106. In some embodiments, each sensor 2-110 may include a plurality of sub-sensors, each sub-sensor being configured to detect emission energy of a different wavelength from the sample.

[0242] Optical elements for directing and coupling excitation energy to the sample well 2-108 are located on both the integrated device 2-102 and the instrument 2-104. Such source-to-well elements may include: grating couplers located on the integrated device 2-102 for coupling excitation energy to the integrated device, waveguides for delivering excitation energy to each pixel 2-112, and lenses, plasmonic elements on the integrated device, and dielectric coatings for directing excitation energy received from the instrument 2-104 to the sample well 2-108. In addition, optical elements located on the integrated device direct emission energy from the sample well toward the sensor. Such well-to-sample elements may include components that direct emission energy into a radiation pattern, wherein the radiation pattern is dependent on the emission energy emitted by the sample in the sample well. The sample well 2-108, a portion of the excitation source-to-well optical components, and the sample well-to-sensor optical components are located on the integrated device 2-102. The excitation source 2-106 and a portion of the excitation source-to-well components are located in the instrument 2-104. In some implementations, a single component may play a role in coupling excitation energy to a sample well 2-108 and transferring emission energy from the sample well 2-108 to the sensor 2-110.

[0243] like Figure 2-1B As shown in , the integrated device includes a plurality of pixels, each pixel 2-112 being associated with its own individual sample well 2-108 and sensor 2-110. The plurality of pixels may be arranged in an array, and any suitable number of pixels may be present. For example, the integrated device 2-102 may include between 100 and 1,000 pixels according to some embodiments, between 1,000 and 10,000 pixels according to some embodiments, between 10,000 and 100,000 pixels according to some embodiments, between 100,000 and 1,000,000 pixels according to some embodiments, and between 1,000,000 and 10,000,000 pixels according to some embodiments. In some embodiments, fewer or more pixels may be present on the integrated device 2-102. The integrated device 2-102 and the instrument 2-104 may include a multi-channel high-speed communication link for processing data associated with a large pixel array (e.g., more than 1000 pixels).

[0244] The excitation source 2-106 can be any suitable excitation source arranged to deliver excitation energy to at least one sample well. In some embodiments, an array of one or more excitation sources is located adjacent to an array of pixels on the same integrated device. In other embodiments, one or more excitation sources are on a second substrate mounted in close proximity to the substrate on which the array of pixels is formed.

[0245] The instrument 2-104 is interconnected with the integrated device 2-102 via an integrated device interface 2-114. The integrated device interface 2-114 may include components for positioning and / or aligning the integrated device 2-102 to the instrument 2-104 to improve the coupling of excitation energy from the excitation source 2-106 to the integrated device 2-102. In some embodiments, the excitation source 2-106 includes a plurality of excitation sources, which are combined to deliver excitation energy to the integrated device 2-102. A plurality of excitation sources may be used to generate a variety of excitation energies or wavelengths. The integrated device interface 2-114 may receive a readout signal from a sensor in a pixel located on the integrated device. In addition, the integrated device interface 2-114 may be designed so that the integrated device is attached to the instrument by fixing the integrated device to the integrated device interface 2-114.

[0246] The instrument 2-104 includes a user interface 2-116 for controlling the operation of the instrument 2-104. The user interface 2-116 is used to allow the user to input the information of the command and / or setting, such as for controlling the operation of the instrument, into the instrument. In some embodiments, the user interface 2-116 may include a button, a switch, a dial, and a microphone for voice commands. In addition, the user interface 2-116 may allow the user to receive feedback (such as correct alignment) about the performance of the instrument and / or the integrated device, and / or information obtained based on the readout signal from the sensor on the integrated device. In some embodiments, the user interface 2-116 may utilize a speaker to provide feedback for providing auditory feedback and an indicator light and / or a display screen for providing visual feedback. In some embodiments, the instrument 2-104 includes a computer interface 2-118 for connecting to a computing device 2-120. Any suitable computer interface 2-118 and computing device 2-120 may be used. For example, the computer interface 2-118 may be a USB interface or a FireWire interface. The computing device 2-120 may be any general-purpose computer, such as a laptop or a desktop computer. The computer interface 2-118 facilitates information communication between the instrument 2-104 and the computing device 2-120. Input information for controlling and / or setting the instrument 2-104 may be provided via the computing device 2-120 connected to the computer interface 2-118 of the instrument. In addition, output information may be received by the computing device 2-120 via the computer interface 2-118. Such output information may include feedback on the performance of the instrument 2-104 and / or the integrated device 2-112, and information based on the readout signal of the sensor 2-110. The instrument 2-104 may also include a processing device 2-122 for analyzing the data received from the sensor 2-110 and / or sending a control signal to the excitation source 2-106. In some embodiments, the processing device 2-122 may include a general-purpose processor, a specialized processor (e.g., a central processing unit (CPU) such as one or more microprocessor or microcontroller cores, a field programmable gate array (FPGA), an application-specific integrated circuit (ASIC), a custom integrated circuit, a digital signal processor (DSP), or a combination thereof). In some embodiments, processing of data from the sensor 2-110 may be performed by both the processing device 2-122 and the external computing device 2-120. In other embodiments, the computing device 2-120 may be omitted and processing of data from the sensor 2-110 may be performed only by the processing device 2-122.

[0247] A schematic cross-sectional view of an integrated device 3-102 showing a row of pixels is shown in Figure 3-1AEach pixel 3-112 includes a sample well 3-108 and a sensor 3-110. The sensor 3-110 may be aligned and positioned with respect to the sample well 3-112. When an excitation source is coupled to the integrated device, excitation energy is provided to one or more pixels. Figure 3-1B is a schematic diagram illustrating coupling of an excitation source 3-106 to an integrated device 3-102. In the integrated device 3-102, the excitation source 3-106 provides excitation energy 3-130 (indicated by a dashed line) in the integrated device 3-102. Figure 3-1B The path of excitation energy from an excitation energy source 3-106 to a sample well 3-108 in a pixel 3-112 is shown. Components located at a location separate from the integrated device may be used to position and align the excitation source 3-106 to the integrated device. Such components may include optical components, including lenses, mirrors, prisms, apertures, attenuators, and / or optical fibers. Other mechanical components may be included in the instrument to allow control of one or more alignment components. Such mechanical components may include actuators, stepper motors, and / or knobs. The integrated device includes components that direct excitation energy 3-130 toward pixels in the integrated device. Within each pixel 3-112, the excitation energy is coupled to the sample well 3-108 associated with that pixel. Although Figure 3-1B Excitation energy coupling to each sample well in a row of pixels is shown, but in some embodiments, the excitation energy may not be coupled to all pixels in the row. In some embodiments, the excitation energy may be coupled to a portion of the pixels or sample wells in a row of pixels in an integrated device. The excitation energy may illuminate the sample located inside the sample well. The sample may reach an excited state in response to being illuminated by the excitation energy. When the sample is in the excited state, the sample may emit emission energy, and the emission energy may be detected using a sensor. Figure 3-1B The path of emission energy 3-140 from a sample well 3-108 to a sensor 3-110 of a pixel 3-112 is schematically shown (illustrated as a solid line). The sensor 3-110 in the pixel 3-112 may be configured and positioned to detect emission energy from the sample well 3-108. In some embodiments, the sensor 3-110 may include one or more sub-sensors.

[0248] A sample to be analyzed may be introduced into a sample well 3-108 of a pixel 3-112. The sample may be a biological sample or any other suitable sample, such as a chemical sample. The sample may include a plurality of molecules, and the sample well may be used to separate single molecules. In some cases, the size of the sample well may function to confine a single molecule within the sample well, thereby allowing the single molecule to be measured. An excitation source 3-106 may be used to deliver excitation energy into the sample well 3-108, thereby exciting the sample or at least one luminescent marker attached to or associated with the sample when the sample is within an excitation region within the sample well 3-108. In a sample of a plurality of molecules, the type of luminescent marker may be uniquely associated with one type of molecule. During or after excitation, the luminescent marker may emit emission energy. When a plurality of markers are used, they may emit light at different characteristic energies. In some embodiments, a plurality of markers may have different characteristic lifetimes. In addition, a plurality of markers may differ in response to a plurality of excitation energies. The markers used for a sample may be distinguished by their excitation response, characteristic energy or wavelength, and / or characteristic lifetime. Emission from the sample may radiate from the sample well 3-108 to the sensor 3-110.

[0249] The components may focus the emission energy toward the sensor, and additionally or alternatively, spatially separate the emission energy having each characteristic energy or wavelength. The excitation energy emitted from the excitation source 3-106 may be directed toward the sample well 3-108 in any suitable manner and used to excite at least one sample received in the sample well 3-108. According to some embodiments, the excitation source 3-106 may excite a sample that may emit light. The excitation source may provide one or more excitation energies to the sample well 3-108. In some embodiments, the excitation source 3-106 may excite one or more markers that emit light or energy in response to the excitation and are attached to or associated with the sample. The light emission or energy generated based on the excitation may be directed to the sensor 3-110, which may be used to detect the intensity and / or timing of the received emission. Non-limiting examples of luminescence are photoluminescence, fluorescence, and phosphorescence.

[0250] In some embodiments, an integrated device may include components to direct emission energy into a radiation pattern that depends on the spectral range of the emission energy. A sensor or a sensor region containing multiple sub-sensors may detect the spatial distribution of the emission energy that depends on the radiation pattern. Signs emitting different emission energies and / or spectral ranges may form different radiation patterns. The sensor or sensor region may detect information about the spatial distribution of the emission energy, which can be used to identify a sign among multiple signs.

[0251] One or more emission energies may be detected by the sensor and converted into at least one electrical signal. The electrical signal may be transmitted along the integrated device interface (e.g. Figure 2-1BThe electrical signals may then be processed and / or analyzed. The processing or analysis of the electrical signals may be performed on a suitable computing device located on or off the instrument 2-104 (e.g., in a computer). Figure 2-1B The process is performed on the computing device 2-120 shown in FIG. 2 .

[0252] The integrated device 2-210 may be Figure 2-2 As shown in . Electronic, optical and related structures can all be incorporated into a single substrate 2-200. The integrated device may include an array of pixels 2-205, and integrated electronic circuits. The integrated electronic circuits may include drive and readout circuits 2-215 of sensors coupled to the pixel array, and signal processing circuits. The signal processing circuits may include an analog-to-digital converter 2-217, and one or more field programmable gate arrays and / or digital signal processors 2-219. Some embodiments may have more circuit components, and some embodiments may have fewer circuit components integrated on a substrate. Although in Figure 2-2 The components of the integrated device are illustrated on a single level, but the components may be fabricated on multiple levels on the substrate 2-200.

[0253] In some embodiments, there may be an optical element (not shown) located on the integrated device that is arranged to direct and couple excitation energy from one or more excitation sources to a sample well. Such source-to-well elements may include plasmonic structures and other micromachined structures located adjacent to the sample well. Additionally, in some embodiments, there may be an optical element located on the integrated device that is used to direct emission energy from the sample well to a corresponding sensor. Such well-to-sample elements may include plasmonic structures and other micromachined structures located adjacent to the sample well. In some embodiments, a single component may function in both coupling excitation energy to a sample well and delivering emission energy from a sample well to a corresponding sensor.

[0254] In some embodiments, an integrated device may include more than one type of excitation source for exciting a sample at a sample well. For example, there may be multiple excitation sources for generating multiple excitation energies or wavelengths for exciting a sample. In some embodiments, a single excitation source emits multiple wavelengths that may be used to emit light to excite a sample in a sample well. In some embodiments, each sensor at a pixel of an integrated device may include multiple sub-sensors for detecting different emission energy signatures from a sample.

[0255] In operation, parallel analysis of samples within the sample wells is performed by exciting the samples within the wells with an excitation source and detecting signals emitted from the samples with sensors. Emission energy from the samples can be detected by corresponding sensors and converted into at least one electrical signal. In some embodiments, one or more of the signals formed can be processed on an integrated device or transmitted to an instrument for processing by a processing device and / or a computing device. Signals from the sample wells can be received and processed independently of signals associated with other pixels.

[0256] When the excitation source delivers excitation energy to the sample well, at least one sample within the well may luminesce, and the resulting emission may be detected by the sensor. As used herein, the phrases "the sample may luminesce" or "the sample may emit radiation" or "emission from the sample" mean that the fluorescent label, marker, or reporter gene, the sample itself, or a reaction product associated with the sample may generate the emitted radiation.

[0257] In some embodiments, samples may be labeled with one or more markers, and an instrument may be used to distinguish emissions associated with the markers. For example, a sensor may be used to convert photons from the emission energy into electrons to form an electrical signal that can be used to distinguish lifetimes that depend on the emission energy from a particular marker. By using markers with different lifetimes to label samples, specific samples may be identified based on the electrical signals formed as detected by the sensors. In some embodiments, components of the integrated device may affect the emission from the sample well to produce a spatial emission distribution pattern that depends on the emission wavelength. A corresponding sensor for the sample well may be used to detect the spatial distribution pattern from the sample well and produce a signal that distinguishes between different emission wavelengths, as described in more detail below.

[0258] II. Integrated Device

[0259] The integrated device can be used to receive excitation energy from an external excitation energy source. In some embodiments, a region of the device can be used to couple to an excitation energy source located remote from the integrated device. Components of the integrated device can guide excitation energy from the excitation source coupling region to at least one pixel. In some embodiments, at least one waveguide can be used to deliver excitation energy to at least one pixel having a sample well. A sample located within the sample well can emit emission energy in response to being irradiated with the excitation energy. One or more sensors located within the pixel are used to receive the emission energy.

[0260] According to some embodiments, Figure 3-2The components and / or layers of the integrated device 3-200 shown in FIG. 3 include a sample well 3-203, a waveguide 3-220, and a sensor 3-275 integrated into one device. The sample well 3-203 may be formed in a sample well layer 3-201 of the integrated device 3-200. In some embodiments, the sample well layer 3-201 may be metal. The sample well 3-203 may have a dimension D that may represent a cross-sectional dimension of the sample well. tv The sample well 3-203 may function as a nanopore and have one or more sub-wavelength dimensions that result in a field enhancement effect that increases the excitation intensity of the sample in the sample well 3-203. The waveguide 3-220 is used to deliver excitation energy from an excitation source 3-230 located at a position remote from the integrated device 3-200 to the sample well 3-203. The waveguide 3-220 may be formed in a layer between the sample well layer 3-201 and the sensor 3-275. The design of the integrated device 3-200 allows the sensor 3-275 to collect luminescence emitted from the sample in the sample well 3-203. At least part of the time, the sample absorbs the excitation energy and emits photons having an energy less than the excitation energy, which is referred to as emission energy or luminescence.

[0261] Having a sample well 3-203 and a sensor 3-275 on an integrated device 3-200 can reduce the optical distance that light travels from the sample well 3-203 to the sensor 3-275. The dimensions of the integrated device 3-200 or components within the integrated device can be used for a certain optical distance. The optical properties of the materials of the components and / or one or more layers of the device can determine the optical distance between the sample well and the sensor. In some embodiments, the thickness of the one or more layers can determine the optical distance between the sample well and the sensor in a pixel. Additionally or alternatively, the refractive index of the material of the one or more layers of the integrated device 3-200 can determine the optical distance between the sample well 3-203 and the sensor 3-275 in a pixel. Such an optical distance between the sample well and the sensor in a pixel can be less than 1 mm, less than 100 microns, less than 25 microns, and / or less than 10 microns. One or more layers may be present between the sample well layer 3-201 and the waveguide layer 3-220 to improve coupling of excitation energy from the waveguide 3-220 to the sample well 3-203. Figure 3-2The integrated device 3-200 shown in FIG. 3 shows only a single layer 3-210, but multiple layers may be formed between the sample well 3-203 and the waveguide 3-220. The layer 3-210 may be formed to have optical properties to improve coupling of excitation energy from the waveguide 3-220 to the sample well 3-203. The layer 3-210 may be used to reduce scattering and / or absorption of excitation energy and / or increase luminescence from a sample in the sample well 3-203. According to some embodiments, the layer 3-210 may be optically transparent so that light may travel to and from the sample well 3-203 with little attenuation. In some embodiments, the layer 3-210 may be formed using a dielectric material. In some embodiments, an excitation energy coupling component may be provided within the layer 3-210 and / or at the interface between the layer 3-210 and the sample well layer 3-201 to improve coupling of excitation energy from the waveguide 3-220 to the sample well 3-203. As an example, an energy harvesting component 3-215 formed at the interface between the sample well layer 3-201 and the layer 3-210 may be used to improve coupling of excitation energy from the waveguide 3-220 to the sample well 3-203. The energy harvesting component 3-215 is optional, and in some embodiments, the configuration of the waveguide 3-220 and the sample well 3-203 may allow for sufficient coupling of excitation energy in the absence of the excitation energy harvesting component 3-215.

[0262] The luminescence or energy emitted from the sample in the sample well 3-203 may be transferred to the sensor 3-275 in a variety of ways, some examples of which are described in more detail below. Some embodiments may utilize optical components to increase the likelihood of directing a particular wavelength of light to an area or portion of the sensor 3-275 dedicated to detecting that particular wavelength of light. The sensor 3-275 may include multiple portions for simultaneously detecting different wavelengths of light that may correspond to emissions from different luminescent markers.

[0263] There may be one or more layers between the sample well 3-203 and the sensor 3-275 that may be used to improve the collection of luminescence from the sample well 3-203 toward the sensor 3-275. A luminescence directing component may be located at the interface between the sample well layer 3-201 and the layer 3-210. An energy harvesting component 3-215 may focus emission energy toward the sensor 3-275 and may additionally or alternatively spatially separate emission energy having different characteristic energies or wavelengths. Such an energy harvesting component 3-215 may include a grating structure for directing luminescence toward the sensor 3-275. In some embodiments, the grating structure may be a series of concentric rings or a "bull's eye" grating structure configuration. Concentric circular gratings may protrude from the bottom surface of the sample well layer 3-201. These circular gratings may function as plasmonic elements that may be used to reduce the spread of signal light and direct signal light toward an associated sensor 3-275. Such a bull's eye grating may more efficiently direct luminescence toward the sensor 3-275.

[0264] A layer 3-225 may be formed adjacent to the waveguide. The optical properties of the layer 3-225 may be selected to improve the collection of luminescence from the sample well to the sensor 3-275. In some embodiments, the layer 3-225 may be a dielectric material. A baffle may be formed between the sample well layer 3-201 and the sensor 3-275. The baffle 3-240 may be configured such that the sensor 3-275 receives luminescence corresponding to the sample well 3-203 and reduces luminescence, and reflected / scattered excitation from other sample wells. The filter element 3-260 may be positioned and configured to reduce excitation energy reaching the sensor 3-275. In some embodiments, the filter element 3-260 may include a filter that selectively passes emission energy of one or more markers used to tag the sample. In embodiments where a sample well array and a sensor array are used and each sample well has a corresponding sensor, a baffle corresponding to each sample well may be formed to reduce luminescence from other sample wells and reflected and / or scattered excitation light collected by the sensor corresponding to the sample well.

[0265] One or more layers may be formed between the waveguide 3-220 and the sensor 3-275 to reduce the transmission of excitation energy to the sensor. In some embodiments, a filter element may be formed between the waveguide 3-220 and the sensor 3-275. Such a filter element may be used to reduce the transmission of excitation energy to the sensor 3-275 while allowing luminescence from the sample well to be collected by the sensor 3-275.

[0266] The one or more emission energies may be detected by the sensor 3-275 and converted into at least one electrical signal. The one or more electrical signals may be transmitted along one or more rows or columns of conductive lines (not shown) to an integrated electronic circuit on the substrate 3-200 for subsequent signal processing.

[0267] Above Figure 3-2 The description is a schematic diagram of the components of a portion of the device according to some embodiments. In some embodiments, Figure 3-2 One or more elements may not be present or may be located in a different position. The components of the integrated device 3-200 and the excitation source 3-230 are described in more detail below.

[0268] A. Excitation source coupling region

[0269] The integrated device may have an excitation source coupling region for coupling with an external excitation energy source and directing excitation toward at least one pixel in a pixel region of the integrated device. The excitation source coupling region may include one or more structures for coupling light into at least one waveguide. Any suitable mechanism for coupling excitation energy into a waveguide may be employed.

[0270] In some embodiments, excitation energy from an external excitation source can be coupled to a waveguide of an integrated device via edge coupling. The edge of the integrated device can include an end of a waveguide such that an external excitation source located near the end of the waveguide can couple light into the waveguide. In such embodiments, fabrication of the excitation source coupling region can include positioning an end of the waveguide at the edge of the integrated device. Figure 4-1A An example of edge coupling is shown. An optical fiber 4-106 for propagating excitation energy is located near an edge of an integrated device 4-102, wherein one end of a waveguide 4-104 of the integrated device 4-102 is located at the edge, so that the optical fiber 4-106 can couple light into the waveguide 4-104. In this embodiment, monitoring the alignment of the optical fiber 4-106 or other excitation source with the waveguide 4-104 can increase the amount of light provided by the optical fiber to the waveguide.

[0271] In some embodiments, a prism can couple light to a waveguide. The light can be directed and refracted by the prism to match the optical phase frequency of the propagating waveguide mode. The refractive index of the material used for the prism can be selected to improve coupling with the waveguide. In some cases, the prism has a higher refractive index and a narrower gap relative to the waveguide. In other embodiments, light can be coupled directly to one end of the waveguide. The edges of the integrated device can be fully polished to allow focusing and alignment of the light to the waveguide.

[0272] The excitation source coupling region of the integrated device may include structural components for coupling with an external excitation source. The integrated device may include a grating coupler for coupling with an external excitation source, which is located adjacent to the surface of the integrated device and guides light toward at least one waveguide of the integrated device. Features of the grating coupler, such as size, shape, and / or grating structure, may be formed to improve the coupling of excitation energy from the excitation source to the waveguide. The grating coupler may include one or more structural components, wherein the spacing between each structural component may function to propagate light. One or more size grating couplers may provide ideal coupling of light having a certain characteristic wavelength.

[0273] The integrated device may also include a waveguide having a tapered region at one end of the waveguide. One or more dimensions of the waveguide perpendicular to the direction of light propagation in the waveguide may be larger at one end of the waveguide, thereby forming a tapered region of the waveguide. In some embodiments, the tapered region of the waveguide may have a dimension perpendicular to the propagation of light and parallel to the surface of the integrated device, which dimension is larger at one end of the waveguide and becomes smaller along the length of the waveguide. In an embodiment including a grating coupler, the tapered region may be located near the grating coupler so that the larger end of the tapered region is located closest to the grating coupler. The size and shape of the tapered region can be designed to improve the coupling of light between the grating coupler and the waveguide by expanding one or more dimensions of the waveguide, thereby allowing improved mode overlap of the waveguide with the grating coupler. In this way, an excitation source located adjacent to the surface of the integrated device can couple light into the waveguide using the grating coupler. This combination of a grating coupler and a waveguide tapered structure can allow for more tolerance in the alignment and positioning of the excitation source with the integrated device.

[0274] An exemplary integrated device with a grating coupler and a waveguide with a tapered region is shown in Figure 4-1B . The integrated device 4-100 has an excitation source coupling region including a waveguide with a tapered region 4-114 and a grating coupler 4-116. The tapered region 4-114 has one end that is parallel to the surface 4-112 of the integrated device 4-100 and perpendicular to the larger dimension of light propagation along the waveguide. The size and shape of the end of the tapered region 4-114 can be designed to provide suitable coupling between the grating coupler 4-116 and the waveguide. An optical fiber 4-120 or other suitable excitation source positioned relative to the grating coupler 4-116 can couple excitation energy to the waveguide.

[0275] The grating coupler may be located in an area of ​​the integrated device outside of a pixel of the integrated device. On the surface of the integrated device, the sample well of each pixel may occupy a surface area separate from the excitation source coupling region. An excitation source located near the surface of the excitation source coupling region may be coupled to the grating coupler. The sample well may be located at a position separate from the excitation source coupling region to reduce interference of light from the excitation source on pixel performance. The grating coupler of the integrated device may be formed inside one or more layers of the integrated device including the waveguide. In this way, the excitation source coupling region of the integrated device may include a grating coupler in the same plane of the integrated device as the waveguide. The grating coupler may be configured for a specific set of beam parameters, including beam width, incident angle, and / or polarization of incident excitation energy.

[0276] A cross-sectional view of the integrated device 4-200 is shown in Figure 4-2 The integrated device 4-200 includes at least one sample well 4-222 formed in a layer 4-223 of the integrated device 4-200. The integrated device 4-200 includes a grating coupler 4-216 and a waveguide 4-220 formed in substantially the same plane of the integrated device 4-200. In some embodiments, the grating coupler 4-216 and the waveguide 4-220 are formed from the same layer of the integrated device 4-200 and may include the same material. The excitation source coupling region 4-201 of the integrated device 4-200 includes the grating coupler 4-216. Figure 4-2 As shown in FIG, a sample well 4-222 is located on a surface of an integrated device 4-200 outside of an excitation source coupling region 4-201. An excitation source 4-214 positioned relative to the integrated device 4-200 may provide excitation energy incident on a surface 4-215 of the integrated device 4-200 within the excitation source coupling region 4-201. By positioning a grating coupler 4-216 within the excitation source coupling region 4-201, the grating coupler 4-216 may couple with the excitation energy from the excitation source 4-214 and couple the excitation energy to a waveguide 4-220. The waveguide 4-220 is used to propagate the excitation energy to the vicinity of one or more sample wells 4-222.

[0277] The grating coupler can be made of one or more materials. In some embodiments, the grating coupler can include alternating regions of different materials parallel to the direction of light propagation in the waveguide. Figure 4-2 As shown in , the grating coupler 4-216 includes a structure surrounded by a material 4-224. The one or more materials comprising the grating coupler may have one or more refractive indices suitable for coupling and propagating light. In some embodiments, the grating coupler may include a structure composed of a material having a larger refractive index surrounded by a material. As an example, the grating coupler may include a structure composed of silicon nitride and surrounded by silicon dioxide.

[0278] Any suitable size and / or grid line spacing may be used to form the grating coupler. The grating coupler 4-216 may have a dimension perpendicular to the propagation of light through the waveguide (e.g., in the y-direction) of about 50 nm, about 100 nm, about 150 nm, or about 200 nm, such as Figure 4-2 The spacing between the structures of the grating coupler parallel to the direction of light propagation in the waveguide (e.g., along the z-direction) is shown in FIG. Figure 4-2 , can have any suitable distance. The grid line spacing can be about 300nm, about 350nm, about 400nm, about 420nm, about 450nm or about 500nm. In some embodiments, the grid line spacing is variable inside the grating coupler. The grating coupler 4-216 can have one or more dimensions roughly parallel to the surface 4-215 of the integrated device 4-200, and this dimension provides suitable area for coupling with the external excitation source 4-214. This area of ​​the grating coupler 4-216 can be consistent with one or more dimensions of the light beam from the excitation source 4-214, so that the light beam overlaps with the grating coupler 4-215. The grating coupler can have a region for a beam diameter of about 10 microns, about 20 microns, about 30 microns or about 40 microns.

[0279] A cross-sectional view of a portion of an exemplary excitation source coupling region of an integrated device is shown in Figure 4-3A The excitation source coupling region includes a grating coupler 4-326 and a reflective layer 4-336, the reflective layer being used to reflect the excitation light back toward the grating coupler 4-326 through the grating coupler 4-326. The grating coupler 4-326 may include a Figure 4-3AThe structures of the grating line spacing represented by Λ in the z-direction shown in . These structures may have linear, curved, or any other suitable shape. In some embodiments, the grating coupler 4-326 may have dimensions along the y-direction similar to the waveguide 4-330, as represented by the arrows on either side of the waveguide 4-330. The grating coupler 4-326 is surrounded by the region 4-324, and the combination of the materials constituting the grating coupler 4-324 and the region 4-324 can provide the desired coupling of light to the waveguide 4-330. The refractive index of the waveguide 4-330, the grating coupler 4-326, and / or the surrounding material 4-324 can affect the coupling of the excitation energy to the waveguide and the total coupling efficiency of the excitation energy to the waveguide 4-330. The excitation source coupling region of the integrated device may have a coupling efficiency greater than about 50%. The grating coupler 4-326 may be used to couple one or more characteristics of the incident beam of excitation energy 4-314, including a characteristic wavelength, a beam diameter (represented by an arrow), and a beam angle of incidence (represented by θ). The grating coupler 4-326 may be configured for a beam having a beam diameter of approximately 10 microns, approximately 20 microns, approximately 30 microns, or approximately 40 microns. The grating coupler 4-326 may be configured for a beam having an angle of incidence of approximately 2 degrees, approximately 5 degrees, or approximately 7 degrees. The grating coupler 4-326 may be used to couple to excitation energy of a certain polarization, such as TM or TE polarized light.

[0280] A simulation of light coupling from a beam into a waveguide using a grating coupler is shown in Figure 4-3B . The waveguide and grating coupler are located approximately at the 0 point on the y-axis along the z-axis. The light beam is at an incident angle of approximately 5 degrees relative to the y-direction and has a diameter of 20 microns. The waveguide used in this simulation has a height of 100nm (along the y-direction) and the graded coupler has a grating line spacing of 420nm. The waveguide and grating coupler structure has a refractive index of approximately 1.87. The material surrounding the waveguide and grating structure has a refractive index of approximately 1.45. Figure 4-3B The light intensity of the light beam coupled to the grating coupler and the waveguide is shown, and the mode of the light in the waveguide is shown as a darker area.

[0281] The tapered region of the waveguide of one or more dimensions and the relative positioning of the tapered region and the grating coupler can provide adequate coupling of excitation energy into the waveguide. The curvature and chirp of the tapered region can accommodate the convergence and / or divergence of the propagation of incident excitation energy into the waveguide. A plan view of an exemplary waveguide layer including a grating coupler 4-316 and a tapered waveguide region 4-318 is shown in Figure 4-3C The tapered waveguide region 4-318 has dimensions perpendicular to light propagation and is Figure 4-3C The grating coupler 4-316 may have a grating coupler 4-316 that is suitable for coupling with an external excitation source. Figure 4-3C The alignment of the beam of excitation energy with the grating coupler 4-316 causes the beam to substantially overlap the region of the grating coupler 4-316, thereby improving coupling of the excitation energy into the waveguide 4-320. The arrangement of the tapered region 4-318 relative to the grating coupler 4-316 may provide a suitable coupling efficiency. The angle of the tapered region 4-318 to the grating coupler 4-316 may be selected to improve the coupling efficiency of the excitation energy from the excitation source to the waveguide 4-320 by reducing the loss of excitation energy due to the reduction in the size of the waveguide perpendicular to the propagation of light.

[0282] The integrated device may include a layer formed on the side of the grating coupler opposite the excitation source for reflecting light. The layer may reflect excitation energy passing through the grating coupler toward the grating coupler. By including the layer in the integrated device, the coupling efficiency of the excitation energy to the waveguide may be improved. An example of a reflective layer is Figure 4-2 The layer 4-218 of the integrated device 4-200 shown in FIG. Figure 4-3A 4-218 is located inside the excitation source coupling region 4-201 of the integrated device 4-200 and is used to reflect light toward the grating coupler 4-216. Layer 4-218 is formed on a side of the grating coupler 4-216 that is close to the incident excitation energy from the excitation source 4-214. Positioning layer 4-218 outside the pixel of the integrated device 4-200 can reduce the interference of layer 4-218 on the performance of each pixel. Layer 4-218 can include any suitable material. Layer 4-218 is substantially reflective to one or more excitation energies. In some embodiments, this layer may include Al, AlCu, and / or TiN.

[0283] B. Waveguide

[0284] The integrated device may include one or more waveguides for delivering a desired amount of excitation energy to one or more sample wells of the integrated device. The waveguide is located near the one or more sample wells so that a portion of the excitation energy is coupled to the one or more sample wells when the excitation energy propagates along the waveguide. The waveguide can couple the excitation energy to multiple pixels and serve as a bus waveguide. For example, a single waveguide can deliver excitation energy to a row or column of pixels of the integrated device. In some embodiments, a waveguide can be used to propagate excitation energy with multiple characteristic wavelengths. A pixel of the integrated device may include other structures (e.g., microcavities) for guiding the excitation energy from the waveguide toward the vicinity of the sample well. In some embodiments, the waveguide may carry an optical mode having an evanescent wave tail for extending into the sample well and / or in an area near the sample well. Other energy coupling structures located near the sample well can couple energy from the evanescent wave tail into the sample well.

[0285] One or more dimensions of a waveguide of an integrated device may provide desired propagation of excitation energy along the waveguide and / or into one or more sample wells. The waveguide may have a dimension perpendicular to light propagation and parallel to the plane of the waveguide, which dimension may be considered a cross-sectional width. The waveguide may have a cross-sectional width of approximately 0.4 microns, approximately 0.5 microns, approximately 0.6 microns, approximately 0.65 microns, approximately 0.8 microns, approximately 1 micron, or approximately 1.2 microns. The waveguide may have a dimension perpendicular to light propagation and perpendicular to the plane of the waveguide, which dimension may be considered a cross-sectional height. The waveguide may have a cross-sectional height of approximately 0.05 microns, approximately 0.1 microns, approximately 0.15 microns, approximately 0.16 microns, approximately 0.17 microns, approximately 0.2 microns, or approximately 0.3 microns. In some embodiments, the waveguide has a cross-sectional width that is greater than the cross-sectional height. The waveguide may be located at a distance from one or more sample wells in the integrated device, such as at Figure 4-2 The distance D between the sample well 4-222 and the waveguide 4-220 shown in FIG. 4 is approximately 0.3 microns, 0.5 microns, or approximately 0.7 microns.

[0286] In one exemplary embodiment, the waveguide may have a cross-sectional width of approximately 0.5 μm and a cross-sectional height of approximately 0.1 μm, and be located approximately 0.5 μm below the sample well layer. In another exemplary embodiment, the waveguide may have a cross-sectional width of approximately 1 μm and a cross-sectional height of 0.18 μm, and be located 0.3 μm below the sample well layer.

[0287] The size of the waveguide can be designed to support a single transverse radiation mode, or the size can be designed to support multiple transverse radiation modes. In some embodiments, one or more sizes of the waveguide can function so that the waveguide maintains only a single transverse mode and can selectively propagate TE or TM polarization modes. In some embodiments, the waveguide may have a high reflective portion formed on its end so that it supports a longitudinal standing wave mode inside the waveguide. By supporting one mode, the waveguide may have a reduced mode interference effect from cross-coupling of modes with different propagation constants. In some embodiments, the high reflective portion includes a single high reflective surface. In other embodiments, the high reflective portion includes a plurality of reflective structures that collectively result in high reflectivity. The waveguide can be used to divide the excitation energy from a single excitation source with a higher output intensity using a waveguide optical beam splitter, thereby forming a plurality of excitation energy beams from a single excitation source. Such an optical beam splitter may include an evanescent wave coupling mechanism. In addition or alternatively, photonic crystals can be used in the waveguide structure to improve the propagation of the excitation energy and / or used in the material surrounding the waveguide to reduce the scattering of the excitation energy.

[0288] The position and arrangement of the waveguide relative to other components in a pixel of the integrated device can be used to improve the coupling of excitation energy toward the sample well, improve the collection of emission energy by the sensor, and / or reduce signal noise introduced by the excitation energy. The size of the waveguide can be designed and / or positioned relative to the sample well to reduce the interference of the excitation energy propagating in the waveguide with the emission energy emitted from the sample well. The positioning and arrangement of the waveguide in the integrated device can depend on the refractive index of the waveguide and the material surrounding the waveguide. For example, the size of the waveguide perpendicular to the direction of light propagation along the waveguide and in the plane of the waveguide can be reduced so that a large amount of emission energy from the sample well passes through the waveguide when the emission energy propagates to the sensor of the pixel. In some embodiments, the distance between the sample well and the waveguide and / or the thickness of the waveguide can be selected to reduce reflections from one or more interfaces between the waveguide and the surrounding material. According to some embodiments, the reflection of the emission energy by the waveguide can be reduced to less than about 5% in some embodiments, less than about 2% in some embodiments, and less than about 1% in some embodiments.

[0289] The ability of a waveguide to propagate excitation energy may depend on the material used for the waveguide and the material surrounding the waveguide. Thus, a waveguide structure may include a core material (e.g., waveguide 4-220), and a cladding material (e.g., Figure 4-2 The materials of both the waveguide and the surrounding material may allow propagation of excitation energy having a characteristic wavelength through the waveguide. The material of the waveguide or surrounding material may be selected for a particular refractive index or combination of refractive indices. The waveguide material may have a lower refractive index than the surrounding waveguide material. Exemplary waveguide materials include: silicon nitride (Si x N y ), silicon oxynitride, silicon carbide, tantalum oxide (TaO 2 ), aluminum dioxide. Exemplary surrounding waveguide materials include silicon dioxide (SiO 2 ) and silicon oxide. The waveguide and / or surrounding material may include one or more materials. In some cases, a desired refractive index for the waveguide and / or surrounding material may be obtained by forming the waveguide and / or surrounding material to include more than one material. In some embodiments, the waveguide includes silicon nitride and the surrounding material includes silicon dioxide.

[0290] In one exemplary embodiment, the waveguide comprises silicon nitride and has a refractive index of about 1.90 and a cross-sectional height of about 100 nm, and the surrounding material comprises silicon dioxide and has a refractive index of about 1.46. In some embodiments, a waveguide may have a refractive index of about 1.88 while the surrounding material has a refractive index of about 1.46. In such an embodiment, the lower refractive index for the waveguide may reduce optical losses. In another exemplary embodiment, the waveguide comprises a silicon nitride core and a silicon dioxide cladding, and is used to propagate excitation energy having a characteristic wavelength of 635 nm. The core may have a refractive index of 1.99 and dimensions of 100 nm x 500 nm.

[0291] The waveguides of an integrated device may be formed to have a desired degree of uniformity within a waveguide and / or in multiple waveguides. Uniformity within an integrated device may be achieved by making a core and / or cladding having a waveguide structure of substantially similar size and / or refractive index along a waveguide and in multiple waveguides. In addition, the waveguides may be formed in different integrated devices in a repeatable manner by ensuring a repeatable manufacturing process so as to achieve a certain degree of compliance in different devices. The variation in the cross-sectional height of a waveguide and / or multiple waveguides may be less than about 2%, less than about 3%, or less than about 4%. The variation in the refractive index of a waveguide and / or multiple waveguides may be less than about 0.5%, less than about 1%, or less than about 2%. The variation in the refractive index of the surrounding material or cladding of a waveguide and / or multiple waveguides may be less than about 0.5%, less than about 1%, or less than about 2%.

[0292] The waveguide may be located in a pixel between the sample well and one or more sensors. Figure 4-2 , the waveguide 4-220 is positioned between a sample well 4-222 and a layer 4-230 comprising at least one sensor. In some embodiments, the sample well 4-222 may be positioned between the waveguide and the sensor. The waveguide may be aligned with the sensor in a center-to-center manner, such as so that the center of the waveguide is approximately aligned with the center of the sample well. In some embodiments, the waveguide may be displaced by a certain distance from a position aligned center-to-center with the sample well. In some embodiments, two substantially parallel waveguides may deliver excitation energy of the same wavelength or different wavelengths to a pixel, and the sample well may be positioned between the two waveguides. In some embodiments, multiple waveguides at different heights within the integrated device may direct excitation energy toward the vicinity of one or more sample wells positioned on the integrated device.

[0293] One or more waveguides of the integrated device may include a bend. The bends of each waveguide may provide a desired arrangement of the waveguides and / or pixels so that a sufficient amount of excitation energy is coupled to one or more sample wells of the integrated device. The design of the waveguide bends may utilize the curvature and spatial dimensions of each bend to balance the excitation energy loss. In addition, with the correct design, the bends inside the waveguide may also be used to filter out a portion of the propagating light. A portion of the waveguide is designed to reduce light of one polarization by filtering provided by a certain radius of curvature provided by the curvature in the waveguide. This bend can be used to select for a specific polarization mode, such as TM and TE. In some embodiments, the bend can be used to filter out and / or weaken the TM mode and maintain the TE mode. Figure 4-4 The optical loss due to bending as a function of the radius of curvature of a waveguide bend having a cross-sectional height of 100 nm and widths of 300 nm, 400 nm, 500 nm, 700 nm, and 1000 nm is plotted. As an example, to achieve a loss of at least 0.1 dB / 90 degree bend, a waveguide having a cross-sectional width of 500 nm may have a bending radius greater than about 35 microns, or a waveguide having a cross-sectional width of 700 nm may have a bending radius greater than about 22 microns.

[0294] Dividing each waveguide from a single waveguide into multiple waveguides can allow excitation energy to reach multiple rows or columns of sample wells in an integrated device. An excitation source can be coupled to an input waveguide, and the input waveguide can be divided into multiple output waveguides, wherein each output waveguide delivers excitation energy to a row or column of sample wells. Any suitable technique for dividing and / or combining waveguides can be used. Such waveguide dividing and / or combining techniques can include star splitters or couplers, Y-splitters, and / or evanescent wave couplers. In addition or alternatively, a multimode interference type beam splitter (MMI) can be used to divide and / or combine waveguides. One or more of these waveguide dividing and / or combining techniques can be used to direct excitation energy to the sample wells.

[0295] An exemplary star coupler 4-500 is shown in Figure 4-5 A single waveguide or two waveguides 4-501 and 4-502 may be used to input two different wavelengths of light into the star coupler, such as Figure 4-5 As shown in . The size and shape of each input waveguide can be used to individually adjust the spread of each excitation beam so as to match at the output waveguide 4-504, thereby increasing the possibility of obtaining similar power distribution in each output waveguide 4-504. The star coupler includes a free propagation region 4-503, which can be embodied as a slab waveguide. The free propagation region 4-503 is a high multimode region that allows the input light to propagate substantially freely in the plane of the slab waveguide. The free propagation region 4-503 can have a width of, for example, 300-400 microns from the first output waveguide to the last output waveguide.

[0296] The coupling of the excitation energy to the output waveguide 4-504 may be adjusted using various parameters of the star coupler, including one or more dimensions of individual components of the star coupler. For example, the dimensions of the transverse cross-section of each input waveguide 4-501 and 4-502, the dimensions of the transverse cross-section of each output waveguide 4-504, and the distance of each output waveguide 4-504 from the input waveguide may be parameters of the star coupler that may be adjusted to improve the coupling of the excitation energy to the output waveguide 4-504. In some embodiments, the star coupler may be formed so that the output waveguides 4-504 have approximately the same power distribution relative to each other. In some embodiments, the output waveguides near the outer edges of the star coupler may have different dimensions than the output waveguides near the center. For example, the waveguides near the edges may have a larger transverse cross-sectional area, thereby collecting more light than the output waveguides near the center of the star coupler (which have a smaller transverse cross-sectional area). In some embodiments, the distance of the output waveguides from the input waveguides may vary. For example, as Figure 4-5 As shown in , the output waveguides near the edge may have a distance from the input waveguides 4-501 and 4-502 that is smaller than the output waveguides near the center of the star coupler.

[0297] The star coupler 4-500 may have any suitable number of output waveguides. In some embodiments, a single star coupler distributes excitation energy to the entire integrated device. Therefore, the number of output waveguides is equal to the number of rows of pixels in the integrated device. For example, there may be 128 output waveguides. In other embodiments, more than one star coupler may be used. In such an embodiment, the first star coupler may have 64 output waveguides and the second star coupler may have 64 output waveguides, such that the combination of the first and second star couplers provides excitation energy to 128 rows of pixels in the integrated device.

[0298] In some embodiments, rather than having an input waveguide, one or more grating couplers may couple excitation energy directly into a free propagation region of a star coupler having multiple output waveguides. A single grating coupler may be used to couple multiple wavelengths into the free propagation region of a star coupler. To do this, light of different wavelengths may be incident on the grating coupler at different angles. In some embodiments, multiple grating couplers may be used to couple excitation energy of different wavelengths. For example, one grating coupler may be used for each excitation wavelength. A grating coupler configured as part of a free propagation region may operate in a manner similar to one of the above-described grating couplers, but instead of coupling light into an input waveguide that propagates light to a star coupler, light is coupled directly into a slab waveguide, such as 4-603, that forms the free propagation region.

[0299] Another exemplary configuration of a star coupler is shown in Figure 4-6. Two wavelengths of light may be input into the star coupler using grating couplers 4-605 and 4-606 connected to waveguides 4-609 and 4-610, respectively. The grating couplers 4-605 and 4-606 and waveguides 4-609 and 4-610 may be used to reduce the loss of excitation energy by reducing the number of bends and / or selecting bend angles for improving the propagation of excitation energy toward the free propagation region 4-607. The output waveguide 4-608 from the star coupler may be configured in any suitable manner to provide excitation energy to a row of pixels. Figure 4-6 In the example shown in , there are 32 output waveguides. The lateral cross-sectional area of ​​the output waveguides 4-608 near the free propagation region 4-607 may vary so that the output waveguides near one end of the free propagation region 4-607 have a larger lateral cross-sectional area than the output waveguides near the center of the free propagation region 4-607. For example, Figure 4-6 As shown in FIG. 4 , the output waveguide 4-608a has a larger transverse cross-sectional area than the output waveguide 4-608b located closer to the center of the free propagation region 4-607. Such variation in transverse cross-sectional area among each output waveguide 4-608 can improve the distribution of excitation energy among each output waveguide 4-608 and, in some embodiments, allow substantially similar amounts of excitation energy to be delivered to a row of pixels by each output waveguide. In this way, multiple rows of pixels can receive substantially similar amounts of excitation energy in each row of pixels.

[0300] The design for waveguide partitioning can be selected based on the efficiency of the partitioning technique for that number of output waveguides. If the partitioning efficiency is higher, then more output waveguides can be generated and only a single partitioning step can be performed. In some embodiments, a single output waveguide from a splitter can correspond to each row of sample wells. Some embodiments include multiple partitioning steps to obtain a sufficient number of waveguides for delivering excitation energy to a portion of the sample wells of an integrated device. In some embodiments, a multi-mode interference splitter (MMI) can be used to further partition the waveguide into multiple waveguides. In an MMI, an input waveguide can be partitioned into multiple output waveguides, wherein one or more sizes of the MMI can determine the number of output waveguides and / or the amount of excitation energy delivered to the output waveguides. For example, as Figure 4-7 As shown in FIG. 4 , an MMI splitter 4-707 is configured to provide an output 4-708 from an input 4-710. The output 4-708 may be coupled to a waveguide for propagating excitation energy to each row of sample wells. In some embodiments, the MMI splitter 4-707 may have 280 outputs that are coupled to waveguides for propagating excitation energy to the 280 rows of sample wells. In some embodiments, the MMI splitter is configured to receive excitation energy from multiple inputs and direct the excitation energy to multiple outputs. Figure 4-7As shown in , the MMI splitter 4-717 is coupled to a plurality of inputs 4-720 and provides a plurality of outputs 4-718. In such an embodiment, the number of outputs 4-718 may be greater than the number of inputs 4-720. In some embodiments, the inputs 4-720 may provide different excitation energies to the MMI splitter 4-717. In other embodiments, the division of each waveguide may be performed in a plurality of division steps. As an example, two sets of multimode interference splitters may be used for division, wherein the output waveguide from the first MMI splitter is used as the input of the second MMI splitter. For example, as Figure 4-7 As shown in FIG. 4 , an MMI splitter 4-727 divides an input waveguide 4-730 into a plurality of outputs, including output 4-728 that is coupled to an MMI splitter 4-737 for providing output 4-738. In some embodiments, the MMI splitter 4-727 may provide 35 outputs and each output may be divided into 8 output waveguides using another MMI splitter (e.g., 4-737). Because each of the intermediate 35 waveguides is divided into 8 waveguides, 280 waveguides are formed in this non-limiting example.

[0301] In some embodiments, multiple input waveguides can be cross-coupled in an MMI splitter to form an output waveguide for coupling light to a sample well of an integrated device. On such an integrated device, there can be multiple grating couplers coupled to multiple excitation sources. Cross-coupling MMI is used to couple multiple excitation sources together and form multiple output waveguides, such as Figure 4-7 As shown in . Each of the multiple input waveguides can begin with one of the grating couplers to couple light from one excitation source to the MMI splitter. This cross-coupling of multiple excitation sources can improve the robustness of the system to prevent excitation source degradation and / or failure. For example, if one of the multiple excitation sources stops generating excitation energy, then other excitation sources can be used to provide sufficient excitation energy to obtain the desired level of performance of the integrated device. A compensation mechanism can also be included that compensates for a reduction in excitation energy caused by one or more of the multiple excitation sources by increasing the intensity provided by the remaining active excitation sources.

[0302] The techniques for dividing and / or combining waveguides may be selected so as to reduce the loss of excitation energy, including insertion loss, when the waveguides are divided and / or composed. The insertion loss due to dividing and / or combining the waveguides may be approximately less than 10%, approximately less than 20%, or approximately less than 30%. The techniques for dividing the waveguides may allow for approximately uniform division of the excitation energy among a plurality of output waveguides, thereby uniformly distributing the excitation energy among each output waveguide. The techniques for combining the waveguides may allow for approximately uniform relative distribution of the excitation energy from a plurality of input waveguides among each output waveguide. In some embodiments, the uniformity among the output waveguides may be approximately less than 10%, approximately less than 20%, or approximately less than 30%. The techniques for designing the waveguides may be selected for certain tolerances of manufacturing parameters, including waveguide cross-sectional height, cross-sectional width, and / or the refractive index of the waveguide.

[0303] One or more dimensions of the MMI splitter affect the number of output waveguides and / or the efficiency of waveguide division and / or combination.Designing an MMI splitter may include determining the dimensions of the MMI splitter to have a certain number of output waveguides and / or a certain division efficiency. Figure 4-8 A simulation of the intensity waveform of light inside an exemplary MMI splitter for receiving light from an input waveguide and directing the light into eight output waveguides is shown. In this example, the input and output waveguides have a cross-sectional width of 500 nm, and the dimensions of the MMI splitter have a width W of 16.35 microns and a length L of 84.28 microns. By measuring the light delivered at each output waveguide, the coupling uniformity and / or efficiency can be measured. Figure 4-8 For the exemplary MMI splitter shown in , the intensities of the eight output waveguides can vary by up to about 0.1%.

[0304] In some implementations, a grating coupler can be configured to direct input light into multiple output waveguides. Figure 4-9A A thin sheet grating coupler is shown that can be used to couple light having one or more wavelengths into multiple output waveguides 4-904. The thin sheet grating is a linear grating structure that is much wider than the wavelength of light (e.g., hundreds of microns wide). It is formed from alternating layers of dielectrics (e.g., silicon nitride and silicon oxide). In some embodiments, multiple wavelengths can be coupled to the thin sheet grating by emitting different wavelengths at the thin sheet grating so that they are incident at different angles. In some embodiments, one or more light beams incident on the grating coupler have a spot size 4-603 that is approximately the size of the grating structure itself, such as Figure 4-9A as shown in .

[0305] Figure 4-9B and Figure 4-9C An exemplary thin-film grating coupler is shown, wherein Figure 4-9Cyes Figure 4-9B A zoomed view of region 4-906 is shown in FIG. 4-906 and includes a thin-film grating coupler 4-903 and an output waveguide 4-905. This configuration provides coupling and division of input power of different excitation wavelengths into multiple output waveguides. Figure 4-9B In the example shown in , there are 128 output waveguides 4-905. In embodiments employing multiple excitation wavelengths, the grating region 4-906 has a grating pitch designed to couple multiple excitation wavelengths to the output waveguides. Power may be provided to the waveguides through a single slice in the grating region, where the width of the slice may be varied to compensate for the varying intensity of the input excitation energy beam at the grating coupler.

[0306] C. Sample Well

[0307] According to some embodiments, a sample well 5-210 may be formed at one or more pixels of an integrated device. The sample well may include a small space or area formed on a surface of a substrate 5-105 and arranged such that a sample 5-101 may diffuse into or out of the sample well from a specimen deposited on the substrate surface, such as Figure 5-1 and Figure 5-2 , which shows a single pixel 5-100 of an integrated device. In various embodiments, a sample well 5-210 may be arranged to receive excitation energy from a waveguide 5-240. A sample 5-101 diffused into the sample well may be temporarily or permanently retained within an excitation region 5-215 of the sample well using an adhesive 5-211. In the excitation region, the sample may be excited using excitation energy (e.g., excitation radiation 5-247) to subsequently emit radiation, which may be observed and evaluated to identify the sample.

[0308] In more detailed operation, at least one sample 5-101 to be analyzed may be introduced into the sample well 5-210, such as from a sample (not shown) of a fluid suspension containing the sample. When the sample is within the excitation region 5-215 within the sample well, excitation energy from the waveguide 5-240 may excite the sample or at least one marker attached to the sample or included in a label associated with the sample. According to some embodiments, the marker may be a luminescent molecule (e.g., a fluorophore) or a quantum dot. In some embodiments, there may be more than one marker for analyzing the sample (e.g., different markers and tags for single molecule gene sequencing, as described in "Real-time DNA sequencing based on a single polymerase molecule" by J. Eid et al., Science 323, 133 (2009), the entire contents of which are incorporated herein by reference). During and / or after excitation, the sample or marker may emit emission energy. When multiple markers are used, they may emit different characteristic energies and / or emit with different time characteristics including different lifetimes. Emission energy from the sample well may radiate or travel to the sensor 5-260, where it is detected and converted into an electrical signal that can be used to identify the sample.

[0309] According to some embodiments, the sample well 5-210 may be a partially enclosed structure, such as Figure 5-2 In some embodiments, the sample well 5-210 includes a sub-micron sized hole or opening (characterized by at least one lateral dimension Dsw) formed in at least one layer of material 5-230. In some cases, this may be referred to as a "nanopore". The lateral dimensions of the sample well may be between about 20 nanometers and about 1 micron in accordance with some embodiments, although larger and smaller dimensions may be used in some embodiments. In some embodiments, the volume of the sample well 5-210 may be between about 10 -21 Liters and about 10 -15 The sample well may be formed as a waveguide that may or may not support a propagation mode. The sample well may be formed as a waveguide that may or may not support a propagation mode. In some embodiments, the sample well may be formed to have a cylindrical shape (or similar shape) and a diameter (or large lateral dimension) D sw A zero mode waveguide (ZMW) can be formed in a single metal layer in the form of a nanoscale hole that does not support a propagating optical mode through the hole.

[0310] Because the sample well 5-210 has a small volume, detection of single sample events (e.g., single molecule events) at each pixel may be feasible, even though the sample may be concentrated in the specimen being examined similar to concentrations found in the natural environment. For example, micromolar concentrations of sample may be present in a specimen placed in contact with the integrated device, but only approximately one sample (or single molecule event) may be inside the sample well at any given time at the pixel height. Statistically, some sample wells may contain no sample and some sample wells may contain more than one sample. However, a significant number of sample wells may contain a single sample (e.g., at least 30% in some embodiments), so that single molecule analysis may be performed in parallel for a large number of pixels. Because single molecule or single sample events may be analyzed at each pixel, the integrated device is able to detect rare events that are overlooked in the ensemble average.

[0311] The lateral dimension D of the sample well sw In some embodiments, the sample well 5-210 may have a depth or height between about 500 nm and about 1 micron, in some embodiments, between about 250 nm and about 500 nm, in some embodiments, between about 100 nm and about 250 nm, and in some embodiments, between about 20 nm and about 100 nm. According to some embodiments, the lateral dimension of the sample well is between about 80 nm and about 180 nm, or between about 1 / 4 and 1 / 8 of the excitation wavelength or emission wavelength. According to other embodiments, the lateral dimension of the sample well is between about 120 nm and about 170 nm. In some embodiments, the depth or height of the sample well 5-210 may be between about 50 nm and about 500 nm. In some embodiments, the depth or height of the sample well 5-210 may be between about 80 nm and about 250 nm.

[0312] A sample well 5-210 having sub-wavelength lateral dimensions can improve the operation of a pixel 5-100 integrated device in at least two ways. For example, excitation energy incident on the sample well from the side opposite the sample can be coupled into the excitation region 5-215 with exponentially decreasing power and does not propagate through the sample well to reach the sample. Thus, the excitation energy is increased in the excitation region, where it excites the sample of interest, and decreased in the sample, where it would excite other samples that would contribute to background noise. Additionally, emission from a sample held at the base of the sample well (e.g., closer to the sensor 5-260) is preferably directed toward the sensor because emission propagating upward through the sample well is highly suppressed. These two effects can improve the signal-to-noise ratio at the pixel. The inventors have recognized several aspects of the sample well that can be improved to further improve the signal-to-noise ratio at the pixel. These aspects relate to the shape and structure of the sample well, and also to adjacent optical and plasmonic structures (described below) that facilitate coupling of excitation energy to the sample well and radiation emitted from the sample well.

[0313] According to some embodiments, the sample well 5-210 may be formed as a nanopore that is configured to not support propagation modes for a particular wavelength of interest. In some cases, the nanopore is configured where all modes are below a threshold wavelength and the pore may be a sub-cutoff nanopore (SCN). For example, the sample well 5-210 may include a cylindrically shaped hole or inner hole in a conductive layer. The cross-section of the sample well need not be circular, and in some embodiments may be elliptical, square, rectangular, or polygonal. Excitation energy 5-247 (e.g., visible or near infrared radiation) may enter the sample well through an inlet aperture 5-212, which may be defined by a wall 5-214 of the sample well at a first end of the well, such as Figure 5-2 . When formed as an SCN, the excitation energy may decay exponentially along the length of the nanohole (e.g., in the direction of the sample). In some embodiments, the waveguide may include an SCN for emitting radiation from the sample, but may not be an SCN for excitation energy. For example, the hole and waveguide formed by the sample well may be large enough to support a propagation mode for the excitation energy because the excitation energy may have a wavelength shorter than the emitted radiation. Emission at longer wavelengths may exceed the cutoff wavelength for the propagation mode in the waveguide. According to some embodiments, the sample well 5-210 may include an SCN for excitation energy such that the maximum intensity of the excitation energy is confined to an excitation region 5-215 of the sample well at the entrance of the sample well 5-210 (e.g., confined near the interface between the layer 5-235 and the layer 5-230, as shown in the figure). Such localization of the excitation energy may improve the localization of the emission energy from the sample and limit the observed emission emitted from a single sample (e.g., a single molecule).

[0314] According to some embodiments, a pixel 5-100 may include other structures. For example, a pixel 5-100 may include one or more excitation coupling structures 5-220 that enable coupling of excitation energy to a sample within a sample well. A pixel may also include an emission directing structure 5-250 that directs emission energy from a sample within a sample well to a sensor 5-260.

[0315] An example of localizing the excitation close to the entrance of a sample well including an SCN is shown in Figure 5-3 . Numerical simulations were performed to determine the intensity of excitation radiation within and near a sample well 5-210 formed as an SCN. The results indicate that the intensity of the excitation radiation is approximately 70% of the incident energy at the sample well entrance aperture and drops to approximately 20% of the incident intensity within approximately 100 nm in the sample well. For this simulation, the characteristic wavelength of the excitation energy is 633 nm and the diameter of the sample well 5-210 is 140 nm. The sample well 5-210 is formed in a layer of gold metal. The horizontal divisions in the figure are 50 nm. As depicted in the figure, more than half of the excitation energy received in the sample well is confined to approximately 50 nm within the entrance aperture 5-212 of the sample well.

[0316] To increase the intensity of the excitation energy localized at the sample well, the present inventors have developed and studied other sample well structures. Figure 5-4 One embodiment of a sample well including a cavity or dimple 5-216 at the excitation end of the sample well is shown. Figure 5-3 As shown in the simulation results of , an area of ​​higher excitation intensity is present in front of the entrance hole 5-212 of the sample well. According to some embodiments, adding a dimple 5-216 to the sample well allows the sample to move into the area of ​​higher excitation intensity. In some embodiments, the shape and structure of the dimple changes the local excitation field (e.g., due to the difference in refractive index between the layer 5-235 and the fluid in the sample well), and can further increase the intensity of the excitation energy in the dimple. The dimple 5-216 can be formed inside the layer 5-235 so that the sample space occupying a portion of the sample well 5-214 and the dimple 5-216 is surrounded by the material constituting the layer 5-216.

[0317] The pit may have any suitable shape. The pit may have a lateral shape that is substantially equivalent to the lateral shape of the sample well, such as a circle, an ellipse, a square, a rectangle, a polygon, etc. In some embodiments, the sidewalls of the pit may be substantially straight and vertical, like the walls of the sample well. In some embodiments, the sidewalls of the pit may be inclined and / or curved, as shown in the accompanying figures. The lateral dimension of the pit may be substantially the same dimension as the lateral dimension of the sample well in some embodiments, may be smaller than the lateral dimension of the sample well in some embodiments, or may be larger than the lateral dimension of the sample well in some embodiments. The pit 5-216 may extend beyond the sample well layer 5-230 by between about 10 nm and about 200 nm. In some embodiments, the pit may extend beyond the sample well layer 5-230 by between about 50 nm and about 150 nm. In some embodiments, the pit may extend beyond the sample well layer 5-230 by between about 150 nm and about 250 nm. By forming the pit, the excitation region 5-215 may extend outside of the sample well, such as Figure 5-4 as shown in .

[0318] Figure 5-5 The increase in excitation energy at the excitation region for a sample well containing a dimple is shown (shown in the left simulated image). For comparison, the excitation field was also simulated for a sample well without a dimple shown on the right. The field magnitude has been converted from colorimetric to field magnitude in these graphs, and the dark area at the base of the dimple represents a higher intensity than the light area inside the sample well. The dark area above the sample well represents the lowest intensity. As can be seen from the figure, the dimple allows the sample 5-101 to move to an area of ​​higher excitation intensity, and the dimple also increases the localization of the area of ​​highest intensity at the excitation end of the sample well. It should be noted that the area of ​​high intensity is more distributed for a sample well without a dimple. In some embodiments, the dimple 5-216 provides an increase in excitation energy at the excitation region of more than two times. In some embodiments, more than a two-fold increase can be obtained based on the shape and depth of the dimple. In these simulations, the layer containing the sample well comprises aluminum and has a thickness of about 100 nm, the dimple has a depth of about 50 nm, and the excitation energy wavelength is 635 nm.

[0319] Figure 5-6A Another embodiment of a sample well 5-210 is shown, wherein the sample well is formed on the surface of the substrate above the protrusion 5-615. Figure 5-1The sample well shown in FIG. 5-620 can increase the excitation energy at the sample by more than two times compared to the sample well shown in FIG. 5-620 , and can focus the emission from the sample well to the sensor 5-260. According to some embodiments, a protrusion 5-615 is patterned in the first layer 5-610 of material. In some embodiments, the protrusion comprises a waveguide. The protrusion may be formed as a ridge having a rectangular cross-section in some embodiments, and the material of the second layer 5-620 may be deposited above the first layer of the protrusion. At the protrusion, a second layer may be formed above the protrusion closest to the cylindrical portion 5-625, as shown in the figure. In some embodiments, a conductive layer 5-230 (e.g., a reflective metal) may be deposited above the second layer 5-620 and patterned to form a sample well 5-210 in the conductive layer above the protrusion. Then, a pit 5-216 may be etched into the second layer. The pit 5-216 may extend below the conductive layer 5-230 to between about 50 nm and about 150 nm. According to some embodiments, the first layer 5-610 and the second layer 5-620 may be optically transparent and may or may not be composed of the same material. In some embodiments, the first layer 5-610 may be composed of an oxide (e.g., SiO 2 ) or nitrides (e.g., Si 3 N 4 ) is composed, and the second layer 5-620 can be composed of oxide or nitride.

[0320] According to some embodiments, the conductive layer 5-230 above the protrusion 5-615 is shaped to be approximately like a cylindrical reflector 5-630. The shape of the cylindrical portion can be controlled by selecting the height h of the protrusion, the width or lateral dimension w of the protrusion, and the thickness t of the second layer 5-620. The position of the excitation zone and the position of the sample relative to the optical focus of the cylindrical reflector can be adjusted by selecting the pit depth d. It will be appreciated that the cylindrical reflector 5-630 can focus the excitation energy in the excitation zone 5-215, and can also collect radiation emitted from the sample and reflect and focus the radiation toward the sensor 5-260.

[0321] Some embodiments relate to an integrated device having a sample well with a pit located proximate to a waveguide. Figure 5-6BAn integrated device having a sample well 5-632 formed in a layer 5-630 and a layer 5-636 is shown. The layer 5-630 may be a metal layer and include one or more metals (e.g., Al). The layer 5-636 may function as a dielectric layer and include one or more dielectric materials (e.g., silicon dioxide). The sample well 5-632 may have a variable dimension in a direction parallel to the layer 5-630 and / or the layer 5-636. The sample well 5-632 may have a dimension D2 along the z-direction at least within the layer 5-630 of the integrated device, which may be considered to be the diameter of the sample well 5-632 in some embodiments. The dimension D2 of the sample well 5-632 may be about 700 nm, about 800 nm, about 900 nm, about 1 micron, or about 1.1 microns. The sample well 5-632 may have a dimension D1 along the z-direction within the layer 5-636 of the integrated device, which may be considered to be the diameter at the surface of the sample well 5-632 in some embodiments. Dimension D1 may be about 100 nm, about 150 nm, about 200 nm, or about 250 nm. A surface of a sample well 5-632 having dimension D1 is located a dimension d1 away from a waveguide 5-634 in the x-direction. Positioning the sample well 5-632 proximate the waveguide 5-634 by a distance d1 may allow for improved coupling of excitation energy from the waveguide 5-634 to the sample well 5-632. Dimension d1 may be about 50 nm, about 100 nm, about 150 nm, about 200 nm, or about 250 nm.

[0322] As described above, the sample well may be formed in any suitable shape, and is not limited to a cylindrical shape. In some embodiments, the sample well may be conical, tetrahedral, pentahedral, or the like. Figure 5-7A – Figure 5-7F Some exemplary sample well shapes and structures that may be employed in some embodiments are shown. According to some embodiments, a sample well 5-210 may be formed with an entrance aperture 5-212that is larger than an exit aperture 5-218 for excitation energy. The sidewalls of the sample well may be tapered or curved. Forming the sample well in this manner may allow more excitation energy to enter the excitation region, but still significantly attenuate the excitation energy traveling toward the specimen. Additionally, radiation emitted by the specimen may preferentially radiate toward the end of the sample well having the larger aperture due to favorable energy transfer in that direction.

[0323] In some embodiments, the dimple 5-216 may have a lateral dimension that is smaller than the base of the sample well, such as Figure 5-7B. The smaller pits can be formed by coating the sidewalls of the sample well with a sacrificial layer before etching the pits, and then removing the sacrificial layer. The smaller pits can be formed to keep the sample in a region more equidistant from the conductive walls of the sample well. Keeping the sample equidistant from the walls of the sample well can reduce undesirable effects of the sample well walls on the radiating sample, such as quenching of emission and / or changes in radiative lifetime.

[0324] Figure 5-7C and Figure 5-7D Another embodiment of a sample well is shown. According to this embodiment, the sample well 5-210 may include an excitation energy enhancement structure 5-711, and an adhesive 5-211 formed adjacent to the excitation energy enhancement structure. According to some embodiments, the energy enhancement structure 5-711 may include a surface plasmon or nano-antenna structure formed in a conductive material on the optically transparent layer 5-235. Figure 5-7C shows an elevation view of a sample well 5-210 and nearby structures, Figure 5-7D A plan view is shown. The shape of the excitation energy enhancement structure 5-711 can be designed and arranged to enhance the excitation energy in a small local area. For example, these structures may include a pointed conductor with a sharp angle at the sample well, which increases the intensity of the excitation energy inside the excitation zone 5-215. In the illustrated example, the excitation energy enhancement structure 5-711 is in the form of a bow tie. The sample 5-101 diffused into the area can be temporarily or permanently retained using an adhesive 5-211 and excited using excitation energy that can be output from a waveguide 5-240 located adjacent to the sample well 5-210. According to some embodiments, the excitation energy can drive surface-plasmon waves in the energy enhancement structure 5-711. The surface plasmon current formed can generate high electric fields at the sharp points of the structure 5-711, and these high electric fields can excite the sample held in the excitation zone 5-215. In some embodiments, Figure 5-7C The sample well 5-210 shown in FIG. 5 may include a dimple 5-216.

[0325] Another embodiment of a sample well is shown in Figure 5-7E , and an excitation energy enhancing structure 5-720 formed along the inner wall of a sample well 5-210 is shown. The excitation energy enhancing structure 5-720 may comprise a metal or conductor and may be formed using an oblique (or shallow) directional deposition, wherein the substrate on which the sample well is formed is rotated during deposition. During deposition, the base of the sample well 5-210 is masked by the upper wall of the well so that the deposited material does not accumulate at the base. The formed structure 5-720 may form an acute angle 5-722 near the bottom of the structure, and this acute angle of the conductor may enhance the excitation energy inside the sample well.

[0326] exist Figure 5-7EIn the embodiment shown in , the material 5-232 constituting the sample well need not be a conductor, and may be any suitable dielectric. According to some embodiments, the sample well 5-210 and the excitation energy enhancement structure 5-720 may be formed at blind holes etched into the dielectric layer 5-235, and there is no need to deposit a separate layer 5-232.

[0327] In some embodiments, the Figure 5-7E Shallow evaporation is performed on the structure shown in Figure 1 to deposit a metal or conductive energy enhancement structure, such as a trapezoidal structure or a pointed cone at the base of the sample well, as represented by the dashed line. The energy enhancement structure can enhance the excitation energy inside the sample well using surface plasmons. After the post-shallow evaporation, a planarization process (e.g., a chemical mechanical polishing step or a plasma etching process) can be performed to remove or etch back the deposited material on the top of the sample well while leaving the energy enhancement structure inside the well.

[0328] In some embodiments, a sample well 5-210 may be formed from more than a single metal layer. Figure 5-7F A sample well formed in a multi-layer structure is shown, where different materials may be used for different layers. According to some embodiments, the sample well 5-210 may be formed in a first layer 5-232 (which may be a semiconductor material or a conductive material), a second layer 5-234 (which may be an insulator or a dielectric material), and a third layer 5-230 (which may be a conductor or a semiconductor). In some embodiments, a degenerate doped semiconductor or graphene may also be used in one layer of the sample well. In some embodiments, the sample well may be formed in two layers, and in other embodiments, the sample well may be formed in four or more layers. In some embodiments, the multi-layer materials used to form the sample well may be selected to increase or suppress the generation of interface excitons using excitation radiation incident on the sample well. In some embodiments, the multi-layer materials used to form the sample well may be selected to increase surface plasmon generation at the base of the sample well or suppress surface plasmon radiation at the top of the sample well. In some embodiments, the multi-layer materials used to form the sample well may be selected to suppress the propagation of excitation radiation beyond the sample well and the multi-layer structure into the bulk sample. In some embodiments, the multiple layers of material used to form a sample well can be selected to increase or suppress interface excitons that may be generated by excitation radiation incident on the sample well.

[0329] Various materials may be used to construct the sample wells described in the previous embodiments. According to some embodiments, the sample well 5-21 may be composed of at least one layer of material 5-230, which may include any one or combination of conductive materials, semiconductors, and insulators. In some embodiments, the sample well 5-210 includes a highly conductive metal layer, such as gold, silver, aluminum, copper. In some embodiments, the layer 5-230 may include a multilayer stack that includes any one or combination of gold, silver, aluminum, copper, titanium, titanium nitride, palladium, platinum, and chromium. Additionally or alternatively, in some embodiments, other metals may be used. According to some embodiments, the sample well may include an alloy, such as AlCu or AlSi.

[0330] In some embodiments, multiple layers of different metals or alloys may be used to form a sample well. In some embodiments, the material comprising the sample well 5-210 may include alternating layers of metals and non-metals, such as alternating layers of a metal and one or more oxides. In some embodiments, the non-metal may include a polymer, such as polyvinylphosphonic acid or polyethylene glycol (PEG)-thiol.

[0331] According to some embodiments, the layer 5-230 in which the sample well is formed may be deposited on or adjacent to at least one optically transparent layer 5-235 so that excitation energy (in the form of optical radiation, such as visible or near infrared radiation) and emission energy (in the form of optical radiation, such as visible or near infrared radiation) may travel to and from the sample well 5-210 without significant attenuation. For example, excitation energy from a waveguide 5-240 may pass through at least one optically transparent layer 5-235 to the excitation region 5-215, and emission from the sample may pass through the same layer or layers to the sensor 5-260. This excitation energy may be an evanescent tail from the excitation light guided by the waveguide.

[0332] In some embodiments, at least one surface of the sample well 5-210 may be coated with one or more layers 5-211, 5-280 of material that enables the function of the sample within the sample well, such as Figure 5-8 , as shown in . For example, a thin dielectric layer 5-280 (e.g., aluminum oxide, titanium nitride, or silicon oxide) may be deposited on the sidewalls of the sample well in the form of a passivation coating. Such a coating may be used to reduce sample adhesion of a sample outside of the excitation region 5-215, or to reduce interaction between the sample and the material 5-230 in which the sample well 5-210 is formed. According to some embodiments, the thickness of the passivation coating inside the sample well may be between about 5 nm and about 50 nm.

[0333] In some embodiments, the material used for the coating 5-280 may be selected based on the affinity of the chemical agent used for the material so that the coating 5-280 can be treated with a chemical or biological substance to further inhibit the adhesion of sample radicals to the coating 5-280. For example, according to some embodiments, the coating 5-280 may include aluminum oxide, which may be passivated with a polyphosphonate passivation layer. In some embodiments, additional or alternative coatings and passivating agents may be used.

[0334] According to some embodiments, at least the bottom surface of the sample well 5-210 and / or the pit 5-216 may be treated with a chemical or biological adhesive 5-211 (e.g., biotin) to facilitate retention of the sample. The sample may be retained permanently or temporarily, for example, for a period of at least between about 0.5 milliseconds and about 50 milliseconds. In another embodiment, the adhesive may facilitate temporary retention of the sample 5-101 for a longer period of time. In various embodiments, any suitable adhesive may be used and is not limited to biotin.

[0335] According to some embodiments, a layer of material 5-235 adjacent to a sample well may be selected based on an affinity of an adhesive for the material of that layer. In some embodiments, passivation of the sidewalls of the sample well may inhibit coating of the adhesive on the sidewalls so that the adhesive 5-211 is preferentially deposited at the base of the sample well. In some embodiments, the adhesive coating may extend upward along a portion of the sidewalls of the sample well. In some embodiments, the adhesive may be deposited using an anisotropic physical deposition process (e.g., evaporation, sputtering) such that the adhesive accumulates at the base of the sample well or pit and is not visibly formed on the sidewalls of the sample well.

[0336] Various fabrication techniques may be used to fabricate the sample well 5-210 for an integrated device. Some exemplary processes are described below, but the present invention is not limited to these examples.

[0337] The sample well 5-210 may be formed using any suitable micro- or nano-fabrication process, which may include, but is not limited to, processing steps associated with photolithography, deep ultraviolet lithography, immersion lithography, near-field optical contact lithography, EUV lithography, X-ray lithography, nanoimprint lithography, interference lithography, step-and-flash lithography, milling, ion beam lithography, ion beam milling, lift-off process, reactive ion etching, and the like. According to some embodiments, the sample well 5-210 may be formed using photolithography and lift-off processes. Exemplary fabrication steps associated with a lift-off process for a sample well are shown in Figure 5-9A - F. Although the fabrication of only a single sample well or structure at one pixel is generally depicted in the figures, it should be understood that a large number of sample wells or structures may be fabricated in parallel on a substrate (eg, at each pixel).

[0338] According to some embodiments, a layer 5-235 (eg, an oxide layer) on the substrate may be covered with an anti-reflective coating (ARC) layer 5-910 and a photoresist 5-920, such as Figure 5-9A The photoresist may be exposed and patterned by photolithography and development of the resist. The resist may be developed to remove exposed portions or unexposed portions (based on the type of resist), leaving a pillar 5-922 having a diameter approximately equal to the desired diameter Dsw of the sample well, as shown in FIG. Figure 5-9B The height of the post can be substantially different than the desired depth of the sample well. For example, the height of the post can be substantially greater than the desired depth of the sample well.

[0339] The pattern of pillars 5-922 may be transferred to the ARC layer 5-910 using anisotropic reactive ion etching (RIE), e.g. Figure 5-9C The region may then be coated with at least one material 5-230 (e.g., a conductor or metal) that is desirable for forming a sample well. A portion of the one or more deposited materials forms a cap 5-232 over the pillar 5-922, such as Figure 5-9D . The photoresist 5-920 and ARC layer 5-910 may then be stripped from the substrate using a selective removal process (e.g., using a chemical bath with or without agitation that dissolves at least the resist and releases or "strips" the cap). If the ARC layer 5-910 remains, it may be stripped from the substrate using a selective etch, thereby leaving the sample well 5-210, as shown. Figure 5-9E According to some embodiments, due to the nature of the deposition of the at least one material 5-230, the sidewalls 5-214 of the sample well may be sloped.

[0340] As used herein, "selective etching" refers to an etching process in which an etchant selectively removes or etches one material at a higher rate (eg, at least twice as high) than the etchant etches other materials that the etchant is not intended to remove.

[0341] Because the photoresist 5-920 and ARC layer 5-910 are typically polymer based, they are considered soft materials that may not be suitable for forming sample wells with high aspect ratios (e.g., aspect ratios greater than about 2:1, in terms of height / width). For sample wells with higher aspect ratios, a hard material may be included in the stripping process. For example, a layer of hard material (e.g., an inorganic material) may be deposited prior to depositing the ARC layer and photoresist. In some embodiments, a layer of titanium or silicon nitride may be deposited. The layer of hard material should show preferential etching above the one or more materials 5-230 in which the sample well is formed. After patterning the photoresist, the pattern of the pillars may be transferred into the ARC layer and the underlying hard material 5-930 to obtain a sample well such as Figure 5-9FThe photoresist and ARC layers may then be stripped, material 5-230 may be deposited, and a lift-off step may be performed to form a sample well.

[0342] According to some embodiments, a sample well including an energy-enhancing structure 5-711 may be formed using a lift-off process, such as Figure 5-7C and Figure 5-7D as shown in .

[0343] An alternative process for forming a sample well is shown in Figure 5-10A -D. In this process, the sample well may be etched directly into the at least one material 5-230. For example, the at least one material 5-230 that constitutes the sample well may be deposited on a substrate. The layer may be covered with an ARC layer 5-910 and a photoresist 5-920, such as Figure 5-10A The photoresist may be patterned to form a hole having a diameter approximately equal to the desired diameter of the sample well, as shown in FIG. Figure 5-10B For example, anisotropic reactive ion etching can be used to transfer the hole pattern to the ARC and through layer 5-230, such as Figure 5-10C The photoresist and ARC layers can be stripped to obtain Figure 5-10D According to some embodiments, the sidewalls of a sample well formed by etching into the layer of material 5-230 may be more vertical than sidewalls formed using a lift-off process.

[0344] In some embodiments, a hard mask (e.g., a silicon nitride or oxide layer, not shown) may be patterned over the material 5-230 using a photoresist and ARC layer. The patterned holes may then be transferred to the hard mask, which is then used to transfer the pattern into the layer of material 5-230. The hard mask may allow for a greater etch depth into the layer of material 5-230, thereby forming a sample well with a higher aspect ratio.

[0345] It should be understood that when multiple layers of different materials are used to form the stack of material 5-230 that constitutes the sample well, the sample well may be formed using the lift-off process and direct etching fabrication techniques described above. An exemplary stack is shown in Figure 5-11 In some embodiments, a stack of materials may be used to form a sample well to improve coupling of excitation energy to an excitation region of the sample well or to reduce transmission or re-radiation of excitation energy into a bulk sample. For example, an absorber layer 5-942 may be deposited over a first layer 5-940. The first layer may comprise a metal or metal alloy, and the absorber layer may comprise a material that suppresses surface plasmons, such as amorphous silicon, TaN, TiN, or Cr. In some embodiments, a surface layer 5-944 may also be deposited to passivate the surface surrounding the sample well (e.g., to inhibit adhesion of molecules).

[0346] The formation of the sample well including the recess 5-216 may be accomplished in any suitable manner. In some embodiments, the recess may be formed by further etching into an adjacent layer 5-235 and / or any intervening layer or layers adjacent to the sample well. For example, after forming the sample well in a layer of material 5-230, the layer 5-230 may be used as an etch mask for patterning the recess, such as Figure 5-12 For example, the substrate may be subjected to selective anisotropic reactive ion etching so that the pit 5-216 may be etched into the adjacent layer 5-235. For example, in an embodiment where the material 5-230 is a metal and the adjacent layer 5-235 is silicon oxide, a substrate having a CHF 3 or CF 4 The reactive ion plasma etching of the feed gas preferentially removes the exposed silicon oxide below the sample well and forms the pit 5-216. For example, "silicon oxide" as used herein generally refers to SiO x , and may include silicon dioxide.

[0347] In some embodiments, conditions within the plasma during etching (e.g., bias toward the substrate and pressure) may be controlled to determine the etch profile of the pit. For example, at low pressure (e.g., less than about 100 mTorr) and high DC bias (e.g., greater than about 20 V), the etch may be highly anisotropic and form substantially straight and vertical sidewalls of the pit, as shown in the accompanying figures. At higher pressures and lower biases, the etch may be more isotropic, resulting in tapered and / or curved sidewalls of the pit. In some embodiments, the pit may be formed using a wet etch, which may be substantially isotropic and form a substantially spherical pit that may extend laterally below the material 5-230, up to or beyond the sidewalls of the sample well.

[0348] Figure 5-13A to Figure 5-13C A method for forming a recess 5-216 having a lateral dimension smaller than that of a sample well 5-210 (eg, as shown in FIG. 5 ) is shown. Figure 5-7B ) in a process step of forming a sample well such as a depression shown in FIG. 1 . In some embodiments, after forming the sample well, a conformal sacrificial layer 5-960 may be deposited over the area including the sample well. According to some embodiments, the sacrificial layer 5-960 may be deposited using a vapor deposition process such as chemical vapor deposition (CVD), plasma enhanced CVD, or atomic layer deposition (ALD). The sacrificial layer 5-960 may then be etched back using a first anisotropic etch that is selective to the sacrificial layer 5-960, removing the layer from horizontal surfaces, leaving a sidewall coating 5-962 on the walls of the sample well, such as Figure 5-13BThe etch back may be selective in some embodiments and stop on the material 5-230 and the adjacent layer 5-235, or may be a non-selective timed etch in some embodiments.

[0349] A second anisotropic etch selective to the adjacent layer 5-235 may be performed to etch the pit 5-216 into the adjacent layer, such as Figure 5-13C . The sacrificial sidewall coating 5-962 may then optionally be removed using a selective wet or dry etch. The removal of the sidewall coating opens up the sample well to have a lateral dimension that is larger than the pit 5-216.

[0350] According to some embodiments, the sacrificial layer 5-960 may comprise the same material as the adjacent layer 5-235. In such embodiments, when etching the pit into the adjacent layer 5-235, the second etch may remove at least a portion of the sidewall coating 5-962. In some embodiments, this etch back of the sidewall coating may form a tapered sidewall of the pit.

[0351] In some embodiments, the sacrificial layer 5-960 may be formed from or include a layer of material used to passivate the sidewalls of the sample well (e.g., to reduce adhesion of the sample to the sidewalls of the sample well). Then, after formation of the recess, at least a portion of the layer 5-960 may remain on the walls of the sample well.

[0352] According to some embodiments, the formation of the sidewall coating 5-962 may be performed after the formation of the pit. In such an embodiment, the layer 5-960 covers the sidewalls of the pit. This process may be used to passivate the sidewalls of the pit and confine the sample to the interior of the central region of the pit.

[0353] The process steps associated with depositing an adhesive 5-211 at the base of the sample well 5-210 and a passivation layer 5-280 are shown in FIG. 5-14. According to some embodiments, the sample well may include a first passivation layer 5-280 on the sample well wall. For example, the sample well may be formed as described above in conjunction with the first passivation layer 5-280. Figure 5-13B or Figure 5-8 In some embodiments, the first passivation layer 5-280 may be formed by any suitable deposition process and etch back. In some embodiments, the first passivation layer 5-280 may be formed by oxidizing the material 5-230 in which the sample well is formed. For example, the sample well may be formed of aluminum, which may be oxidized to form a coating of aluminum oxide on the sidewalls of the sample well.

[0354] Adhesive 5-980 or an adhesive precursor (eg, a material that preferentially binds to an adhesive) may be deposited on a substrate using an anisotropic physical deposition process, such as evaporative deposition, such as Figure 5-14AThe adhesive or adhesive precursor may form an adhesive layer 5-211 at the base of the sample well, such as Figure 5-14B , and may cover an upper surface of a material 5-230 in which a sample well is formed. Figure 5-14C The subsequent oblique directional deposition (sometimes referred to as a shallow deposition or shallow evaporation process) shown in FIG. 1 deposits a second passivation layer 5-280 of the passivation material 5-990 above the upper surface of the material 5-230 and not covering the adhesive layer 5-211. During the shallow deposition process, the substrate may be rotated about an axis perpendicular to the substrate so that the second passivation layer 5-280 is more uniformly deposited near the upper edge of the sample well. According to some embodiments, the resulting structure is shown in FIG. Figure 5-14D As an alternative to depositing a second passivation layer, the adhesive may be removed from the upper surface of the material 5-230 using a planarization etch (eg, a CMP step).

[0355] According to some embodiments, the adhesion agent layer 5-211 may be deposited centrally at the base of the tapered sample well, such as Figure 5-15 For example, Figure 5-14A As shown in , the adhesive or adhesive precursor can be directionally deposited in the conical sample well formed in the above manner. Before or after the deposition of the adhesive layer 5-211, the sample well wall is passivated using an oxidation process. Figure 5-14D In the manner described, the adhesive or precursor remaining on the surface of the material 5-230 is passivated. In some embodiments, the adhesive on the upper surface of the material 5-230 may be removed by a chemical mechanical polishing step. By forming the adhesive layer or adhesive layer precursor centrally at the base of the sample well, deleterious effects on emission from the sample (e.g., suppression or quenching of sample radiation from sample walls, unfavorable radiation distribution from the sample because it is not centrally located relative to energy coupling structures formed around the sample well, negative effects on sample luminescence lifetime) may be effectively reduced.

[0356] In some embodiments, the lift-off patterning, etching, and deposition processes used to form the sample well and the pits can be compatible with CMOS processes used to form integrated CMOS circuits on the integrated device. Thus, the integrated device can be fabricated using conventional CMOS equipment and fabrication techniques, although custom or dedicated fabrication equipment can be used in some embodiments.

[0357] Variations of the above process steps may be used to form alternative embodiments of the sample well. Figure 5-14C The inclined deposition process shown in FIG. forms, for example, Figure 5-7A or Figure 5-7B The tapered sample well shown in Figure 5-7BFor a sample well of this embodiment, the angle of deposition may be varied during the deposition process. For such an embodiment, a sample well having substantially straight and vertical sidewalls may be first formed, and then other materials 5-230 may be deposited using an angled deposition to tapered the sidewalls of the sample well.

[0358] In some implementations, a sample well can be formed from a multilayer stack including multiple layers. Figure 5-16 A sample well having a pit formed in a substrate layer 5-105 is shown. In this embodiment, the sample well has a diameter of about 140-180 nm with a pit depth of about 40-90 nm. The substrate 5-105 may be formed of any suitable material, such as silicon oxide. A first layer 5-1001 may be formed on a surface of the substrate 5-105. The first layer 5-1001 may be formed of any suitable metal, such as aluminum, and may have a thickness of, for example, about 60 nm. A second layer 5-1003 may be formed on the first layer 5-1001. The second layer 5-1003 may be formed from any suitable metal, such as titanium, and may have a thickness of, for example, 10 nm. A third layer 5-1005 may be formed on the second layer 5-1003. The third layer 5-1005 may be formed from any suitable ceramic, such as titanium nitride, and may have a thickness of, for example, 30 nm. A fourth layer 5-1007 may be formed on top of the third layer 5-1005 and coat the vertical walls of the sample well. This fourth layer 5-1007 may be formed from any suitable material, such as aluminum oxide, and may have a thickness of approximately 5 nm.

[0359] Figure 5-16 The sample well may be formed about 350 nm above the waveguide 5-240 for carrying excitation energy in the form of light pulses to the sample well. The waveguide may have a width of, for example, 250 nm-700 nm. In some embodiments, the waveguide has a width of about 500 nm.

[0360] The sample well may be formed in any suitable manner. For example, the first three layers (5-1001, 5-1003, and 5-1005) may be formed on the substrate 5-105, as described above. In addition, a thin layer (approximately 5 nm) of aluminum oxide may be formed over the first three layers. The sample well and the pit may then be chemically etched into these layers. A second layer of aluminum oxide may be deposited to conformally coat the edges of the sample well, including the bottom of the pit. According to some embodiments, the second layer of aluminum oxide may be deposited using atomic layer deposition. The second layer of aluminum oxide may then be anisotropically etched from the bottom of the pit to expose the silicon oxide substrate.

[0361] D. Coupling the excitation energy into the sample well

[0362] Excitation energy is coupled to one or more sample wells of an integrated device using one or more techniques. As previously described, in some embodiments, a waveguide is positioned to couple an excitation source to one or more sample wells. As the excitation energy propagates along the waveguide, a portion of the excitation energy can be coupled to one or more sample wells using a variety of optical coupling techniques. For example, the waveguide can guide the excitation energy generally in one direction, and an evanescent wave or tail can be formed in a manner perpendicular to this one direction, and in some cases is located outside the waveguide structure. This evanescent wave tail can guide a portion of the excitation energy toward one or more sample wells. In some embodiments, a sample well layer can be designed and used to guide the excitation energy to a localized area inside the sample well. The sample well can be used to hold the sample inside the localized area of ​​the sample well so as to guide the excitation energy toward the sample.

[0363] Figure 6-1A and Figure 6-1B is a cross-sectional view of an integrated device and provides an exemplary illustration of utilizing a waveguide to couple excitation energy into a sample well. Figure 6-1A FIG. 6 is a schematic cross-sectional view of a waveguide 6-104 positioned adjacent to a sample well 6-108 in a sample well layer 6-116. Figure 6-1A Positioning the sample well near the waveguide may allow the excitation energy to couple into the sample well. Figure 6-1B A close-up view of the sample well 6-108 and the sample well layer 6-116 is shown, and excitation energy located within the sample well 6-108 is shown.

[0364] In addition, one or more components may be formed in the integrated device to improve or enhance the coupling of excitation energy into the sample well. These other components may be formed in a pixel and provide coupling of excitation energy from the waveguide into the pixel and toward the sample well. One or more components located in a pixel may draw a portion of the excitation energy from the waveguide into the pixel. Such components may include optical structures, such as grating structures, scattering structures, microcavities and / or nanoantennas. The characteristics or configurations of one or more of these components may be selected to couple each amount of excitation energy to each sample well within a row or column of sample wells. A waveguide for passing excitation energy to a row of pixels may be connected to a component in each pixel region, thereby providing a portion of the excitation energy to each pixel in each row of pixels. When a waveguide is used to guide excitation energy from an excitation source toward one or more pixels, the waveguide may be referred to as a bus waveguide.

[0365] A component located adjacent to the sample well can improve the coupling of excitation energy from the waveguide to the sample well. Such a component can be referred to as an optical tap and / or a microcavity. The microcavity can deflect a portion of the excitation energy from the waveguide so that the excitation energy reaches the sample well. One or more microcavities can be used to couple the excitation energy to the sample well. One or more microcavities can reduce the excitation energy from the waveguide, including metal losses. One or more microcavities can act as a lens for focusing the excitation energy to the sample well. In some embodiments, one or more microcavities can improve the guidance of light from the marker toward the sensor in the sample well. The microcavity can have a cylindrical, convex, concave, rectangular, spherical, or elliptical configuration or any other suitable shape. The microcavity can be made of any suitable material. In some embodiments, the microcavity can include silicon nitride.

[0366] When viewed from the top of an integrated device in which a sample well is present, one or more microcavities may overlap at least a portion of a waveguide to direct excitation energy toward the sample well. The thickness of the waveguide may be configured to reduce the loss of excitation energy and improve the coupling of excitation energy to the one or more microcavities. In some embodiments, the microcavities along a row of sample wells may have a variation in coupling strength between the waveguide and each sample well. The microcavity may enhance coupling along the direction of propagation of the excitation energy to accommodate the reduced power in the waveguide when directing the excitation energy from the waveguide to each sample well. In some embodiments, one or more microcavities are adjacent to the sample well. There may be an offset distance between the location of the center of the sample well and the center of the microcavity. In other embodiments, a microcavity is located below the sample well such that at least a portion of the sample well overlaps a portion of the microcavity when viewed from the top of the integrated device in which the sample well is present.

[0367] Figure 6-2A A plan view of an exemplary pixel region having a waveguide 6-204 is shown, the waveguide 6-204 being located proximate to a sample well 6-208 such that the waveguide 6-204 and the sample well 6-208 do not overlap. Figure 6-2AAs shown in , the microcavity 6-218b has a smaller cross-sectional diameter than the microcavity 6-218a. The microcavities 6-218a and 6-218b are positioned relative to the waveguide 6-204 and the sample well 6-208 so as to couple the excitation energy from the waveguide 6-204 to the sample well 6-208. Together, the microcavities 6-218a and 6-218b deflect a portion of the excitation energy inside the waveguide to the sample well. A portion of the microcavity 6-218b is positioned to overlap the waveguide 6-204. The microcavity 6-218b is positioned proximate to the microcavity 6-218b to provide sufficient coupling between the two microcavities. The microcavity 6-218a is located closer to the sample well 6-208 than the microcavity 6-218b. In this configuration, the microcavities 6-218a and 6-218b may function as optical taps to couple a portion of the excitation energy from the waveguide 6-204 to the sample well 6-208. Figure 6-2B Shows the Figure 6-2A 6-204 and 6-206. The microcavities 6-218a and 6-218b are located adjacent to the layer 6-216, thereby forming the sample well 6-208. The layer 6-216 may include a metal (e.g., aluminum). In this exemplary embodiment, the microcavities 6-218a and 6-218b have a cylindrical shape, wherein one end of the cylinder is located at or at least close to the surface of the layer 6-218. The distance between the waveguide 6-204 and the edge of the microcavity 6-218a and / or the microcavity 6-218b may allow a desired amount of excitation energy to be coupled into the sample well 6-218. Figure 6-2C shows the Figure 6-2A The microcavity 6-218b is positioned to overlap the waveguide 6-204, while the microcavity 6-218a is positioned not to overlap the waveguide 6-204.

[0368] Figure 6-3A -D shows a plan view of another exemplary configuration of one or more microcavities relative to a sample well and a waveguide in an integrated device. One or more microcavities may overlap a portion of a waveguide. In some cases, the microcavity is located below the sample well. Figure 6-3A A microcavity 6-306 is shown overlapping a sample well 6-308 with respect to a waveguide 6-304. Figure 6-3B The cavity 6-316b is shown overlapping the sample well 6-318 relative to the waveguide 6-314. In other embodiments, the microcavity is located at an offset distance from the sample well such that the microcavity does not overlap the sample well. Figure 6-3B A microcavity 6-318a is shown located apart from the sample well 6-318 such that there is no overlap region between the microcavity 6-316a and the sample well 6-318. Figure 6-3CThe microcavity 6-326 is shown positioned separate from the sample well 6-328 relative to the waveguide 6-324 such that there is no overlap region between the microcavity 6-316a and the sample well 6-318. In some implementations, multiple microcavities may be positioned offset from the sample well. Figure 6-3D Microcavities 6-336a and 6-336b are shown positioned to be separated from sample well 6-338 relative to waveguide 6-334 so that there is no overlap area between sample well 6-338 and 6-336a or 6-336b. The microcavity design, position relative to the sample well, and / or position relative to the waveguide may be determined based on reducing overall coupling losses and improving coupling efficiency between the waveguide and the sample well.

[0369] Figure 6-4 A cross-sectional view of a microcavity 6-418 for coupling excitation energy from a waveguide 6-404 to a sample well 6-408 in a layer 6-416 is shown. One or more dimensions of the microcavity 6-418 and / or waveguide 6-404 may provide a desired amount of coupling. A waveguide of an integrated device (e.g., waveguide 6-404) has a cross-sectional height t, which may be about 50 nm, about 100 nm, about 150 nm, about 160 nm, about 170 nm, or about 200 nm. The microcavity 6-418 has a cross-sectional dimension D, such as a diameter if the microcavity has a cylindrical shape. The cross-sectional dimension D may be about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, or about 800 nm. The microcavity 6-418 has a cross-sectional height h, which may be about 450 nm, about 500 nm, about 550 nm, or about 600 nm. The location of the microcavity 6-418 relative to the waveguide 6-408 and the sample well 6-408 may allow excitation energy to be coupled from the waveguide 6-408 to the sample well 6-408. The distance between the microcavity 6-418 and the waveguide 6-408 perpendicular to the propagation of light in the waveguide 6-408 may be Figure 6-4 The distance that the microcavity 6-418 is offset from the sample well 6-408 is shown as x in FIG. Figure 6-4 d, may be about 500 nm, about 550 nm, about 600 nm, about 650 nm, or about 700 nm. The layer 6-416 including the sample well 6-408 may have a cross-sectional height of about 50 nm, about 100 nm, or about 150 nm. The sample well 6-408 may have a cross-sectional dimension, such as a diameter of about 75 nm, about 100 nm, about 125 nm, about 150 nm, or about 175 nm.

[0370] In one exemplary embodiment, the waveguide 6-404 has a cross-sectional width of 667 nm and a cross-sectional height of 100 nm. The microcavity 6-418 has a dimension h of 591 nm, a cross-sectional diameter of 750 nm, and is positioned such that the distance x from the waveguide 6-404 is 398 nm and the distance d from the sample well 6-408 is 693 nm. This pixel configuration may have a total coupling loss of 0.28%, which if embodied in an integrated device having 256 pixels may have a transmission loss of approximately 51% and a metal loss of approximately 11%.

[0371] In another exemplary embodiment, the waveguide 6-404 has a cross-sectional width of 639 nm and a cross-sectional height of 100 nm. The microcavity 6-418 has an ellipsoidal shape having one cross-sectional dimension of 600 nm, another cross-sectional dimension of 639 nm, and a dimension h of 512 nm. This pixel configuration may have a total coupling loss of about 0.77%, which if embodied in an integrated device having 64 pixels may have a transmission loss of about 30% and a metal loss of about 35%.

[0372] Figure 6-5 The results of a simulation of the propagation of light with respect to a sample well 6-538 and a waveguide 6-534 in a configuration having two microcavities 6-536a and 6-536b are shown. Figure 6-5 As shown in FIG. 6 , the dark regions correspond to higher intensity light extending from the waveguide 6-534 to the sample well 6-538 and supported by the microcavities 6-536a and 6-536b.

[0373] In another exemplary embodiment, the waveguide has a cross-sectional width of 700 nm and a cross-sectional height of 100 nm. The microcavity is cylindrical with a diameter of 600 nm and has a dimension h of 650 nm. The microcavity is positioned close to the waveguide and the sample well, so there is a coupling of 0.148%, a metal loss of 0.09856%, a return loss of 0.1225%, and a radiation loss of 0.1270%. Figure 6-6A to Figure 6-6C A simulation of light propagation according to this embodiment is shown. Figure 6-6A A cross-sectional view of a sample well 6-638, a microcavity 6-636, and a waveguide 6-634 is shown. The waveguide 6-634 supports a mode of light propagating along the waveguide 6-634, and the microcavity 6-636 is positioned to act as a light tap, thereby coupling a portion of the excitation energy into the sample well 6-638. Figure 6-6A , darker areas represent areas with higher light intensity. Figure 6-6B A plan view of a microcavity 6-636 and a sample well 6-638 is shown, wherein light is directed through the microcavity 6-636 toward the sample well 6-638. Figure 6-6CA transverse cross-sectional view showing light being coupled from a waveguide 6-634 to a sample well 6-638 is shown.

[0374] Figure 6-6D A plan view of a simulation for another exemplary configuration having a waveguide 6-654, a microcavity 6-656, and a sample well 6-658 is shown. In this configuration, the sample well 6-658 overlaps the microcavity 6-656, and the microcavity 6-658 overlaps the waveguide 6-654. Figure 6-6D Light is shown, with dark regions representing higher intensity, propagation toward the sample well 6-658 supported by the microcavity 6-656.

[0375] Some embodiments relate to a microcavity positioned between a sample well layer and a waveguide such that the microcavity is offset from a surface of the sample well layer. The microcavity may be offset from a surface of the sample well layer in a direction perpendicular to light propagation along the waveguide. In some embodiments, the microcavity may also be offset from the location of the sample well such that the sample well and the microcavity do not overlap. The size and formation of the microcavity may be designed to provide a desired amount of excitation energy coupling from the waveguide to the sample well. In some embodiments, the microcavity may have a longer dimension in a direction parallel to the direction of light propagation through the waveguide than in a dimension perpendicular to the light propagation through the waveguide.

[0376] Figure 6-7A A cross-sectional view of an integrated device having a microcavity 6-718 located proximate to a sample well 6-708 and a waveguide 6-704 is shown. The sample well 6-708 is formed in a sample well layer 6-716. The microcavity has a dimension D, which is parallel to the direction of light propagation through the waveguide 6-704; and a dimension h, which is perpendicular to the direction of light propagation through the waveguide 6-704. In some embodiments, the dimension D of the microcavity 6-718 is greater than the dimension h of the microcavity 6-718. The microcavity 6-718 may have a dimension h of approximately 100 nm, approximately 150 nm, or approximately 200 nm. The microcavity 6-718 may have a dimension D of approximately 500 nm, approximately 750 nm, or approximately 1000 nm. The microcavity 6-718 is located at a distance in the y-direction from a surface of the waveguide 6-704 having a dimension x1 and at a distance in the y-direction from a surface of the sample well layer 6-716 having a dimension x2. In some embodiments, dimension x2 is less than dimension x1. In some embodiments, the microcavity 6-718 may be offset from the sample well layer 6-716 such that x2 is approximately 200 nm, approximately 300 nm, or approximately 400 nm.

[0377] In some embodiments, the microcavity 6-718 is offset from the sample well 6-708 by a distance in a direction parallel to the direction of light propagation through the waveguide 6-704. The microcavity 6-718 may be offset from the sample well 6-708 by a center-to-center distance d. In some embodiments, the microcavity 6-718 is offset from the sample well 6-708 such that the microcavity 6-718 and the sample well 6-708 do not overlap. In some embodiments, the microcavity 6-718 may be positioned such that an edge of the microcavity 6-718 proximate to the sample well 6-708 is offset from the sample well 6-708 by a distance. The offset distance between the edge of the microcavity 6-718 and the sample well 6-708 may be about 50 nm, about 100 nm, about 150 nm, or about 200 nm.

[0378] Figure 6-7B A cross-sectional view showing the intensity of light as excitation energy propagates through the waveguide 6-704 and is coupled to the sample well 6-708 in the sample well layer 6-716 using the microcavity 6-718, similar to FIG. Figure 6-7A The excitation energy travels through the waveguide 6-704 in the z-direction. Figure 6-7B As shown in FIG, a portion of the excitation energy (illustrated as a black line) reaches the sample well 6-708 by coupling into the microcavity 6-718. Since the excitation energy continues to propagate along the waveguide 6-704, the microcavity 6-718 acts as an optical tap by directing a portion of the excitation energy from the waveguide 6-704 toward the sample well 6-708.

[0379] Figure 6-7C A plan view of a microcavity 6-718 located proximate to a sample well 6-708 is shown. Excitation energy (shown as a dark area) propagates through the microcavity and is directed into the interior of the sample well 6-708. Figure 6-7C As shown in , the microcavity 6-718 may have a rectangular shape with curved edges that function to direct light toward the sample well 6-708. The edges of the microcavity 6-718 may have a radius of curvature to allow coupling of a desired level of excitation energy to the sample well 6-708. A first edge of the microcavity 6-718 proximate the sample well 6-708 may have a radius of curvature that is smaller than a second edge of the microcavity 6-718 that is opposite the radius of curvature of the first edge.

[0380] Some embodiments relate to a microcavity positioned between a sample well and a waveguide such that the microcavity overlaps the sample well and is offset from the sample well by a distance. The microcavity and the sample well may overlap in a direction perpendicular to the direction of light propagation along the waveguide. The microcavity may function to enhance coupling of excitation energy into the sample well. In some embodiments, the microcavity may be aligned with the sample well by taking advantage of a significant center-to-center alignment between the microcavity and the sample well. In some embodiments, the microcavity may be positioned closer to the waveguide than the sample well. In some embodiments, the microcavity may be located on a surface of the waveguide.

[0381] Figure 6-7D A cross-sectional view of an integrated device having a sample well 6-728, a waveguide 6-734, and a microcavity 6-738 located between the sample well 6-728 and the waveguide 6-734 is shown. The sample well 6-728 is a recess formed in the sample well layer 6-738 and may extend beyond the sample well layer 6-738 to include a layer 6-732 of the integrated device. The layer 6-732 may have a dimension h along the x-direction, such as Figure 6-7D As shown in . The microcavity 6-738 may be located within the layer 6-732. Such that a portion of the layer 6-732 exists between the microcavity 6-738 and the sample well 6-728, and a portion of the layer 6-732 exists between the microcavity 6-738 and the waveguide 6-734. Figure 6-7D , the microcavity 6-738 is located a distance in the y-direction from the sample well 6-728 having a dimension d1. The microcavity 6-738 is located a distance in the y-direction from the waveguide 6-734 having a dimension d2. In some embodiments, dimension d2 is less than d1. In some embodiments, the microcavity 6-738 is located at the surface of the sample well 6-728 and within the layer 6-732 such that dimension d1 is equal to zero.

[0382] One or more dimensions of a waveguide of an integrated device may vary along the length of the waveguide in a direction of light propagation through the waveguide. One or more dimensions that vary along the waveguide may improve coupling efficiency and approximate uniformity in the amount of excitation energy provided by the waveguide to a plurality of sample wells. In some embodiments, a cross-sectional width of the waveguide may vary along a row or column of pixels. The waveguide may include a tapered portion such that a cross-sectional width of the waveguide decreases along a direction of propagation of the excitation energy through the waveguide. Figure 6-8A A plan view of a waveguide 6-804 in an integrated device is shown. An excitation source 6-802 is coupled to the waveguide 6-804 using one or more of the techniques described herein (e.g., grating couplers, star couplers, MMI splitters). The waveguide 6-804 is tapered so that along Figure 6-8A804, the dimension of the waveguide 6-804 is reduced in the x-direction shown in , along the dimension of the waveguide 6-804, along the z-direction, or in the direction of light propagation through the waveguide 6-804. Thus, the waveguide 6-804 has a greater cross-sectional width (in the x-direction) near the incident excitation source 6-802 than at a location further along the length of the waveguide 6-804 (in the z-direction). Positioning the waveguide 6-804 to couple with the sample wells in a row of pixels can provide a configuration sufficient to couple a desired amount of excitation energy into each sample well. Because a portion of the excitation energy is coupled out of the waveguide 6-804 for each sample well, the amount of excitation energy propagated by the waveguide 6-804 is reduced in the z-direction. As the excitation energy is coupled out of the sample well, the amount of power along the waveguide 6-804 is reduced. By reducing the cross-sectional width of the waveguide 6-804, the excitation energy can be propagated further along the waveguide 6-804 than a waveguide without such a tapered portion.

[0383] Figure 6-8B 8A shows a cross-sectional view of the waveguide 6-804 shown in FIG. 8A. The dimensions of the waveguide 6-804 along the y-direction remain substantially similar such that the distance between the sample well 6-808 and the waveguide 6-804 along the y-direction is substantially constant along the length of the waveguide 6-804. The dashed curve represents the spread of the excitation energy as the excitation energy propagates along the waveguide 6-804 in the z-direction. As the cross-sectional width of the waveguide 6-808 narrows, the spread of the excitation energy becomes wider and can compensate for the reduced power. In this way, the cross-sectional width of the waveguide 6-802 can balance the reduction in the power of the excitation energy propagated by the waveguide 6-802 so that a sufficient amount of excitation energy is delivered to each sample well in a row of pixels to obtain the desired performance of the integrated device.

[0384] In some embodiments, a tapered waveguide can be configured for similar coupling efficiency for a row of pixels, where each pixel in the row includes a microcavity and a sample well. A combination of varying one or more dimensions of the tapered waveguide and / or microcavity can accommodate a reduction in excitation energy power along the length of the waveguide when excitation energy is coupled to each sample well in the row.

[0385] In some embodiments, one or more sensors may be positioned relative to the waveguide to measure the excitation energy propagating along the waveguide. Other structures (e.g., gratings) may be used to draw at least a portion of the excitation energy to the sensors. Figure 6-8B8, monitoring sensors 6-812a and 6-812b are located near the ends of the waveguides. Gratings 6-810a and 6-810b are located on a side of the waveguide 6-808 opposite the sensors 6-812a and 6-812b. Gratings 6-810a and 6-810b may be used to direct excitation energy in the waveguide 6-808 toward sensors 6-812a and 6-812b, respectively. The combination of grating 6-810a and sensor 6-812a may monitor the excitation energy input into the waveguide 6-804. The combination of grating 6-810b and sensor 6-812b may monitor the excitation energy, if any, that remains at one end of the waveguide 6-804 after coupling the excitation energy to a row of sample wells 6-808. Thus, the sensors 6-812a and 6-812b may monitor the power in the waveguide 6-804 at the input, and output, and / or at any suitable location along the waveguide 6-808. The sensors 6-812a and 6-812b may detect information including the power level at a location along the waveguide 6-808. This information may be used to control aspects of components for aligning the excitation source with the integrated device, and / or the power of the excitation source.

[0386] In some embodiments, a waveguide can be coupled to a sample well via an evanescent wavefield. In some embodiments, a bull's eye grating structure having concentric grating rings located near the sample well can improve coupling of excitation energy from the waveguide to the sample well. In some embodiments, a waveguide can include a region having a reduced cross-sectional width near the sample well so that the evanescent wavefield from the excitation energy propagating in the waveguide couples to the sample well. For a row of pixels, the waveguide can include multiple regions having a reduced cross-sectional width along the length of the waveguide to improve coupling uniformity and efficiency in each sample well in the row. In this way, the waveguide can be considered to have a pinched portion at certain locations along the waveguide.

[0387] A layer having one or more sample wells in an integrated device can interfere with the propagation of light through a waveguide. In some embodiments, the sample well layer is made of a metal (e.g., aluminum). It is desirable to position the sample well layer at a certain distance from the waveguide to reduce losses in the device and improve performance. These techniques can allow the desired performance to be achieved by positioning the sample well layer at a certain distance from the waveguide while allowing the sample wells in the layer to receive a sufficient amount of excitation energy.

[0388] Figure 6-9AA plan view of a waveguide 6-904 relative to a bull's eye grating structure 6-910 is shown. The bull's eye grating structure 6-910 includes a plurality of concentric circular gratings centered at a sample well. The waveguide 6-904 includes a constricted region, wherein the cross-sectional width of the waveguide 6-904 near the center of the bull's eye grating structure 6-910 is smaller. The constricted region of the waveguide 6-904 may be formed by forming a taper along a certain distance of the waveguide 6-904. The length of the tapered waveguide 6-904 may be a suitable amount for reducing excitation energy loss and / or improving coupling efficiency. The waveguide may include a plurality of constricted regions, wherein each constricted region is associated with a sample well. The plurality of constricted regions may have one or more dimensions that vary along the length of the waveguide so as to provide appropriately uniform power and coupling efficiency to each sample well. Figure 6-9B A cross-sectional view of a waveguide 6-904 for coupling excitation energy to a sample well 6-908 is shown. A bull's eye grating 6-910 is positioned approximately at the sample well 6-908 and is used to couple excitation energy from the waveguide 6-904 to the sample well 6-908. The cross-sectional width of the waveguide 6-904 has a constricted region proximate the sample well 6-908 such that the spread of the excitation energy field widens as the cross-sectional width narrows. The dashed line represents the extent to which the field of excitation energy extends vertically from the waveguide 6-904 in the y-direction. At locations along the waveguide 6-904 that overlap with the sample well 6-908, the field of excitation energy is at a location that allows the excitation energy to be coupled to the sample well 6-908.

[0389] The taper in one or more dimensions in the pinch and / or the shape of the taper in the waveguide pinch may provide a desired level of coupling efficiency. Figure 6-10 A graph of total loss for different tapered waveforms as a function of the length over which the tapered waveform exists, or the length of the tapered portion representing the amount of loss due to the tapering process, is shown. As shown in FIG. 10 , a tapered portion with a smaller slope in the constriction region can improve coupling efficiency by reducing the total coupling loss. For a linear or S-shaped configuration, tapering the cross-sectional width from 0.5 microns to 0.3 microns has a lower total loss than tapering the cross-sectional width from 0.5 microns to 0.2 microns. In addition, increasing the length of the reduction in cross-sectional width can improve the total coupling loss. In some embodiments, the waveguide may include a constriction region where the cross-sectional width of the waveguide changes from about 0.3 microns to about 0.5 microns over a length along the waveguide of about 20 microns, about 40 microns, about 50 microns, about 60 microns, about 80 microns, or about 100 microns.

[0390] In some implementations, the waveguide can be coupled to the sample well by reducing the distance between the sample well and the waveguide within the sample well region. This configuration can allow portions of the waveguide to be located at a distance from the sample well layer to provide desired efficiency and loss of excitation energy. Figure 6-11A A cross-sectional view of a waveguide 6-1104 positioned relative to a layer 6-1102 having a sample well 6-1108 is shown. A bull's eye grating is formed adjacent to the layer 6-1102 and approximately centered at the sample well 6-1108. In this embodiment, the waveguide has a substantially uniform cross-sectional thickness, and the field of excitation energy extending from the waveguide 6-1104 is substantially uniform along the length of the waveguide 6-1104. By forming the layer 6-1102 so that the sample well 6-1108 and the waveguide 6-1104 have a certain distance along the y-direction, the excitation energy can be coupled to the sample well 6-1108. The bull's eye grating 6-1110 can further couple the excitation energy from the waveguide 6-1104 to the sample well 6-1108. Other regions along the waveguide 6-1104 separated from the sample well 6-1108 may have a layer 6-1102 at a greater distance along the y-direction than near the sample well 6-1108. This configuration may be considered a sample well descender configuration and in some embodiments provides a coupling efficiency of approximately 0.3%.

[0391] Some embodiments involve forming one or more layers of material on a waveguide to improve coupling of excitation energy from the waveguide to a sample well. Figure 6-11B A cross-sectional view of a sample well 6-1112 formed by a sample well layer 6-1116 is shown. Figure 6-11B As shown in , the sample well layer 6-1116 includes a region having an opening of the sample well layer 6-1116, which forms a sample well 6-1112. The region of the sample well layer 6-1116 is offset from the surface of the integrated device by a dimension h. The dimension h may be greater than about 200 nm, about 250 nm, about 300 nm, about 350 nm, or about 400 nm. The offset region of the sample well layer 6-1116 may be formed by etching a portion of the layer 6-1120 and forming the sample well layer 6-116 over the layer 6-1120. The region of the sample well layer 6-1116 including the sample well 6-1112 is located a distance d1 from the waveguide 6-1118 in the y-direction. The distance d1 may be about 250 nm, about 300 nm, about 350 nm, about 400 nm, or about 450 nm. A layer 6-1114 is formed adjacent to the waveguide 6-1118. The layer 1114 may have a higher refractive index than the layer 6-1120 and a lower refractive index than the waveguide 6-1118. In some embodiments, the layer 6-1114 includes multiple layers having different materials. In an exemplary embodiment, the waveguide 6-1118 includes silicon nitride and the layer 6-1114 includes aluminum oxide and / or titanium oxide. The layer 6-1114 is located at a distance d2 from the sample well layer 6-1116a. The distance d2 may be about 50 nm, about 100 nm, about 150 nm, about 200 nm, or about 250 nm. Figure 6-11C Shown along Figure 6-11B A cross-sectional view of the line AA' shown in FIG. Figure 6-11C As shown in , the waveguide 6-1118 may have a lateral transverse dimension along the x-direction that overlaps an offset region of the sample well layer 6-1116 including the sample well 6-1112. The layer 6-1114 may be formed on the waveguide 6-1118 such that the layer 6-1114 contacts multiple sides of the waveguide 6-111C.

[0392] Additionally or alternatively, the spacing between pixels in a row along a waveguide can be selected to reduce waveguide losses. Each pixel can include a sample well, one or more sensors, and / or a bull's eye grating. The spacing between rows of pixels can be adjusted to accommodate other integrated device components, such as circuitry. Figure 6-12 A plan view of an integrated device having pixels 6-1208 arranged in a rectangular array with waveguides 6-1204 for delivering excitation energy to a row of pixels 6-1208 is shown. Figure 6-12 A configuration of a pixel array is shown where the spacing x between pixels in a row is less than the spacing y between rows. By positioning pixels at a certain distance in a row, waveguide losses along the waveguide can be reduced. Having a larger spacing between rows can reduce interference of pixels in one row with another row and / or provide suitable spacing to accommodate other components on the device, including circuit components.

[0393] The entire pixel array of the integrated device can have any suitable number of pixels and any suitable pixel arrangement. In some embodiments, a pixel array can have a square and / or rectangular configuration. In some embodiments, the pixel array can be rectangular and have a length parallel to the waveguide that is longer than a length perpendicular to the waveguide. In other embodiments, there can be more rows of waveguides and / or pixels, and the length of the pixels in each row parallel to the waveguide can be shorter than the length perpendicular to the waveguide.

[0394] Some embodiments relate to an integrated device with a waveguide having a variable size in a direction perpendicular to light propagation through the waveguide and perpendicular to a surface of the integrated device having one or more sample wells. In some embodiments, the size of the waveguide can vary to be larger in a region proximate to but not overlapping the sample well. In some embodiments, the size of the waveguide can decrease in a region of the waveguide overlapping the sample well. The integrated device can include a waveguide for coupling to a row of sample wells, wherein the variation in the waveguide size allows for coupling of substantially similar amounts of excitation energy to each sample well.

[0395] Figure 6-13A cross-sectional view of an integrated device having a sample well 6-1308 formed in a sample well layer 6-1316 is shown. The waveguide 6-1304 includes a region 6-1310 having a dimension x1 along the y-direction x1 that is larger than a portion of the waveguide 6-1304 that overlaps the sample well 6-1308 having a dimension x2 along the y-direction. Figure 6-13 As shown in FIG. 6A , the region 6-1310 is offset from the sample well 6-1308 and forms a ridge in the waveguide 6-1304. The size and shape of the region 6-1310 is designed to provide a desired amount of excitation energy from the waveguide 6-1304 to the sample well 6-1308. Figure 6-14 A cross-sectional view of an integrated device having a sample well 6-1408 formed in a sample well layer 6-1416 is shown. The waveguide 6-1404 includes a region 6-1410 that overlaps the sample well 6-1408. The waveguide in the region 6-1410 has a dimension x2 along the y-direction that is smaller than the waveguide regions outside of the region 6-1410, which have a dimension x1 along the y-direction. In this configuration, the waveguide 6-1404 includes a smaller dimension inside the region 6-1410 that overlaps the sample well 6-1408.

[0396] although Figure 6-13 and Figure 6-14 A single sample well is shown, but the waveguides 6-1304 and 6-1404 may be configured to couple to a row of sample wells and have areas similar to the areas 6-1310 and 6-1404 located proximate to each sample well. In some embodiments, the size and / or shape of the areas located proximate to multiple sample wells in the row may vary so that a substantially similar amount of excitation energy is coupled to each sample well. Thus, the areas of the waveguide may be configured to accommodate for reducing the propagation of excitation energy along the waveguide and along a row of sample wells.

[0397] E. Direct the transmitted energy to the sensor

[0398] The integrated device may include one or more components located between a sample well and a sensor of a pixel to improve the collection of luminescence from a sample in the sample well by the sensor. Such components may increase the signal-to-noise ratio of the luminescence signal to the background signal, thereby providing improved detection of luminescence signatures. Some components may direct luminescence from the sample well to a corresponding sensor in a pixel. In some embodiments, a component may provide coupling of appropriate excitation energy to the sample well and coupling of luminescence from the sample well. Other components (e.g., filters) may reduce excitation energy and other light not associated with the sample, and / or signatures that contribute to the signal acquired by the sensor.

[0399] The bull's eye grating may consist of concentric grating rings around a sample well. The bull's eye grating may be coupled to the sample well to improve the propagation of luminescence from the sample well. The bull's eye grating structure may direct luminescence toward the sensor in a pixel having a sample well. In some embodiments, the effective diameter of the luminescence directed by the bull's eye grating is about 5 microns.

[0400] Figure 7-1A A plan view of a bull's eye 7-110 configured to direct luminescence from a sample well 7-108 is shown. The bull's eye structure 7-110 includes a plurality of concentric gratings. These concentric gratings may be substantially aligned with the center of the sample well 7-108. The waveguide 7-104 may be located overlapping at least a portion of the bull's eye structure 7-110 and the sample well 7-108. The waveguide 7-104 includes a tapered region that may allow luminescence to reach a sensor located within a pixel having a sample well 7-108, and a desired degree of collection of luminescence by the sensor. The tapered region may allow for reduced interference of the waveguide 7-104 with luminescence collection by the sensor. In some embodiments, the bull's eye grating may be configured to direct more than one characteristic luminescence energy or wavelength toward the sensor. Figure 7-1B A cross-sectional view of a bull's eye grating for directing light emission from a sample well 7-108 is shown. The dashed lines represent the diffusion of two characteristic wavelengths λ1 and λ2 that may be coupled out of the sample well 7-108. The two characteristic wavelengths λ1 and λ2 may be characteristic wavelengths emitted from different markers for labeling a sample. Figure 7-1C A cross-sectional view of a bull's eye grating 7-110 in a transverse cross-sectional view of a waveguide 7-104 is shown. The bull's eye grating 7-110 and waveguide 7-104 are configured such that luminescence emitted from a sample well 7-108 is diffused thereby allowing luminescence energy to pass through regions of the integrated device other than the waveguide 7-104. The bull's eye grating 7-110 may direct luminescence over a distance from the sample well 7-108 that is greater if the bull's eye grating is not present. In some embodiments, the bull's eye grating 7-110 may reduce on any scattering of luminescence. The bull's eye grating 7-110 may be configured for coupling excitation energy into the sample well 7-108 and directing luminescence energy out of the sample well 7-108.

[0401] The microcavity provides coupling of the waveguide and the sample well, thereby allowing the excitation energy to propagate to the sample well and also directing luminescence from the sample well to the sensor. Figure 7-2A A cross-sectional view of the waveguide 7-204 is shown displaced from the sample well 7-208. The microcavity 7-211 is configured to guide light emission from the sample well 7-208. Figure 7-2A The intensity of the luminescence emitted by the sample well 7-208 is shown, where areas with higher luminescence intensity are darker. The microcavity 7-211 may direct the luminescence at an angle away from the waveguide 7-204, thereby reducing scattering of the luminescence by the waveguide 7-204. Figure 7-2B Graphs showing the luminescence emitted from a sample well as a function of angle are shown. Figure 7-2B As shown in , the microcavity 7-211 can direct the luminescence at an angle of about 15 degrees from the y-direction. One or more microcavities can direct the luminescence at an angle of about 5 degrees, about 10 degrees, about 15 degrees, about 20 degrees, about 25 degrees from the y-direction. In some embodiments, the sensor can be offset from the sample well to receive the luminescence directed at such an angle. The microcavity 7-211 can also be used to couple excitation energy from the waveguide to the sample well, so that the microcavity 7-211 provides coupling of excitation energy to the sample well and guiding of luminescence from the sample well. In some embodiments, more than one microcavity can be formed to couple luminescence from the sample well.

[0402] A baffle having an opening centered over the sensor may be formed between the sample well and the sensor. Figure 4-2 As shown in FIG. 4 , the baffle layer 4-226 is located between the sample well 4-22 and the sensor layer 4-230. The baffle in a pixel may be designed to inhibit the collection of energy other than the luminescence of the pixel. The baffle associated with the sample well and the sensor may allow luminescence from the sample well to reach the sensor while reducing luminescence from adjacent pixels, excitation energy, and other energy not associated with luminescence from the sample well associated with the sensor. The size of the opening of the baffle may be configured to allow luminescence directed by a bull's eye on the same pixel. Figure 4-2 As shown in FIG. 4 , the baffle 4-226 has an opening in the z-direction to allow light to pass through the sensor located in layer 4-230. The material of the baffle may be selected for certain optical properties (e.g., reducing the transmission of certain wavelengths of light or energy at certain angles of incidence). In some embodiments, the baffle may be formed of multiple layers of materials having different refractive indices. The baffle may include one or more layers of silicon, silicon nitride (Si 3 N 4 ), silicon, titanium nitride (TiN), and aluminum (Al). The layer used to form the baffle may have a cross-sectional height of about 20 nm, about 20 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, or about 90 nm.

[0403] In one exemplary embodiment, the baffle includes four layers: a silicon nitride layer having a cross-sectional thickness of 22.34 nm, an amorphous silicon layer having a cross-sectional thickness of 81.34 nm, a titanium nitride layer having a cross-sectional thickness of 30 nm, and an aluminum layer having a cross-sectional thickness of 50 nm. The absorption and reflection of the exemplary baffle design are shown in Figure 7-3 , which shows the absorbance and reflectance at different incident angles of 0, 20 and 35 degrees for p and s polarization. Figure 7-3As shown in , the baffle has varying levels of absorptivity for different angles of incidence to the baffle, with greater absorptivity at larger angles of incidence.

[0404] A filter component may be formed between the waveguide and the sensor to reduce the collection of excitation energy by the sensor. Any suitable means for filtering the excitation energy may be provided. Techniques for filtering the excitation energy may include filtering based on one or more characteristics of the light. Such characteristics may include wavelength and / or polarization. The filter component may selectively suppress scattered excitation energy while allowing light emission to pass through the sensor. Figure 4-2 The layer 4-228 shown in FIG. 4 may include one or more filtering components described herein.

[0405] Polarization filters for reflecting and / or absorbing light of a specific polarization can act as filters for integrated devices. Polarization filters can be used in systems where the excitation energy propagating through the waveguide has a certain polarization and the luminescence emitted by the mark can have a polarization different from the excitation energy. The polarization excitation filter can be configured to filter the polarization of the excitation energy and thereby reduce the collection of the excitation energy by the sensor. In some embodiments, the polarization filter can be configured to absorb TE polarized light when the excitation energy provided by the excitation is TE polarized. The polarization filter can be composed of a patterned wire mesh polarizer. In some embodiments, the wires in the wire mesh are aluminum. The orientation of the wires can be aligned parallel to a direction perpendicular to the propagation direction of one or more waveguides. When the excitation energy is TE polarized, the oscillating electric field of the TE polarized excitation energy is used to align the orientation of the wires. The manufacture of such a wire mesh polarizer can provide a suitable filter while negligibly causing the thickness of the overall integrated device. Such a wire mesh polarizer can have a loss of scattered excitation energy of approximately greater than 10 dB and a luminescence loss of approximately 3 dB. In some embodiments, the openings in the wire mesh filter in which the wires are absent can be aligned with one or more sensors below. The openings may have dimensions that are smaller or larger than the dimensions of one or more sensors.The openings may allow luminescence from a sample well of a pixel to be collected by the sensor in the pixel while the surrounding wire mesh pattern may provide filtering of the excitation energy.

[0406] Figure 7-4A A plan view of a polarizing excitation filter having a mesh including a plurality of lines 7-406 is shown. The mesh of lines has openings 7-408 forming a pixel region in a pixel array of an integrated device. A waveguide 7-404 may be positioned through a row of openings 7-408. Figure 7-4B A cross-sectional view along line A-A' is shown, as shown in FIG. Figure 7-4A. A line 7-406 is positioned between the waveguide 7-404 and the sensor 7-410. An opening 7-408 in the line 7-406 is positioned to at least partially overlap the sensor 7-410. In this way, the opening 7-408 and the sensor 7-410 can be included in the same pixel of the integrated device. The opening 7-408 can allow luminescent energy to pass so that the sensor 7-410 can detect luminescence while reducing the amount of excitation energy detected by the sensor 7-410.

[0407] The integrated device may include a wavelength filter that reflects light of one or more characteristic wavelengths and allows transmission of light having different characteristic wavelengths. In some embodiments, light reflected by the wavelength filter may have a characteristic wavelength shorter than light transmitted by the wavelength filter. Such a wavelength filter may reflect excitation energy and allow transmission of luminescence because the excitation energy used to excite the marker generally has a characteristic wavelength shorter than the luminescence emitted by the marker in response to reaching an excited state using the excitation energy.

[0408] The wavelength filter may include one or more layers of one or more materials. The wavelength filter may include titanium dioxide (TiO 2 ) and / or silicon dioxide (SiO 2 ). In some embodiments, a wavelength filter may include a stack of multiple layers having alternating layers of high refractive index material and low refractive index material. The cross-sectional thickness of each layer may be any suitable thickness, and in some cases may be used to reflect and / or transmit light of a particular wavelength. The cross-sectional thickness of each layer may be approximately 1 / 4 of the wavelength of light transmitted by the wavelength filter. The thickness of a layer of a wavelength filter may be approximately 10 nm, approximately 40 nm, approximately 50 nm, approximately 60 nm, approximately 70 nm, approximately 80 nm, approximately 90 nm, approximately 100 nm, approximately 110 nm, approximately 130 nm, or approximately 150 nm. In some embodiments, a layer of a wavelength filter has a variation of less than 1% in thickness throughout the layer, thereby providing suitable thickness uniformity within the wavelength filter.

[0409] In some embodiments, the different materials may include titanium dioxide (TiO 2 ) and silicon dioxide (SiO 2 ). Using titanium dioxide and silicon dioxide as alternating layers in a wavelength filter can reduce the overall thickness of the structure of the wavelength filter.

[0410] The stack of alternating layers may have any suitable thickness so as to provide a desired level of filtering of excitation energy while allowing transmission of luminescence. The wavelength filter may have a thickness of about 3 microns, about 3.5 microns, about 4 microns, or about 4.5 microns. The wavelength filter may be formed in multiple pixels of an integrated device.

[0411] Figure 7-5 A cross-sectional view of a wavelength filter 7-500 of an integrated device is shown. The wavelength filter includes alternating layers of layers 7-501 and 7-502. The layers 7-501 and 7-502 have different refractive indices. For example, the layer 7-501 may have a higher refractive index than the layer 7-502. The different refractive indices for the layers 7-501 and 7-502 may be achieved by using materials with different refractive indices. In some embodiments, the layer 7-501 may include silicon dioxide and the layer 7-502 may include titanium dioxide. The wavelength filter 7-500 is formed within a pixel region 7-508 having a sensor 7-510. The wavelength filter 7-500 may be configured to allow transmission of light having a characteristic wavelength λ2 while substantially reflecting a different characteristic wavelength λ1. The sign may emit cold light having a wavelength of λ2, which may pass through the excitation filter 7-500 and be detected by the sensor 7-510. The excitation energy may include λ1. In this way, the sensor 7-510 can detect a luminous signal roughly relative to the mark.

[0412] In one exemplary embodiment, the waveguide filter may include 49 layers of alternating TiO 2 and SiO 2 , where SiO 2 The layer is in TiO 2 and SiO 2 The total thickness of the entire stack is approximately 3.876 microns. Figure 7-6 The transmittance for p-polarization, average polarization, s-polarization, and transmittance for about 0 degrees of incidence are plotted as a function of wavelength for about 10 degrees of incidence. The extinction loss can vary significantly over a wavelength range of about 21 nm and is about 30 dB at about 646 nm for about 10 degrees of incidence. The thickness variation of such a filter can be less than about 1% to reduce artifacts due to thickness uniformity.

[0413] Changing the thickness of the layers within a multilayer stack of a wavelength filter can change its transmission properties. Changing the thickness of one or more layers in a wavelength filter can change the wavelength of light passed by the filter. Using this technique, portions of the filter element can pass light of one wavelength while other portions can pass light of a different wavelength, thereby forming a multi-wavelength filter. A portion of the multi-wavelength filter can overlap with a sensor for receiving light passed through the portion. The stack of alternating layers of the multi-wavelength filter can have two portions, wherein the thickness of the alternating layers is approximately 1 / 4 of the wavelength of the light, and the spacer has a half wavelength between the two 1 / 4 wavelength portions. The spacer can function as a Fabry-Perot resonator. The spacer can have a variable thickness such that it has a thickness in a first region that is greater than the thickness in a second region. Due to the variation in the thickness of the spacer, the wavelength of light passed by the wavelength filter can vary throughout the filter. The first region can allow transmission of light having a first wavelength, and the second region can allow transmission of light having a second wavelength, wherein the first wavelength is longer than the second wavelength. In addition, the first region of the filter may substantially reflect light having the second wavelength, and the second region of the filter may substantially reflect light having the first wavelength. In this way, the long filter may provide a change in the wavelength of light transmitted in the wavelength filter due to the change in the thickness of the spacer. In embodiments where more than one emission wavelength is detected, such a multi-wavelength filter may be used. The multi-wavelength filter may reflect excitation energy by greater than 10 dB.

[0414] In making such a wavelength filter, the spacer may be formed to a thickness for transmission of longer wavelength light and etched in a region where shorter wavelength light is transmitted. The high refractive index layer in the region where shorter wavelength light is transmitted may have a patterned etched area.

[0415] Figure 7-7 A cross-sectional view of a multi-wavelength filter 7-700 is shown. A pixel 7-707 includes a region 7-708 and a region 7-709 of the multi-wavelength filter 7-700 and sensors 7-710 and 7-711. The multi-wavelength filter 7-700 includes alternating layers of 7-701 and 7-702 and a spacer 7-706. The layers 7-701 and 7-702 have different refractive indices. For example, the layer 7-701 may have a higher refractive index than the layer 7-702. The different refractive indices of the layers 7-701 and 7-702 may be achieved by using materials having different refractive indices. In some embodiments, the layer 7-701 may include silicon dioxide and the layer 7-702 may include titanium dioxide. Figure 7-7As shown in , the spacer 7-706 has a variable thickness such that region 7-708 has a thickness that is less than region 7-709. By varying the thickness of the spacer 7-706, the wavelength of light transmitted by the filter 7-700 varies from region 7-708 to region 7-709. Based on the region, the thickness of the spacer 7-706 may be equal to approximately half the wavelength of the light being transmitted. The spacer 7-706 has a thickness in region 7-708 that is less than that in region 7-709 such that light transmitted through region 7-708 has a wavelength that is less than light transmitted through region 7-709. The variation in thickness of regions 7-708 and 7-709 may be approximately 5 nm, approximately 10 nm, approximately 15 nm, approximately 20 nm, or approximately 25 nm. As shown in FIG. Figure 7-7 As shown in FIG. 7.7 , region 7-708 allows transmission of light of λ1 and region 7-709 allows transmission of light of λ2, where λ2 is greater than λ1. Thus, a sensor 7-710 located in region 7-708 may detect light of λ1 and a sensor 7-711 located in region 7-709 may detect light of λ2. In addition, a multi-wavelength filter 7-706 may be configured to utilize regions 7-708 and 7-709 to reflect excitation energy. As shown in FIG. 7.7 , regions 7-708 and 7-709 both reflect light of λ3, which may have a wavelength longer than both λ1 and λ2.

[0416] Figure 7-8 shows a 13-layer alternating TiO 2 and SiO 2 Graph of the transmittance of polarized light as a function of wavelength for an exemplary wavelength filter having SiO on either side of the stack 2 layer and TiO with variable thickness 2 Spacer. TiO that constitutes the 1 / 4 wavelength layer 2 and SiO 2 The alternating layers of TiO have thicknesses of 76.0 nm and 118.51 nm, respectively. 2 The spacers have a thickness of 137.16 nm in one region and a thickness of approximately 122.16 nm in another region. The total thickness of the entire stack is 1.3 microns. The spacer layer has an etched region that is approximately 15 nm thinner for the bandpass transmission region of the filter than for the longer wavelength bandpass transmission region. The transmission region is centered at approximately 660 nm and 685 nm. Other cavity spacers can broaden the transmission peak into a square wave pulse generator. The extinction loss can be approximately in the range of 10 dB to 15 dB. The thickness variation of such a filter can be less than approximately 1%, thereby reducing artifacts caused by thickness uniformity.

[0417] F. Sensor

[0418] Any suitable sensor capable of acquiring time bin information may be used for the measurement in order to detect the lifetime of the luminescent marker. For example, U.S. Provisional Patent Application 62 / 164,506, entitled "Integrated Device for Time Division of Receiving Photons," filed on May 20, 2015, which is incorporated herein by reference in its entirety, describes a sensor capable of determining the time of arrival of photons. The sensor is aligned so that each sample well has at least one sensor region for detecting luminescence from the sample well. In some embodiments, the integrated device may include a Geiger mode avalanche photodiode array, and / or a single photon avalanche diode array (SPAD).

[0419] Described herein is an integrated photodetector that can accurately measure or "time-slice" the arrival timing of incident photons and can be used for a variety of applications, such as nucleic acid sequencing (e.g., DNA sequencing). In some embodiments, the integrated photodetector can measure the arrival of photons with nanosecond or picosecond resolution, which can simplify the time-domain analysis of the arrival of incident photons.

[0420] Some embodiments relate to an integrated circuit having a photodetector that generates carriers in response to incident photons and is capable of distinguishing the timing of the generation of carriers with the arrival of incident photons relative to a reference time (e.g., a triggering event). In some embodiments, a carrier segregation structure separates carriers generated at different times and directs the carriers into one or more carrier storage areas (referred to as "bins") that gather carriers generated in different time periods. Each bin stores carriers generated in a selected time interval. Reading out the charge stored in each bin can provide information about the number of photons that arrived in each time interval. Such an integrated circuit can be used in any of a variety of applications, such as those described herein.

[0421] An example of an integrated circuit having a photodetection region and a carrier segregation structure is described below. In some embodiments, the integrated circuit may include an array of pixels, and each pixel may include one or more photodetection regions and one or more carrier segregation structures, as described below.

[0422] Figure 7-9A A diagram of a pixel 7-900 according to some embodiments is shown. The pixel 7-900 includes: a photon absorption / carrier generation region 7-902 (also referred to as a photodetection region), a carrier traveling / trapping region 7-906, a carrier storage region 7-908 having one or more carrier storage regions, also referred to herein as a "carrier storage bin" or simply a "bin", and a readout circuit 7-910 for reading out a signal from the carrier storage bin.

[0423] The photon absorption / carrier generation region 7-902 may be a region of semiconductor material (e.g., silicon) that may convert incident photons into photogenerated carriers. The photon absorption / carrier generation region 7-902 may be exposed to light and may receive incident photons. When photons are absorbed by the photon absorption / carrier generation region 7-902, the photons may generate photogenerated carriers, such as electron-hole pairs. Photogenerated carriers are also referred to herein as simply "carriers."

[0424] An electric field may be established in the photon absorption / carrier generation region 7-902. In some embodiments, the electric field may be "static," as distinguished by a changing electric field in the carrier traveling / trapping region 7-906. The electric field in the photon absorption / carrier generation region 7-902 may include a lateral component, a vertical component, or both a lateral component and a vertical component. The lateral component of the electric field may be in the Figure 7-9A This lateral separation results in a force acting on the photogenerated carriers in a downward direction, as represented by the arrows, which drives the photogenerated carriers toward the carrier traveling / trapping region 106. The electric field can be generated in a variety of ways.

[0425] In some embodiments, one or more electrodes may be formed above the photon absorption / carrier generation region 7-902. The electrodes may have a voltage applied thereto to establish an electric field in the photon absorption / carrier generation region 7-902. Such electrodes may be referred to as "photogates." In some embodiments, the photon absorption / carrier generation region 7-902 may be a region of silicon that is completely carrier-poor.

[0426] In some embodiments, a junction (e.g., a PN junction) may be used to establish an electric field in the photon absorption / carrier generation region 7-902. The semiconductor material of the photon absorption / carrier generation region 7-902 may be doped to form a PN junction having an orientation and / or shape that generates an electric field, which results in a force acting on the photogenerated carriers that drives the photogenerated carriers toward the carrier travel / capture region 7-906. In some embodiments, the diode P terminal of the PN junction may be connected to a terminal that sets its voltage. Such a diode may be referred to as a "pinned" photodiode. Due to the terminal that sets its voltage and attracts carriers, the pinned photodiode may promote carrier recombination at the surface, thereby reducing dark current. It is expected that the captured photogenerated carriers may pass under the recombination zone at the surface. In some embodiments, a gradient doping concentration in the semiconductor material may be used to establish a lateral electric field.

[0427] like Figure 7-9AAs shown in FIG. 1 , a photon may be captured and a carrier 7-901A (e.g., an electron) may be generated at time t1. In some embodiments, a potential gradient may be established along the photon absorption / carrier generation region 7-902 and the carrier travel / capture region 7-906, which causes the carrier 7-901A to be generated at time t1. Figure 7-9A Moving in the downward direction (such as Figure 7-9A 7-901A). In response to the potential gradient, the carrier 7-901A may move from its position at time t1 to a second position at time t2, a third position at time t3, a fourth position at time t4, and a fifth position at time t5. Thus, in response to the potential gradient, the carrier 7-901A moves into the carrier traveling / trapping region 7-906.

[0428] The carrier traveling / trapping region 7-906 may be a semiconductor region. In some embodiments, the carrier traveling / trapping region 7-906 may be a semiconductor region of the same material (e.g., silicon) as the photon absorption / carrier generation region 7-902, except that the carrier traveling / trapping region 7-906 may be shielded from incident light (e.g., with an overlying opaque material, such as a metal layer).

[0429] In some embodiments and as further described below, a potential gradient may be established in the photon absorption / carrier generation region 7-902 and the carrier traveling / trapping region 7-906 using electrodes located above these regions. However, the techniques described herein are not limited to the specific locations of the electrodes used to generate the potential gradient. The techniques described herein are also not limited to using electrodes to establish the potential gradient. In some embodiments, the potential gradient may be established using a spatially graded doping profile. Any suitable technique may be used to establish a potential gradient that causes carriers to travel along the photon absorption / carrier generation region 7-902 and the carrier traveling / trapping region 7-906.

[0430] A carrier segregation structure may be formed in the pixel so that carriers generated at different times can be separated. In some embodiments, at least a portion of the carrier segregation structure may be formed above the carrier traveling / trapping region 7-906. As will be described below, the carrier segregation structure may include: one or more electrodes formed above the carrier traveling / trapping region 7-906, the voltage of which may be controlled by a control circuit to change the potential in the carrier traveling / trapping region 7-906.

[0431] The potential in the carrier traveling / trapping region 7-906 may be changed to enable trapping of carriers. The potential gradient may be changed by changing the voltage on one or more electrodes covering the carrier traveling / trapping region 7-906 to create a potential barrier that can confine the carriers within a predetermined spatial region. For example, the covering region 7-906 may be changed at time t5. Figure 7-9A The voltage on the dotted electrode of the carrier traveling / trapping region 7-906 increases along Figure 7-9A The dashed potential barrier in the carrier traveling / trapping region 7-906 thereby trapping the carrier 7-901A. Figure 7-9A As shown in FIG. 1 , the carriers captured at time t5 are transferred to bin “bin 0” of the carrier storage region 7-908. The transfer of carriers to the carrier storage bins may be performed by changing the potential in the carrier traveling / trapping region 7-906 and / or the carrier storage region 7-908 (e.g., by changing the voltage of electrodes covering these regions) thereby causing the carriers to enter the carrier storage bins.

[0432] Changing the potential within a predetermined spatial region of the carrier traveling / capture zone 7-906 at a certain point in time may enable the capture of carriers generated by absorption of photons occurring within a specific time interval. By capturing photogenerated carriers at different times and / or locations, the time at which carriers are generated by absorption of photons may be distinguishable. In this sense, carriers may be "time partitioned" by capturing carriers at a certain point in time and / or spatial point after a triggering event occurs. The time partitioning of carriers within a specific bin provides information about the time at which photogenerated carriers are generated by absorption of incident photons, and thus likewise about the arrival of incident photons that generate photogenerated carriers relative to the "time bin" of the triggering event.

[0433] Figure 7-9B The captured carriers are shown at different time points and spatial points. Figure 7-9B As shown in FIG. 1 , the voltage on the electrode covering the dashed line in the carrier traveling / trapping region 7-906 can be changed at time t9 to increase the Figure 7-9B The dashed potential barrier in the carrier travel / capture region 106 thereby captures the carrier 7-901B. Figure 7-9B , the carrier captured at time t9 may be transferred to bin "bin 1" of carrier storage region 7-908. Because carrier 7-901B was captured at time t9, it represents a photon absorption event that occurred at a different time (i.e., time t6) than the photon absorption event of carrier 7-901A that was captured at time t5 (i.e., at t1).

[0434] Performing multiple measurements based on the time at which a carrier is captured and separating the carriers in the carrier storage bins of the carrier storage region 7-908 can provide information on the time at which a photon is captured in the photon absorption / carrier generation region 7-902. This information can be used for a variety of purposes as described above.

[0435] In some embodiments, the time period captured by each time element after the excitation pulse can be varied. For example, a shorter time element can be used to detect luminescence immediately after the excitation pulse, while a longer time element can be used at a time further away from the excitation pulse. By changing the time source interval, the signal-to-noise ratio of the electrical signal measurement associated with each time element can be improved for a given sensor. Because the probability of a photon emission event is high shortly after the excitation pulse, the time element can have a shorter time interval during this time, thereby resulting in the possibility of more photons being detected. At the same time, at a longer time, the probability of photon emission will be lower and the time element detected during this time can be longer, thereby resulting in the possibility of a smaller number of photons. In some embodiments, a time element with a significantly longer time period can be used to distinguish between multiple lifespans. For example, most of the time elements can capture a time interval within the range of about 0.1-0.5ns, while the time element can capture a time interval within the range of about 2-5ns. The number of time elements and / or the time interval of each element can depend on the sensor used to detect the photons emitted from the sample object. Determining the time interval of each element can include identifying the time interval required for the number of time elements provided by the sensor, so as to distinguish between each luminescent mark used for sample analysis. The distribution of the recorded histogram can be compared to known histograms of markers under similar conditions and time bins to identify the type of marker in the sample well. Various embodiments of the present application can measure the lifetime of each marker, but with variations in the excitation energy used to excite the marker, the number of sensor areas in each pixel, and / or the wavelength detected by the sensor.

[0436] III. Excitation Source

[0437] According to some embodiments, one or more excitation sources may be located outside the integrated device and may be arranged to deliver pulses of light to the integrated device having a sample well. For example, U.S. Provisional Patent Application No. 62 / 164,485, entitled "Pulsed Laser," filed on May 20, 2015, describes a pulsed laser source that may be used as an excitation source, the entire contents of which are incorporated herein by reference. For example, pulses of light may be coupled to a plurality of sample wells and used to excite one or more markers inside the wells. According to some embodiments, one or more excitation sources may deliver pulses of light at one or more characteristic wavelengths. In some cases, the excitation source may be packaged as an exchangeable module installed in or coupled to a base instrument, and the integrated device may be loaded into the exchangeable module. Energy from the excitation source may be delivered radiatively or non-radiatively to at least one sample well or at least one sample in at least one sample well. In some embodiments, an excitation source having a controllable intensity may be arranged to deliver excitation energy to a plurality of pixels of the integrated device. The pixels may be arranged in a linear array (eg, rows or columns), or in a 2D array (eg, a sub-region of a pixel array or the entire pixel array).

[0438] Any suitable light source may be used for the excitation source. Some embodiments may use an incoherent light source and other embodiments may use a coherent light source. By way of non-limiting example, different types of light emitting diodes (LEDs) may be included in the coherent light source according to some embodiments, such as organic LEDs (OLEDs), quantum dots (QLEDs), nano-LEDs, and (inorganic) organic semiconductor LEDs. By way of non-limiting example, coherent light sources according to some embodiments may include different types of lasers, such as semiconductor lasers (e.g., vertical cavity surface emitting lasers (VCSELs), edge emitting lasers, and distributed feedback (DFB) laser diodes). In addition or alternatively, a slab-coupled optical waveguide laser (SCOWL) or other asymmetric single-mode waveguide structures may be used. In some embodiments, the coherent light source may include an organic laser, a quantum dot laser, and a solid-state laser (e.g., a Nd:YAG or ND:glass laser, pumped by a laser diode or a flash lamp). In some embodiments, a laser diode-pumped fiber laser may be used. The coherent light source may be passively mode-locked to produce ultrashort pulses. For an array of pixels on an integrated device, there may be more than one type of excitation source. In some embodiments, different types of excitation sources may be combined. The excitation sources may be manufactured according to conventional techniques for manufacturing the selected type of excitation source.

[0439] By way of illustration and not limitation of the present invention, an exemplary arrangement of coherent light sources is shown in Figure 8-0A. The figure shows an analytical instrument 8-100, which may include an ultrashort pulse laser excitation source 8-110 as an excitation source. The ultrashort pulse laser 8-110 may include: a gain medium 8-105 (which may be a solid material in some embodiments), a pumping source for exciting the gain medium (not shown), and at least two cavity mirrors 8-102, 8-104 that define the ends of an optical laser cavity. In some embodiments, one or more other optical elements may be present in the laser cavity for the purposes of beam shaping, wavelength selection, and / or pulse forming. When operating, the pulsed laser excitation source 8-110 may generate ultrashort optical pulses 8-120 that circulate back and forth in the laser cavity between the end mirrors 8-102, 8-104 of the cavity and pass through the gain medium 8-105. One of the cavity mirrors 8-104 may partially pass a portion of the circulating pulses so that a train of optical pulses 8-122 is emitted from the pulsed laser 8-110 and reaches subsequent components 8-160, such as optical components and integrated devices. The emitted pulses may clear the beam (indicated by the dashed line) as identified by the beam waist w.

[0440] The measured time intensity waveform of the emitted pulse 8-122 may be as follows Figure 8-0B In some embodiments, the peak intensity values ​​of the emitted pulses may be approximately the same, and each waveform may have a Gaussian time waveform, although other waveforms (e.g., a hyperbolic secant waveform) are also possible. In some cases, the pulses may not have a symmetrical time waveform and may have other temporal shapes. In some embodiments, gain and / or loss dynamics may result in pulses with asymmetric waveforms. The duration of each pulse may be identified by a full width half maximum (FWHM) value, such as Figure 8-0B As shown in . Ultrashort optical pulses can have a FWHM value of less than 100 picoseconds.

[0441] The pulses emitted from the laser excitation source may be spaced at regular intervals T. In some embodiments, T may be determined by the active gain and / or loss modulation rate in the laser. For a mode-locked laser, T may be determined by the round trip time between the cavity end mirrors 8-102, 8-104. According to some embodiments, the pulse interval time T may be between about 1 ns and about 100 ns. In some cases, the pulse interval time T may be between about 0.1 ns and about 1 ns. In some embodiments, the pulse interval time T may be between about 100 ns and about 2 ns.

[0442] In some embodiments, the optical system 8-140 may operate on a beam of pulses 8-122 from a laser excitation source 8-110. For example, the optical system may include one or more lenses for shaping the beam and / or changing the divergence of the beam. Shaping of the beam may include increasing or decreasing the value of the beam waist and / or changing the cross-sectional shape of the beam (e.g., from elliptical to circular, from circular to elliptical, etc.). Changing the divergence of the beam may include converging or diverging the beam. In some embodiments, the optical system 8-140 may include an attenuator or amplifier for changing the amount of energy of the beam. In some cases, the optical system may include a wavelength filtering element. In some embodiments, the optical system may include a pulse shaping element, such as a pulse stretcher and / or a pulse compressor. In some embodiments, the optical system may include one or more nonlinear optical elements, such as a saturable absorber for reducing pulse length. According to some embodiments, the optical system 8-140 may include one or more elements that change the polarization of pulses from the laser excitation source 8-110.

[0443] In some embodiments, the optical system 8-140 may include a nonlinear crystal for utilizing frequency doubling or converting the output wavelength from the excitation source 8-110 to a shorter wavelength, or a longer wavelength utilizing parametric amplification. For example, the output of a laser may be frequency doubled in a nonlinear crystal (e.g., in periodically poled lithium niobate (PPLN)) or other non-polarized nonlinear crystal. Such a frequency doubling process may allow a more efficient laser to produce a wavelength more suitable for excitation of a selected fluorophore.

[0444] The phrase "characteristic wavelength" or "wavelength" may refer to a central wavelength or dominant wavelength within a limited bandwidth of radiation generated by an excitation source. In some cases, it may refer to a peak wavelength within the bandwidth of radiation generated by an excitation source. For example, the characteristic wavelength of the excitation source may be selected based on the selection of luminescent markers or probes used in a bioanalysis device. In some embodiments, the characteristic wavelength of the excitation energy source is selected for direct excitation (e.g., single photon excitation) of a selected fluorophore. In some embodiments, the characteristic wavelength of the excitation source is selected for indirect excitation (e.g., multiphoton excitation or harmonic conversion to a wavelength that will provide direct excitation). In some embodiments, the excitation radiation may be generated by a light source configured to generate excitation energy at a specific wavelength for application to a sample well. In some embodiments, the characteristic wavelength of the excitation source may be less than the characteristic wavelength of the corresponding emission from the sample. For example, the excitation source may emit radiation having a characteristic wavelength between 500 nm and 700 nm (e.g., 515 nm, 532 nm, 563 nm, 594 nm, 612 nm, 632 nm, 647 nm). In some embodiments, for example, the excitation source can provide excitation energy centered at two different wavelengths, such as 532 nm and 593 nm.

[0445] In some embodiments, a pulsed excitation source may be used to excite a luminescent marker to measure the emission lifetime of the luminescent marker. This may be used to distinguish luminescent markers based on emission lifetime rather than emission color or wavelength. As an example, a pulsed excitation source may periodically excite a luminescent marker, thereby generating and detecting subsequent photon emission events, which are used to determine the lifetime of the marker. When an excitation pulse from an excitation source changes from a peak pulse power or intensity to a lower (e.g., almost extinguished) power or intensity in a time period less than the lifetime of the luminescent marker, lifetime measurement of the luminescent marker may be feasible. It is advantageous that the excitation pulse terminates quickly so that it does not excite the luminescent marker again in the post-excitation stage when the lifetime of the light marker is evaluated. For example, but not limited to, after 250 picoseconds, the pulse power may drop to about 20dB, about 40dB, about 80dB, or about 120dB less than the peak power. In some embodiments, after 100 picoseconds, the pulse power may drop to about 20dB, about 40dB, about 80dB, or about 120dB less than the peak power.

[0446] Another advantage of using ultrashort excitation pulses to excite luminescent markings is reduced photofading of the markings. Applying continuous excitation energy to the markings can fade and / or damage the luminescent markings over time. Even though the peak pulse power of the excitation source can be significantly higher than a level that would quickly damage the markings in continuous exposure, the use of ultrashort pulses can increase the amount of time and number of useful measurements before the markings become damaged by the excitation energy.

[0447] When a pulsed excitation source is used to distinguish the lifetime of a luminescent marker, the time between pulses of excitation energy can be as long as or longer than the longest lifetime of the marker in order to observe and evaluate emission events following each excitation pulse. Figure 8-0B ) may be longer than any emission lifetime of the fluorophores being examined. In this case, a subsequent pulse may not arrive before the excited fluorophores from the previous pulse have had a reasonable amount of time to fluoresce. In some embodiments, the interval T needs to be long enough to determine the time between an excitation pulse that excites a fluorophore and the subsequent emission of a photon by the fluorophore after termination of the excitation pulse and before the next excitation pulse.

[0448] Although the interval T between each excitation pulse should be long enough to observe the decay properties of the fluorophore, it is also desirable that T is short enough to allow multiple measurements to be made in a short period of time. For example, but not limited to, the emission lifetime of the fluorophores used in some applications may be in the range of about 100 picoseconds to about 10 nanoseconds. Therefore, the excitation pulses used to detect and / or distinguish such lifetimes may have a duration (FWHM) in the range of about 25 picoseconds to about 2 nanoseconds, and may be provided at a pulse repetition frequency in the range of about 20 MHz to about 1 GHz.

[0449] In more detail, any suitable technique for modulating the excitation energy to form a pulsed excitation source for lifetime measurement may be used. Direct modulation of an excitation source (e.g., a laser) may include modulating an electrical drive signal of the excitation source so that the emitted power is in the form of pulses. The input power of the light source (including optical pump power) and the injection of excited state carriers and / or the removal of carriers from a portion of the gain region may be modulated to achieve the gain of the gain medium, thereby allowing the formation of excitation energy pulses through dynamic gain shaping. In addition, the quality factor (Q) of the optical resonant cavity may be modulated in various ways to form pulses using Q-switching techniques. Such Q-switching techniques may be active and / or passive. The longitudinal mode of the laser resonant cavity may be phase-locked, thereby generating a series of pulses of emitted light through mode locking. Such mode locking techniques may be active and / or passive. The laser cavity may include a separate absorption portion to allow modulation of the carrier density and control of the absorption loss of the portion, thereby providing other mechanisms for shaping the excitation pulses. In some embodiments, an optical modulator can be used to modulate a beam of continuous wave (CW) light so as to adopt a pulsed form of excitation energy. In other embodiments, a signal sent to an acousto-optic modulator (AOM) connected to an excitation source can be used to change the deflection, intensity, frequency, phase, and / or polarization of the output light to produce pulsed excitation energy. The AOM can also be used for continuous wave beam scanning, Q-switching, and / or mode locking. Although the above-mentioned techniques are used to form a pulsed excitation source, any suitable method of generating a pulsed excitation source can be used to measure the lifetime of a luminescent marker.

[0450] In some embodiments, a technique for forming a pulsed excitation source suitable for lifetime measurement may include modulation of an input electrical signal that drives photon emission. Some excitation sources (e.g., diode lasers and LEDs) convert electrical signals (e.g., input current) into optical signals. The characteristics of the optical signal may depend on the characteristics of the electrical signal. In generating a pulsed optical signal, the electrical signal may vary over time to generate a variable optical signal. Modulating the electrical signal to have a specific waveform may generate an optical signal with a specific waveform. The electrical signal may have a sinusoidal waveform with a certain frequency and the formed optical pulses may appear in a time interval associated with the frequency. For example, an electrical signal with a frequency of 500 MHz may generate a pulsed optical signal every 2 nanoseconds. The combined light beams generated by different pulsed excitation sources, whether similar or different from each other, may have a relative path difference of less than 1 mm.

[0451] In some excitation sources (such as laser diodes), an electrical signal changes the carrier density and generates photons by recombination of electron and hole pairs. The carrier density is related to the optical signal so that a large number of coherent photons are generated using simulated emission when the carrier density exceeds a threshold. The current provided to the laser diode can inject electrons or carriers into the device, thereby increasing the carrier density. When the carrier density exceeds the threshold, photons can be generated at a faster rate than the current providing the carriers, so the carrier density can be reduced to below the threshold and the photon generation is reduced. As the photon generation decreases, the carrier density begins to increase again due to the continued current introduction and absorption of photons, and finally increases again to exceed the threshold. This cycle causes the carrier density to oscillate around the threshold for photon generation, resulting in oscillations of the optical signal. Due to the oscillations of the carrier density, these dynamics (called relaxation oscillations) can cause artifacts in the optical signal. When current is initially provided to the laser, there may be oscillations due to oscillations in the carrier density before the optical signal reaches a stable power. When a pulsed excitation source is formed, oscillations in the carrier density can introduce artifacts into the pulsed optical signal. For example, Figure 8-1 The diagram in shows how the carrier density can be modulated by the gain switch to have relaxation oscillations and a corresponding optical signal with oscillating power. Artifacts from such relaxation oscillations can broaden the pulsed optical signal and / or produce tails in the optical signal, thereby limiting the lifetime that can be detected by such a pulsed light source, since the excitation signal can overlap with photons emitted by the luminescent marker.

[0452] In some embodiments, techniques for shortening the time period of an excitation pulse can be used to reduce the excitation energy required to detect a luminescent mark and thereby reduce or delay fading and other damage to the luminescent mark. Techniques for shortening the time period of an excitation pulse can be used to reduce the power and / or intensity of the excitation energy after the maximum or peak value of the excitation pulse, thereby allowing for shorter lifetime detection. Such techniques can electrically drive the excitation source to reduce the excitation power after the peak power. This can suppress pulse tailing, such as Figure 8-2 The electrical drive signal can be trimmed so that the pulse intensity of the excitation energy is driven to zero as quickly as possible after the peak pulse. An example of a combination of a trimmed electrical drive signal and a gain switch is shown in Figure 8-2 This technique may include inverting the sign of the electrical drive signal after the peak power is generated. This trimmed electrical drive signal may produce Figure 8-2 The electrical signal can be shaped to reduce the carrier density quickly after the first relaxation oscillation or first oscillation of the optical signal. By reducing the carrier density after the first oscillation, a light pulse of only the first oscillation can be generated. The electrical signal can be used to generate a short pulse that quickly turns off the optical signal by reducing the number of photons emitted after a peak in the signal, such as the optical output of such an electrical signal. Figure 8-3 According to some embodiments, a picosecond laser diode system can be designed to emit light pulses. Figure 8-4 A graph is shown of an exemplary light pulse having a peak of 985 mW, a width of 84.3 picoseconds, and a signal that decreases by about 24.3 dB at about 250 picoseconds after the peak. In some embodiments, saturable absorbers, including semiconductor saturable absorbers (SESAMs), can be used to suppress optical tailing. In such embodiments, the use of saturable absorbers can suppress optical tailing by 3-5 dB, or in some cases greater than 5 dB. Reducing the effects of tailing in the excitation pulse can reduce and / or eliminate the need for further filtering of the excitation energy, extend the range of lifetimes that can be measured, and / or obtain faster pulse frequencies. Increasing the excitation pulse frequency can make it possible to conduct more experiments in a given time, thereby reducing the time required to obtain sufficient statistical data to identify the lifetime of a marker that marks a sample object.

[0453] Furthermore, two or more of these techniques may be used together to generate pulsed excitation energy. For example, optical modulation techniques may be used to further adjust the pulsed excitation energy emitted from a directly modulated source. Techniques for modulating the excitation pulse and adjusting the electrical pulse drive signal may be combined in any suitable manner to maximize the pulsed excitation energy used to perform lifetime measurements. A trimmed electrical drive signal may be provided to the pulsed excitation energy from the directly modulated source.

[0454] In some embodiments, a laser diode with a certain number of wire bonds can be used as a pulsed excitation source. A laser diode with more wire bonds can reduce the inductance of the excitation source. A laser diode with lower inductance can enable the current entering the laser to operate at a higher frequency. As shown in Figure 8-5, when driven by an 18V pulse in a 50 ohm transmission line, a laser source with a 3 ohm series resistor and 36 wire bonds has a higher current at a higher frequency than a laser source with fewer wire bonds. Selecting a packaging method to minimize the inductance can increase the power provided to the excitation source at higher frequencies, thereby enabling shorter excitation pulses, faster optical power reduction after the peak, and / or an increased pulse repetition frequency for detecting light-emitting signs.

[0455] In some embodiments, a transmission line in combination with an excitation source can be used to generate light pulses. The transmission line can match the impedance of the laser diode to improve the performance and / or quality factor of the light pulses. In some embodiments, the transmission line impedance can be 50 ohms. In some cases, the terminal resistance can be similar to the resistance of the line to avoid reflections. Alternatively or in addition, the terminal impedance can be similar to the resistance of the line to avoid reflections. The terminal resistance can be less than the impedance of the line in order to reflect negative pulses. In other embodiments, the diagnostic impedance can have a capacitive or inductive component to control the shape of the negative reflected pulse. In other embodiments, the transmission line can allow higher frequency pulses. Figure 8-6A An exemplary prototype of a transmission line pulsar is shown in Figure 8-6B An exemplary optical pulse obtained using such a transmission line is shown. The transmission line can be used to generate an electrical pulse having a frequency in the range of 40 MHz to 500 MHz. The transmission line is used for the above-mentioned trimming of the electrical signal, thereby generating a pulsed light source having an optical pulse having a certain time period and a specific time interval.

[0456] Techniques for adjusting electrical signals to improve generation of optical pulses may include connecting the excitation source to a circuit with negative offset capability. In some implementations, a negative offset may be provided on the excitation source after the optical pulse is emitted to reduce emission of tails in the optical pulse. Figure 8-7An exemplary circuit comprising a power supply, a diode laser, a resistor, a capacitor, and a switch that can be used to reduce the presence of tails in light pulses is shown. Such a circuit can form a constant current that bypasses the diode laser when the switch is closed or in a conductive state. When the switch is open, the switch can have a high resistance and current can flow through the diode laser. Light pulses can be generated by opening and closing the switch to provide intermittent current to the diode laser. In some cases, the resistance can be sufficiently high and the capacitor sufficiently small so that there is a voltage across the capacitor when the switch is open and the diode laser emits light. When the switch is closed, the voltage across the capacitor will reverse bias the diode laser. This reverse bias can reduce or eliminate the presence of tails in the light pulse. In this case, the switch can be configured to close after the peak of the light pulse to reduce the laser power immediately after the peak light pulse. The value of the resistor in the circuit can be selected so that the charge on the capacitor will discharge before the switch is subsequently opened and / or a light pulse is subsequently generated by the laser diode.

[0457] Other circuit components may be provided to adjust the electrical signal of the laser diode to generate the light pulse. In some embodiments, multiple capacitors, resistors, and voltages may be connected in a network circuit to control the waveform of the electrical signal provided to the laser diode. When there are N capacitor subcircuits, a controlled waveform may be formed by switching the number of voltages V1, V2, ..., VN with corresponding signals S1, S2, ..., SN. An exemplary network circuit of four capacitor subcircuits is shown in Figure 8-8 , wherein the controlled electrical waveform can be formed by switching voltages V1, V2, V3, and V4 having signals S1, S2, S3, and S4, respectively. In some embodiments, voltages V1, V2, V3, and V4 can be variable. Figure 8-8 In the example shown in , V4 for the laser is negative and reverse biased based on signal S4. The timing of the frequency of the light pulses emitted by the laser, the duration of each light pulse, and the characteristics of each light pulse can be adjusted using signal inputs S1, S2, S3, and S4. In some embodiments, additional resistors can be added to reduce the peak current. In this case, resistors can be added after one or more switches S1, S2, S3, S4. Although Figure 8-8 One configuration is shown with four capacitors and four voltages, but any suitable configuration and any suitable number of other circuit components may be provided for generating the trimmed electrical signals to the laser diodes to generate the optical pulses for lifetime measurement.

[0458] In some embodiments, the signal used to generate the optical pulses may use a circuit having discrete components, including radio frequency (RF) and / or microwave components. Discrete components that may be included in such a circuit are DC blockers, adapters, logic gates, terminal couplers, phase shifters, delays, attenuators, combiners, and / or RF amplifiers. Such components may be used to form a positive electrical signal having a certain amplitude and then generate a negative electrical signal having another amplitude. There may be a delay between the positive electrical signal and the negative electrical signal. Figure 8-9A An exemplary circuit having an RF amplifier is shown, which can be used to generate a conditioned electrical signal as an output pulse, such as in Figure 8-9B The electrical signal can be provided to an excitation source (e.g., a laser diode) to generate a light pulse. In other embodiments, the circuit can generate multiple electrical signals, which are combined to form an electrical pulse signal for driving the excitation source. Such a circuit can generate a differential output, which can be used to increase the power of the light pulse. By adjusting the discrete components of the circuit, the electrical output signal can be adjusted to generate a light pulse suitable for lifetime measurement. Figure 8-10A In the example shown in FIG. 1 , two RF amplifiers are used to generate outputs having Figure 8-10B The pulse signal of the waveform shown in , the pulse signal is composed of a positive electric signal pulse and a corresponding negative electric signal pulse, wherein the positive and negative electric signal pulses overlap and have similar widths.

[0459] In some embodiments, the excitation sources may be combined to produce light pulses for lifetime measurement. Synchronous pulsed excitation sources may be coupled to a circuit or load at a distance. In some embodiments, the excitation sources may be connected to the circuit in parallel. The excitation sources may be from the same source or from multiple sources. In some embodiments using multiple excitation sources, the types of the multiple excitation sources may vary. When combining the excitation sources, it is important to consider the impedance of the circuit and the excitation sources so as to have sufficient power provided to the excitation sources. The combination of the excitation sources may be achieved by employing one or more of the above-mentioned techniques for making pulsed excitation sources. Figure 8-11A Schematic diagrams are shown for combinations of four different excitation sources having one or more impedance values. Figure 8-11B Graphs of current, power efficiency and voltage as a function of impedance are shown.This exemplary embodiment shows 4 excitation sources delivering power to a 50 ohm transmission line and achieving optimal power delivery when the impedance of the load is equal to the ratio of the impedance of each individual line to the number of excitation sources.

[0460] The excitation source may include a battery or any other power source for providing power to the excitation source. For example, the excitation source may be located in the base instrument, and its operating power may be received through an integrated bioanalytical device coupled to the excitation source (e.g., via conductive leads). The excitation source may be controlled independently of or in conjunction with the integrated bioanalytical device. As just one example, control signals for the excitation source may be provided to the excitation source wirelessly or via a wired interconnection (e.g., a USB interconnection) with a personal computer and / or the integrated bioanalytical device.

[0461] In some embodiments, the excitation source for one or more sensors of the integrated device can be operated in a time-gated and / or synchronized manner. For example, the excitation source can be turned on to excite the luminescent mark and then turned off. The sensor can be turned off while the excitation source is turned on, and then can be turned on for a sampling interval after the excitation source is turned off. In some embodiments, the sensor can be turned on for a sampling interval while the excitation source is turned on.

[0462] IV. Alignment of the Excitation Source and Integrated Device

[0463] The positioning of excitation energy from one or more excitation sources to the grating coupler on the integrated device can be performed using any suitable technique. In some embodiments, the excitation energy can be directed from the excitation source to the grating coupler through one or more optical components. In this embodiment, the excitation energy can be projected onto the integrated device. This alignment of the excitation source can be actively performed by positioning the excitation source and / or the optical components. In other embodiments, the excitation source can be aligned with the grating coupler using components that position the excitation source relative to the grating coupler. This positioning of the excitation energy source can be performed through passive alignment of the optical fiber, thereby providing excitation energy inside the ferrule. In this embodiment, the excitation source can be directly or indirectly connected to the integrated device.

[0464] A. Active Alignment

[0465] Prior to delivering excitation energy to the sample well and / or at multiple times when the integrated device is used for analysis, alignment of one or more excitation sources with the integrated device is performed once. Any suitable technique for alignment and / or stabilization of one or more excitation sources may be used to couple excitation energy to the integrated device. In some embodiments, the external excitation source may be aligned to the integrated device and then fixed in position without further adjustment for a duration while the integrated device is used for analysis. In other embodiments, a feedback mechanism is provided to improve the alignment of the excitation source with the integrated device. The feedback mechanism may be used by an operator to align the excitation source based on a feedback signal using a manual alignment mechanism. In other cases, an automatic alignment mechanism may automatically adjust the alignment of the excitation source with the integrated device based on the feedback signal. Automatic alignment may be performed before operation of the integrated device is performed and / or before measurements are collected. Automatic alignment may also be performed during operation of the integrated device while measurements are being acquired. In some cases, the time at which automatic alignment occurs may correspond to the time at which the sensor is not actively collecting measurements. Re-adjusting the alignment of the excitation source with the integrated device may improve the stability of the excitation source and / or the consistency of sensor measurements over time in one or more samples.

[0466] The alignment can be actively controlled based on the feedback signal in multiple dimensions. Multiple dimensions can include the xy lateral position of the excitation energy beam to the grating coupler, the incident angle of the beam along the direction of the grating coupler, the z direction of the focusing of the beam, and / or the orthogonal incident angle of the beam perpendicular to the direction of the grating coupler. The excitation source can be connected to the integrated device via a frame....

Claims

1. An integrated device comprising: a sample well disposed on the substrate and configured to receive a sample; a waveguide configured to propagate a plurality of pulses of optical excitation energy to the sample well; as well as at least one sensor positioned to receive light from the sample well, the at least one sensor configured to obtain lifetime measurements and spectral measurements of received photons of luminescence emitted by the sample, wherein the at least one sensor comprises a first photosensitive region and a second photosensitive region, the first photosensitive region and the second photosensitive region are configured to detect a first wavelength range and a second wavelength range, respectively, and Wherein, the at least one sensor is configured to gather carriers generated by photons incident on the first photosensitive region into a first plurality of time bins, and to gather carriers generated by photons incident on the second photosensitive region into a second plurality of time bins.

2. The integrated device of claim 1, wherein the first wavelength range includes a red region of the electromagnetic spectrum and the second wavelength range includes a green region of the electromagnetic spectrum.

3. The integrated device of claim 1, wherein the optical excitation energy has a single characteristic wavelength.

4. The integrated device of claim 1, wherein the sample is associated with a plurality of markers having different characteristic wavelengths of light emission and different emission lifetimes, the plurality of markers being distinguishable based on the lifetime measurement and the spectral measurement.

5. The integrated device of claim 4, wherein the plurality of markers have at least two different characteristic wavelengths of light emission, and the portion of the plurality of markers that emits light at one of the at least two different characteristic wavelengths of light emission has at least two different emission lifetimes.

6. The integrated device of claim 1, further comprising at least one optical filter positioned between the waveguide and the at least one sensor.

7. The integrated device of claim 6, wherein the at least one optical filter is configured to selectively reduce transmission of optical excitation energy for luminescence emitted by the sample.

8. An integrated device comprising: a sample well disposed on the substrate and configured to receive a sample; a waveguide configured to propagate a plurality of pulses of optical excitation energy to the sample well; as well as at least one sensor positioned to receive light from the sample well, the at least one sensor configured to obtain lifetime measurements and spectral measurements of received photons of luminescence emitted by the sample, wherein the at least one sensor is configured to gather charge carriers generated by received photons into at least two time bins, and Wherein the at least one sensor is configured to generate at least one signal based at least in part on the focusing to obtain the lifetime measurement.

9. A sample analysis system comprising: Integrated device, including: a sample well disposed on the substrate and configured to receive a sample; a waveguide configured to propagate a plurality of pulses of optical excitation energy to the sample well; and at least one sensor positioned to receive light from the sample well, the at least one sensor configured to obtain lifetime and spectral measurements of received photons of luminescence emitted by the sample; and at least one excitation energy source configured to generate the plurality of pulses of optical excitation energy, wherein the at least one sensor comprises a first photosensitive region and a second photosensitive region, the first photosensitive region and the second photosensitive region are configured to detect a first wavelength range and a second wavelength range, respectively, and Wherein, the at least one sensor is configured to gather carriers generated by photons incident on the first photosensitive region into a first plurality of time bins, and to gather carriers generated by photons incident on the second photosensitive region into a second plurality of time bins.

10. The system of claim 9, wherein the optical excitation energy has a single characteristic wavelength.

11. The system of claim 9, wherein the at least one sensor is configured to aggregate carriers generated by received photons into at least two time bins to obtain the lifetime measurement.

12. An integrated device comprising: A plurality of pixels, wherein a pixel of the plurality of pixels comprises: a sample well configured to receive excitation energy from an excitation source external to the integrated device; and At least one sensor is positioned to receive luminescence from a sample located in the sample well and to generate a signal providing identification information of the sample based on the received luminescence, wherein one of the at least one sensor is configured to focus carriers generated by received photons of luminescence emitted from the sample well into at least two time bins.

13. The integrated device of claim 12, wherein the signal is indicative of a temporal parameter of the received light emission.

14. An integrated device as claimed in claim 13, wherein the time parameter is a lifetime associated with luminescence from the sample.

15. The integrated device of claim 12, wherein the signal is indicative of a spectrum of the luminescent light.

16. The integrated device of claim 15, wherein the signal is indicative of a characteristic wavelength of the luminescence.

17. The integrated device of claim 12, wherein the at least one sensor is further configured to generate a signal indicative of an intensity of the emitted luminous energy.

18. A sample analysis system comprising: an excitation source module, comprising an excitation source configured to emit a pulse of excitation energy having a first time period; and An integrated device comprising: a sample well configured to receive a sample, the sample well emitting luminescence when coupled to the pulse of excitation energy; a sensor that detects the luminescence during a second time period, wherein the second time period occurs after the first time period, wherein the sensor is configured to gather carriers generated by received photons of luminescence emitted from the sample well into at least two time bins; a first energy path along which the pulse of excitation energy moves from the excitation source to an energy source coupling component; a second energy path along which the pulse of excitation energy moves from the energy source coupling component to the sample well; and A third energy path along which the luminescence moves from the sample well to the sensor.

19. The system of claim 18, further comprising a base instrument to which the excitation source module and the integrated device are attached.

20. The system of claim 19, wherein the excitation source module is hingedly attached to the base instrument using a hinge pin.

21. The system of claim 20, wherein: The integrated device also includes a plurality of steel balls; and The excitation source module includes a plurality of magnetized radial V-shaped grooves configured to be placed on the plurality of steel balls.

22. The system of claim 19, wherein the excitation source module is removably attached to the base instrument.

23. The system of claim 19, wherein the excitation source module is attached to the base instrument via a sliding mechanism.

24. The system of claim 18, wherein the excitation source module comprises at least one tiltable window configured to control an incident angle and / or position of the pulse of excitation energy to the integrated device.

25. The system of claim 18, wherein the integrated device further comprises at least one monitoring sensor.

26. The system of claim 18, wherein the excitation source module is aligned to the energy source coupling component via an active alignment mechanism.

27. A method for detecting the presence of a molecule in a sample, the method comprising: introducing a sample labeled with one of a plurality of luminescent markers into a sample well, wherein at least a portion of the plurality of luminescent markers have different luminescent lifetime values; irradiating the sample well with a pulse of light; collecting carriers generated by photons emitted from the sample well and received by the sensor into at least two time bins; and An identity of one of the plurality of luminescent signatures is determined based on at least one signal indicative of the accumulated carriers.

28. The method of claim 27, further comprising: measuring the wavelength of photons emitted from the sample well; and Wherein determining the identity of the luminescent marker is also based on the measured wavelength of the photons.

29. The method of claim 27, wherein irradiation of the sample well is accomplished using a waveguide proximate the sample well such that an evanescent tail of the light is coupled to the sample well.

30. The method of claim 29, wherein the light is coupled to the waveguide using a star coupler.

31. The method of claim 29, wherein the light is coupled to the waveguide using a thin-sheet grating.

32. The method of claim 29, wherein the light is coupled to the waveguide using a multimode interference beam splitter.

33. An integrated device comprising: a sample well for receiving a sample labeled with at least one of a plurality of luminescence signatures, each of at least some of the plurality of luminescence signatures having a different luminescence lifetime value; and A sensor is configured to detect luminescence from one of the plurality of luminescent signs during a plurality of time periods and to aggregate carriers generated by the detected luminescence into time bins associated with the plurality of time periods, wherein the plurality of time periods are selected to distinguish between at least some of the plurality of luminescent signs.

34. The integrated device of claim 33, further comprising a waveguide for delivering excitation energy to a vicinity of the sample well.

35. The integrated device of claim 34, further comprising a grating coupler for receiving excitation energy from an external energy source and coupling the excitation energy into the waveguide.

36. An integrated device as claimed in claim 35, wherein the grating coupler has a width that gradually decreases in the direction of the waveguide.

37. The integrated device of claim 33, wherein the sample well comprises a nanopore formed in a metallic material.

38. The integrated device of claim 37, further comprising a concentric ring grating comprising a plurality of rings formed from the metal material, wherein the nanopore is located in an innermost ring of the plurality of rings.

39. The integrated device of claim 34, further comprising at least one microcavity proximate the sample well, wherein the at least one microcavity is configured to couple excitation energy from the waveguide to the sample well.

40. An integrated device comprising: a sample well configured to receive a sample labeled with one of a plurality of luminescent markers, wherein each marker of the plurality of luminescent markers emits luminescence within one of a plurality of spectral ranges, and a portion of the plurality of luminescent markers that emit luminescence within the one of the plurality of spectral ranges each have a different luminescence lifetime value; and A plurality of sensors, wherein each of the plurality of sensors is configured to detect luminescence for one of the plurality of spectral ranges in a plurality of time periods and to aggregate carriers generated by the detected luminescence into time bins associated with the plurality of time periods, the plurality of time periods being selected to be distinguishable in a portion of the plurality of luminescent signs.

41. The integrated device of claim 40, further comprising: A patterned color filter is disposed between the sample well and the plurality of sensors.

42. An integrated device as described in claim 41, wherein the patterned color filter passes light within a first spectral range of the multiple spectral ranges to a first sensor of the multiple sensors and passes light within a second spectral range of the multiple spectral ranges to a second sensor of the multiple sensors.

43. A sample analysis system comprising: a plurality of excitation sources configured to emit a plurality of excitation energies, wherein each excitation source of the plurality of excitation sources emits a pulse of one of the plurality of excitation energies; Integrated device, including: a sample well configured to receive a sample labeled with one or more luminescent markers, wherein a portion of the plurality of luminescent markers emit luminescent light after being illuminated with one of a plurality of excitation energies, the portion of the plurality of luminescent markers each having a different lifetime value; and A sensor is configured to detect luminescence from one of the plurality of luminescent marks in a plurality of time periods after a pulse of one of the plurality of excitation energies, and to gather carriers generated in the plurality of time periods into different time bins, wherein the timing of the pulse of the one of the plurality of excitation energies and the plurality of time periods are distinguished among the plurality of luminescent marks.

44. A method of forming an integrated device, comprising: forming a plurality of sensor regions, wherein a sensor region of the plurality of sensor regions comprises a plurality of sensors, and at least some of the plurality of sensors are configured to focus carriers generated by received photons into different time bins; forming a plurality of sample wells, wherein a sample well of the plurality of sample wells is aligned with a corresponding one of the plurality of sensor regions; and At least one waveguide is formed that is configured to couple excitation energy from at least one excitation source and direct the excitation energy to at least one sample well.

45. The method of forming an integrated device of claim 44, further comprising: A grating coupler is formed in a region separated from the plurality of sample wells and configured to receive excitation energy from the at least one excitation source external to the integrated device and couple the excitation energy to the at least one waveguide.

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