Systems, devices, and methods for improved optical waveguide transmission and alignment

Abstract

This paper provides systems, apparatuses, and methods for improved optical waveguide transmission and alignment in analytical systems. Waveguides in optical analytical systems exhibit variable and increasing back reflections over time from single-wavelength illumination, limiting their effectiveness and reliability. Systems are also susceptible to optical interference under conditions designed to overcome back reflections. New systems and methods using broadband illumination light with multiple longitudinal modes have been developed to improve light transmission and analysis in these systems. New systems and methods for aligning target waveguide apparatuses and light sources are also disclosed.

Description

[0001] This application is a divisional application of patent application No. 201880085486X, filed on November 3, 2018, entitled "System, Apparatus and Method for Improving Optical Waveguide Transmission and Alignment". Background Technology

[0002] As multiplexed analysis systems continue to miniaturize, scale up, and increase in power, the need to develop improved systems with this capability becomes increasingly important. For example, in optical analysis, increasing multiplexing often presents additional challenges, as it may require more complex optical systems, increased illumination or detection capabilities, and novel containment strategies. In some cases, systems attempt to increase multiplexing by many times, or even orders of magnitude, further complicating these considerations. Similarly, in some cases, the analytical environment in which the system is used is so sensitive that variations between different analyses within a given system may be unacceptable. These objectives often run counter to coarse approximations that simply make the system larger and more powerful, as these steps typically introduce greater consequences, such as crosstalk between systems, reduced signal-to-noise ratios due to lower signal and higher noise, or both. Therefore, it is desirable to provide analytical systems with substantially increased multiplexing for the desired analysis, particularly for highly sensitive reactive systems, and in many cases, to do so while minimizing the negative impacts of this increased multiplexing.

[0003] Conventional optical systems employ complex optical sequences to guide, focus, filter, separate, isolate, and detect light from or originating from sample material. Such systems typically utilize a variety of different optical elements to guide, modify, and otherwise manipulate light entering and leaving the reaction site. These systems are often complex and expensive, and frequently have significant space requirements. For example, a typical system uses mirrors and prisms to guide light from its source to a desired destination. Additionally, such systems may include beam-splitting optics, such as beam-splitting prisms or diffraction gratings, to generate two or more beams from a single original beam.

[0004] Alternatives to conventional optical systems have been described, particularly those with integrated optical components designed and manufactured in highly constrained environments. However, there remains a persistent need to improve the performance of analytical systems and to enhance light energy transmission through waveguides. There is also a persistent need to provide improved alignment between the light source and the target waveguide device, especially in integrated analytical apparatuses that include these components. Summary of the Invention

[0005] This disclosure addresses these and other needs by providing a system for optical analysis, the system comprising:

[0006] A light source configured to emit a broadband multi-longitudinal optical excitation beam into free space, and

[0007] A removable target waveguide device includes:

[0008] At least one optical coupler configured to receive an optically excited beam from a light source passing through free space;

[0009] At least one optical waveguide, the at least one optical waveguide including a first end; wherein the first end of the optical waveguide is configured to receive an optical excitation signal from at least one optical coupler;

[0010] At least one reaction region, optically coupled to at least one waveguide; and

[0011] At least one detector region configured to detect optical emission signals from at least one reaction region.

[0012] In some embodiments of these systems, the target waveguide device further includes:

[0013] At least one optical splitter;

[0014] At least one optical splitter includes an optical input and multiple optical outputs, wherein the optical input of the at least one optical splitter is optically coupled to at least one optical coupler; and

[0015] The first end of the optical waveguide is configured to receive an optical excitation signal from at least one optical coupler through an optical input of at least one optical splitter and a first optical output of at least one optical splitter.

[0016] More specifically, in some of these systems, at least one optical splitter includes 2 to 512 optical outputs.

[0017] In some system embodiments, at least one optical waveguide includes a second end; wherein the second end of at least one optical waveguide is configured to receive an optical excitation signal from a light source.

[0018] In more specific systems, the target waveguide device also includes:

[0019] At least one optical splitter;

[0020] At least one optical splitter includes an optical input and multiple optical outputs, wherein the optical input of the at least one optical splitter is optically coupled to at least one optical coupler; and

[0021] In this configuration, a first end of at least one optical waveguide is configured to receive an optical excitation signal from a first optical output of at least one optical splitter, and a second end of at least one optical waveguide is configured to receive an optical excitation signal from a second optical output of at least one optical splitter.

[0022] In even more specific embodiments, at least one optical splitter includes 2 to 512 optical outputs.

[0023] In an alternative embodiment, the target waveguide device includes:

[0024] First optical coupler and second optical coupler

[0025] The first end of the optical waveguide is configured to receive an optical excitation signal from a first optical coupler, while the second end of the optical waveguide is configured to receive an optical excitation signal from a second optical coupler.

[0026] According to another aspect, this disclosure provides a target waveguide device, comprising:

[0027] At least one optical coupler configured to receive an optically excited beam from a light source passing through free space;

[0028] At least one optical splitter, the at least one optical splitter including an optical input and a plurality of optical outputs, wherein the optical input of the optical splitter is optically coupled to at least one optical coupler;

[0029] At least one optical waveguide, the at least one optical waveguide including a first end and a second end, wherein the first end of the at least one optical waveguide is configured to receive an optical excitation signal from a first optical output of at least one optical splitter, and the second end of the at least one optical waveguide is configured to receive an optical excitation signal from a second optical output of at least one optical splitter.

[0030] At least one reaction region, optically coupled to at least one optical waveguide; and

[0031] At least one detector region configured to detect optical emission signals from at least one reaction region.

[0032] In a particular embodiment, the device includes a single optical coupler for transmitting an optical excitation signal to a first end and a second end of at least one optical waveguide.

[0033] In other specific embodiments, at least one optical splitter includes 2 to 512 optical outputs.

[0034] On the other hand, a system for optical analysis is provided, the system comprising:

[0035] A light source configured to emit an optically excited beam into free space, and

[0036] Movable target waveguide device, comprising:

[0037] At least one optical coupler configured to receive an optically excited beam from a light source passing through free space;

[0038] At least one optical waveguide, the at least one optical waveguide including a first end; wherein the first end of the optical waveguide is configured to receive an optical excitation signal from at least one optical coupler;

[0039] At least one reaction region, optically coupled to at least one waveguide; and

[0040] At least one detector region, optically coupled to at least one reaction region and configured to detect an optical alignment signal from at least one reaction region;

[0041] In this configuration, the target waveguide device or the optical excitation beam is movable relative to each other; and

[0042] The system is configured to monitor the optical alignment signal and move the target waveguide device or optical excitation beam relative to each other to increase the optical alignment signal.

[0043] In some embodiments, the system does not include an alignment camera.

[0044] In other embodiments, the system is configured to move the target waveguide or optical excitation beam relative to each other to maximize the optical alignment signal.

[0045] In other embodiments, the target waveguide device or optical excitation beam can be moved in two dimensions.

[0046] In some system embodiments, at least one detector region is configured to detect both optical alignment signals and optical emission signals from at least one reaction region.

[0047] In other system embodiments, the optical excitation beam is configured to project at least a coarse illumination point and a fine illumination point onto the target waveguide device, wherein the coarse illumination point is larger than the fine illumination point. More specifically, the optical excitation beam is configured to project the coarse illumination point onto the target waveguide device while the target waveguide device or light source is moved.

[0048] In some system embodiments, the target waveguide device further includes at least one auxiliary alignment sensor or at least one auxiliary alignment coupler positioned on the surface of the target waveguide device at a distance from at least one optical coupler. More specifically, this distance is defined using photolithography during the fabrication of the target waveguide device, or the at least one auxiliary alignment sensor or the at least one auxiliary alignment coupler is configured for a coarse alignment process, or the target waveguide device includes multiple auxiliary alignment sensors or multiple auxiliary alignment couplers. Even more specifically, the multiple auxiliary alignment sensors or multiple auxiliary alignment couplers are arranged in a geometric pattern, such as a geometric pattern including at least one optical coupler optically coupled to at least one reaction region.

[0049] In some system embodiments, the target waveguide device includes at least one alignment feature at a defined location on the device, and the location of the alignment feature is stored on the device in a readable format. In other system embodiments, the system includes at least one optical element located between the light source and the target waveguide device, wherein the at least one optical element modulates an optically excited beam between the light source and the target waveguide device, such as a lens, polarizer, wedge, filter, prism, mirror, or grating.

[0050] On the other hand, this disclosure provides methods for using all of the above-described systems and apparatus.

[0051] For example, in some embodiments, this disclosure provides a method for aligning a light source with a target waveguide device, including the following steps:

[0052] A system for optical analysis is provided, the system comprising:

[0053] A light source configured to emit an optically excited beam into free space, and

[0054] Movable target waveguide device, comprising:

[0055] At least one optical coupler configured to receive an optically excited beam from a light source passing through free space;

[0056] At least one optical waveguide configured to receive an optical excitation signal from at least one optical coupler;

[0057] At least one reaction region, optically coupled to at least one waveguide; and

[0058] At least one detector region, optically coupled to at least one reaction region and configured to detect an optical alignment signal from at least one reaction region;

[0059] In this context, the target waveguide device or the optical excitation beam is movable relative to each other;

[0060] Monitoring optical alignment signals; and

[0061] The target waveguide device or optical excitation beam is moved relative to each other to increase the optical alignment signal.

[0062] On the other hand, this disclosure provides a system for optical analysis, the system comprising:

[0063] The light source is configured to emit an optically excited beam into free space.

[0064] Movable target waveguide device;

[0065] The target waveguide device includes:

[0066] At least one optical coupler configured to receive an optically excited beam from a light source passing through free space; and

[0067] At least one optical waveguide, the at least one optical waveguide including a first end; wherein the first end of the optical waveguide is configured to receive an optical excitation signal from at least one optical coupler; and

[0068] At least two translation lenses are located between the light source and the movable target waveguide device; wherein the translation lenses control the transmission of the optical excitation beam from the light source to the target waveguide in multiple directions.

[0069] Specifically, in some system embodiments, the transmission of the optical excitation beam to the target waveguide device is controlled along a first direction, a second direction, and a first angle.

[0070] In some embodiments, the at least two translation lenses include a collimating lens and an objective lens, and more specifically, the collimating lens modulates the optical excitation beam relative to the objective lens at a first angle to the target waveguide device, or the objective lens modulates the optical excitation beam relative to the target waveguide device at a first spatial direction to the target waveguide device.

[0071] In some embodiments, the translational modulation of the objective lens relative to the target waveguide device along a first spatial direction and a second spatial direction transmits the optically excited beam to the target waveguide device along the first spatial direction and the second spatial direction.

[0072] On the other hand, a system for optical analysis is provided, the system comprising:

[0073] The light source is configured to emit an optically excited beam into free space.

[0074] Movable target waveguide device;

[0075] The target waveguide device includes:

[0076] At least one optical coupler configured to receive an optically excited beam from a light source passing through free space; and

[0077] At least one optical waveguide, the at least one optical waveguide including a first end; wherein the first end of the optical waveguide is configured to receive an optical excitation signal from at least one optical coupler;

[0078] A first translation lens is located between the light source and the movable target waveguide device; and

[0079] The first eccentric cam controls the position of the first translation lens along the first direction.

[0080] In one embodiment, the first eccentric cam is driven by a first stepper motor, more specifically, wherein the first stepper motor is fixed.

[0081] In other embodiments, the first eccentric cam controls the position of the first translation lens by being attached to the first translation lens, or the first lens has a stroke of up to 10 mm, or the first lens has a motion resolution of at least 1 μm.

[0082] In some embodiments, the system further includes a second eccentric cam, wherein the second eccentric cam controls the position of the first translation lens along a second direction, more specifically, wherein the second direction is orthogonal to the first direction, or wherein the second eccentric cam is driven by a second stepper motor, and more specifically, wherein the second stepper motor is fixed, and particularly, wherein movement of the second eccentric cam does not affect the position of the first translation lens along the first direction. In a particular embodiment, the second eccentric cam controls the position of the first translation lens by means of a second mount attached to the first translation lens.

[0083] Some system embodiments also include a third eccentric cam, wherein the third eccentric cam controls the position of the second translation lens, and more specifically, a fourth eccentric cam, wherein the fourth eccentric cam controls the position of the second translation lens, particularly wherein the first translation lens is a collimating lens and the second translation lens is an objective lens. Attached Figure Description

[0084] Figure 1 A system, apparatus, or part thereof including an optical waveguide is shown.

[0085] Figure 2 A flowchart outlining the proposed mechanism for time-dependent back reflection is shown.

[0086] Figure 3 A block diagram of the integrated analysis device is shown.

[0087] Figure 4 An example of a device architecture for performing optical analysis is shown.

[0088] Figure 5 An example of an integrated device for four-color analysis is shown.

[0089] Figure 6 A representative planar optical circuit (PLC) is shown.

[0090] Figure 7 It shows how standing waves are formed by backpropagating light beams and how the resulting interference is mitigated by using light beams with different wavelengths.

[0091] Figure 8A The design of a DNA sequencing waveguide device is shown, in which excitation is provided by backpropagating beams emitted from two single-space-mode lasers with different average output wavelengths (530 nm and 532 nm).

[0092] Figure 8B The design of a DNA sequencing waveguide device is shown, wherein excitation is provided by a beam emitted from a single-beam, multi-longitudinal-mode laser via backpropagation, the beam having, for example, an output wavelength of about 522 nm.

[0093] Figure 9 The setup for sending light into a waveguide and measuring the amount of light transmitted over time and the amount of light reflected over time is shown.

[0094] Figures 10A to 10D The design and use of the light source are shown, in which the output beam is controlled in multiple dimensions by two translation lenses.

[0095] Figures 11A to 11F The design features of a high-precision lens actuation system including a motor-driven eccentric cam are shown.

[0096] Figure 12 A set of measurements is shown, which demonstrates the time-dependent back reflection and reduction in transmit power.

[0097] Figure 13 The spectrum of illumination light with multiple longitudinal modes near 520 nm is shown to reduce or eliminate link efficiency variation (LEV) in optical waveguides.

[0098] Figure 14 The transmitted power over time through the waveguide is shown, indicating that LEV was not observed in a system using light with multiple longitudinal modes.

[0099] Figure 15It shows having with Figure 13 A control experiment with the same waveguide was conducted by irradiation with a laser having a single or only a few longitudinal modes.

[0100] Figure 16 A signal-to-noise ratio heatmap of a DNA sequencing waveguide device is shown, in which excitation is provided by back-propagating light from a single-beam multimode laser source.

[0101] Figure 17 The effect of laser power on the spectral bandwidth of the illumination light is shown.

[0102] Figure 18 The effect of laser power on the signal-to-noise ratio heatmap from a DNA sequencing waveguide device is shown.

[0103] Figure 19 The relationship between laser coherence, optical mode, spectral width, and optical path difference is shown.

[0104] Figure 20 This illustrates the effect of laser power and spectral width on sequencing accuracy in DNA sequencing reactions using waveguide devices.

[0105] Figures 21A to 21C This demonstrates the requirement for achieving coarse alignment between a laser beam and an input coupler on a target waveguide without using an alignment camera. Detailed Implementation

[0106] Overview

[0107] In some respects, this disclosure provides systems, apparatus, and methods for improved fluorescence analysis using waveguide irradiation. We have found that some waveguides exhibit time-varying back reflections when irradiated with a laser of a single wavelength. This has been observed, for example, with SiON waveguides irradiated with lasers in the visible wavelength range. This back reflection is undesirable because it reduces the amount of light transmitted through the waveguide. Furthermore, this back reflection can be detrimental because it can cause other problems, such as light being transmitted back to the laser, resulting in instability and laser damage, as well as temporal and spatial variations in the effective irradiation area. This variable back reflection is referred to as link efficiency variation, or LEV.

[0108] This link efficiency variation problem has previously been described, for example, in U.S. Patent Application Publication No. 2016 / 0216538, which describes a solution for LEVs involving time-varying modulation of the light illuminating the waveguide. It has been found that while time-varying wavelength modulation can address the LEV problem, it has several drawbacks. For one hand, lasers capable of time-varying wavelength modulation are expensive, and because they may be non-standard for their intended application, time and effort are required to obtain lasers that possess all the desired characteristics, such as wavelength and power, and also have the ability to modulate wavelength over time, as described in U.S. Patent Application Publication No. 2016 / 0216538, the entire contents of which are incorporated herein by reference.

[0109] The inventors are seeking to solve the LEV problem without using a light source with time-modulated wavelengths. They have discovered that LEV can be reduced or eliminated by other methods, such as illuminating the waveguide with a light source exhibiting multiple longitudinal modes over a narrow wavelength range. The inventors have also found that using a light source with multiple longitudinal modes allows the use of a single light source in a target device having a waveguide illuminated by a backpropagating beam, with minimal impact on system performance.

[0110] Although not bound by theory, it is believed that LEVs can be induced by illumination light forming within the waveguide of a Bragg grating. That is, illumination light from a laser with a single wavelength "writes" the grating into the waveguide at intervals corresponding to the wavelength of the light. A grating with this interval will cause some back reflection of the light passing through the waveguide. In some cases, it is thought that the back-reflected light further enhances the grating, thus producing even more back-reflected light. Furthermore, evidence has been seen that a grating "written" into the waveguide as described above can be erased by exposure to illumination light of a wavelength different from the wavelength at which it was formed.

[0111] It has been found that LEVs are generally more prevalent in lower wavelength light (with higher energy photons) and more prevalent in waveguides with higher laser power. It has also been found that LEVs can be reduced, and in some cases, effectively eliminated, by using laser irradiation with the appropriate characteristics described herein, including the output of multiple longitudinal modes.

[0112] Although not bound by theory, it is believed that by providing illumination light with multiple longitudinal modes, the formation of a Bragg grating in the waveguide by a single mode can be suppressed by the presence of other modes with slightly different wavelengths. That is, each longitudinal mode itself tends to write into the Bragg grating, resulting in LEV and back reflection. However, the presence of another longitudinal mode with a smaller wavelength interval will tend to suppress the development of the Bragg grating caused by that other longitudinal mode. In some cases, it is even possible to reduce the LEV of visible light by separating two longitudinal modes by 10 pm or more. In some cases, having more than two modes can result in a greater reduction in LEV. In some cases, there are four or more longitudinal modes; in some cases, there are eight or more longitudinal modes; in some cases, there are more than 20 longitudinal modes. In some cases, there are 2 to 500 longitudinal modes. For example, the number of longitudinal modes can be at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 30, at least 50, or even more.

[0113] Regarding LEV suppression, any suitable number of longitudinal modes can be used. However, if the number of longitudinal modes results in an excessively wide range of illumination wavelengths, it can have other detrimental effects on the system. For example, in the case of emitting light into a target waveguide (e.g., a waveguide on a target analysis device) via a coupler, the coupler is typically designed to emit light into the waveguide within a specific range of wavelengths. If the wavelength range of light entering the coupler from free space is too wide, some wavelengths will not be effectively coupled into the waveguide, thus reducing the efficiency of the system. For example, some optical couplers will accept light within a one-nanometer wavelength range without significant light loss, but if the wavelength range is 20 nm or larger, significant loss may occur. Although such light loss may not affect the overall performance of the optical system for some applications, it is generally desirable to minimize light loss. The inventors have discovered that by keeping the wavelength range of the longitudinal modes below approximately 20 nm, it is possible to significantly reduce or even eliminate LEV while maintaining acceptable optical coupling efficiency. The chosen wavelength range can depend on various aspects of the system, including the design of the coupler. In some cases, the wavelength range of the longitudinal mode is below about 10 nm; in some cases, the wavelength range of the longitudinal mode is below about 5 nm; in some cases, the wavelength range of the longitudinal mode is below about 2 nm; in some cases, the wavelength range of the longitudinal mode is below about 1 nm, or even lower.

[0114] While the light sources of this disclosure, such as lasers or laser diodes, preferably have multiple longitudinal modes, these sources also typically have a substantially single transverse mode. In other words, the light source provides a Gaussian beam with only the lowest-order fundamental mode (i.e., TEMoo). Such sources are preferred because waveguides typically used in the fluorescence analysis systems of this disclosure are designed to carry a single spatial mode. For these single-mode waveguides, light arriving at the coupler in other spatial modes will not couple into the waveguide or will dissipate rapidly in the waveguide because the waveguide will not support its transmission. In some cases, the light sources of this disclosure will emit more than one transverse mode, and therefore the loss of efficiency will need to be permissible. However, it has been found desirable for light sources to emit a large amount of light in only one mode (often referred to as the Gaussian or fundamental mode). In some cases, greater than 10% of the light intensity is in a single transverse mode; in some cases, greater than 50% of the light intensity is in a single transverse mode; and in some cases, greater than 80% of the light intensity is in a single transverse mode.

[0115] Because LEV back reflection tends to increase over time, the degree to which LEV needs to be controlled for a given application can depend on the duration of the measurement. For example, for single-molecule nucleic acid sequencing, irradiation typically lasts longer than 10 minutes, and in some cases, for several hours, such as 10 hours or more. Within the teachings of this disclosure, parameters of the light source (e.g., a laser source) can be adjusted to achieve the desired LEV suppression with the desired performance. For example, those skilled in the art can set the number of longitudinal modes and the intervals between modes to achieve optimal overall performance.

[0116] The method described herein can be used with any suitable waveguide susceptible to this time-dependent back reflection phenomenon. For example, the method of the present invention can be used with, for example, a target waveguide device for analysis, wherein the waveguide provides illumination light to a reaction region or wells to perform fluorescence measurements on a sample in the reaction region or wells.

[0117] Although described herein in the context of fluorescence analysis systems, the disclosed systems and methods can also be used to improve the effectiveness of any type of device that carries light energy via waveguides. This can be particularly useful for devices that include waveguides that transmit light in the visible light range (e.g., light transmitting from about 450 nm to about 700 nm). It can be useful for waveguides fabricated on semiconductor chips such as silicon chips, especially those with SiON cores. Examples include analytical devices for measuring fluorescence levels and planar optical circuits (PLCs) used in the visible light range, where the waveguide provides excitation illumination in the visible light range for the analytical device.

[0118] Figure 1A system or apparatus, or a portion thereof, relating to this disclosure is shown. A laser or laser system 110, serving as a light source, emits illumination light 115, also referred to as an optical excitation signal or optical excitation beam, into free space. As shown in the figure, in some cases, the laser 110 may emit light 115 directly into free space. In other cases, the laser 110 includes other optical elements through which the light travels before being emitted into free space. For example, other optical elements accompanying the laser before emitting illumination light 115 into free space may include optical fibers, a PLC, or a combination of both. In some cases, the illumination light emitted from the laser is sent directly to a target, such as a target device 170. Typically, the illumination light 115 will pass through one or more optical elements 120, which are used to shape, manipulate, or otherwise control the properties of the illumination light before reaching the target. Illumination light 117, shaped by one or more optical elements 120, is coupled into a waveguide 140. Light is transmitted through waveguide 140 to region of interest 150 on the target device. Typically, as shown here, a coupler 130 (e.g., a grating coupler) is used to direct the illumination light into the waveguide. Although a grating coupler is shown, it should be understood that any type of coupler, prism, or other interface optics or method, including, for example, direct mating coupling, can be used to guide the optical excitation signal from the light source into the waveguide.

[0119] The region of interest 150 has multiple reaction regions 155, such as nanowells or zero-mode waveguides (ZMWs). Waveguides 140 typically extend below the reaction regions 155, thereby illuminating the reaction regions from below using extinction wave illumination. Preferably, the reaction regions contain a fluorescent reactant that emits fluorescence 190 when excited by extinction wave illumination. Fluorescence 190 can be detected for desired analysis (e.g., nucleic acid sequencing). In some cases, as shown herein, the target device also has an integrated sensor 180. Fluorescence emitted from the reaction regions passes downwards through the device for detection at a single pixel or group of pixels 185. Such integrated target devices for fluorescence analysis are described in, for example, U.S. Patent Publications Nos. 2008 / 0128627, 2012 / 0085894, 2016 / 0334334, 2016 / 0363728, 2016 / 0273034, 2016 / 0061740, and 2017 / 0145498, all of which are incorporated herein by reference in their entirety. Target devices including integrated sensors typically also include electrical outputs 175. For example, the integrated sensor detects and processes optical emission signals, and then transmits electronic data related to the detected signals outside the device via one or more electrical outputs. The electrical outputs can be, for example, bonding pads on a silicon chip, which are typically wire-bonded to a chip package, and the chip package will have electronic outputs for transmitting electronic signals from the chip. The electronic signals are typically sent to a computer (not shown), which processes the received signals to perform the desired analysis.

[0120] Typically, it is expected that when a waveguide transmits light to a region of interest, the waveguide (e.g., Figure 1 Waveguide 140 in the target device will have a high level of optical transmission, i.e., a low level of optical loss. As mentioned above, it has been found that in some cases, when continuously irradiated with light of a single wavelength from the laser, one or more waveguides within the target device will exhibit time-dependent back reflection under certain conditions. This back reflection has been observed to be significant, leading to optical losses exceeding 90% in some cases. Even in cases where optical loss is not so large, back reflection can be problematic from the perspective of the increased irradiation power required to achieve a given level of transmitted light, as well as from the adverse effects of redirected light. For example, LEV can cause temporal and spatial variations in irradiation, making it difficult to reliably detect fluorescence signals from the reactive region irradiated by the waveguide. In some cases, variations in link efficiency may be due to coupling to radiation modes without back reflection.

[0121] As described above, the inventors have usefully discovered that back reflections in a waveguide can be reduced or eliminated by using illumination light from a light source, wherein the illumination light comprises multiple longitudinal modes within a narrow wavelength range. As background art, it should be understood that using a waveguide that is a single-mode waveguide in the target device can be useful. Typically, in the case of using such a single-mode waveguide, the light introduced into these waveguides comes from a laser having a single longitudinal mode (i.e., a single wavelength). The inventors have discovered that by using a light source having multiple longitudinal modes, the transmission of light through a single-mode waveguide can be improved. Although the presence of multiple longitudinal modes can be problematic in some applications, in the case of a fluorescence analysis system, the inventors have found that using illumination light with multiple longitudinal modes can be done without significantly degrading performance because the excitation spectrum of a fluorescent label typically spans a relatively large wavelength range; therefore, illumination wavelength ranges on the order of 20 nm or larger can be used without significantly reducing the excitation of the fluorescent label. Furthermore, the emission spectrum of a fluorescent label is generally relatively insensitive to the wavelength of the illumination light; therefore, the dispersion of the illumination wavelengths as described in this disclosure does not introduce any new problems for the detection of the emitted fluorescence.

[0122] The waveguide on the target device can be any suitable waveguide, including optical fiber, planar waveguide, or channel waveguide. Typically, a channel waveguide is used. This waveguide is preferably a single-mode waveguide, but for some applications it can be a multimode waveguide.

[0123] exist Figure 1 In the diagram, waveguide 140 is shown on a target device, which may be a semiconductor chip, such as a silicon chip. The specific system of interest in this invention is a SiON waveguide, such as those formed on a silicon chip. This SiON waveguide will have a SiON core and is typically surrounded by a coating material with a lower refractive index, such as silicon dioxide (SiO2). As is known in the art, SiON can be formed in a deposition process, and the proportions of the elements can be adjusted to control the optical properties of the waveguide. For example, the ratio of oxygen to nitrogen can be changed to alter the refractive index of the film. For the SiON waveguide of this invention, the composition is typically controlled to have a refractive index greater than about 1.6, greater than about 1.7, or greater than about 1.8. The refractive index can be measured, for example, at the sodium D line.

[0124] Figure 2A flowchart outlining the proposed mechanism for the observed time-dependent backscattering, or LEV, is shown. Unbound by theory, it is assumed that a standing wave pattern is formed within the waveguide (step I). The standing wave pattern can be formed, for example, by backscattering from defects or by reflection from a portion of the waveguide wiring pattern (e.g., the exit facet). In step II, a material response affecting the UV color center occurs (e.g., 2-photon absorption). This change in UV absorption then causes a change in the refractive index at the illumination wavelength. The material response leads to a change in the refractive index, resulting in a volumetric phase Bragg grating for light recording (step III). The Bragg grating causes more light to return via reflection (step IV). The increased back-traveling light enhances the standing wave pattern (step V). It can be seen that this step self-feeds in such a way that the initial grating becomes increasingly effective, resulting in an increasingly higher proportion of light returning. This possible mechanism is consistent with the time-dependent backscattering experimentally observed in such systems.

[0125] Understanding the above mechanism provides the inventors with insights into how to address the LEV problem by using multiple closely spaced longitudinal modes for illumination to prevent the accumulation of reflective gratings. Imagine a laser with a single longitudinal mode as a starting point. As described herein, one or more adjacent longitudinal modes are added, each with a different wavelength relatively close to the first wavelength. Each of the added longitudinal modes has a slightly different wavelength, so these modes will not combine with the first mode to write the same grating, and to some extent, each mode alone will create a grating, with each mode tending to erase the grating of another nearby mode. Furthermore, it has been observed that the formation of Bragg gratings in waveguides, as described herein, depends on the power of the light source. When an optical excitation beam is provided with multiple longitudinal modes, the power of the optical signal is distributed. Distributing the power of the optical excitation signal results in each mode having lower power compared to the case where all energy is in a single mode, so each mode is less likely to write into the grating in the first place. In some cases, the effect of the grating on power is more than linear. For example, it has been observed that LEV appears to depend on the square of the power. In such cases, it can be seen that by dividing the power into multiple modes, the formation of Bragg gratings can be significantly reduced. For example, compared to having all power in a single longitudinal mode, in this situation, changing from one longitudinal mode to four longitudinal modes (e.g., each mode spaced 30 pm apart, but each mode having essentially the same intensity) reduces the likelihood of each mode forming a grating by 16 times.

[0126] Waveguide Addressing Analysis System

[0127] In some aspects, this disclosure relates to improved systems, apparatuses, and methods for performing analytical operations, particularly optical analysis of chemical, biochemical, and biological reactions, for use in chemical, biological, medical, and other research and diagnostic applications. The systems, apparatuses, and methods of this disclosure are particularly suitable for applications in integrated analytical components, such as those where multiple functional components of the entire analytical system are integrated into a single modular component. However, as will be apparent from the following disclosure, many aspects of the invention will have wide applications beyond such integrated apparatuses and systems.

[0128] Typically, optical analysis, the subject of this disclosure, seeks to collect and detect one or more optical emission signals from a reaction of interest, the presence or absence of which, or its location, indicates the presence of a given substance in a given chemical or biological reaction and / or sample material. In some cases, reactants, their products, or other substances of interest (referred to herein all as reactants) inherently possess optically detectable signals. In other cases, exogenous labeling groups are provided to reactants to facilitate their detection.

[0129] Nucleic acid sequencing

[0130] As understood by those skilled in the art, fluorescently labeled nucleotides are used in a variety of different nucleic acid sequencing analyses. For example, in some cases, such labeling is used to monitor polymerase-mediated, template-dependent binding of nucleotides in primer extension reactions. Specifically, labeled nucleotides can be introduced into the primer-template polymerase complex, and the binding of labeled nucleotides to primers can be detected. If a specific type of nucleotide is bound at a given position, it indicates the basic and complementary nucleotides in the template molecule sequence. In conventional Sanger sequencing, the detection of bound labeled nucleotides utilizes a termination reaction, in which the labeled nucleotide carries a termination group that prevents further primer extension. By mixing labeled termination nucleotides with unlabeled native nucleotides, a set of nested fragments terminated at different nucleotides is produced. These fragments can then be separated by capillary electrophoresis or other suitable techniques to distinguish those that differ by a single nucleotide, and the labels on the fragments can then be read in ascending order of fragment size to provide the fragment sequence (as indicated by the last added, labeled termination nucleotide). Different nucleotides in a sequence can be easily distinguished by providing different fluorescent labels on each type of added nucleotide (see, for example, U.S. Patent No. 5,821,058, which is incorporated herein by reference in its entirety for all purposes).

[0131] In some sequencing technologies, an array of primer-template complexes is immobilized on the surface of a substrate, allowing individual molecules or homogeneous groups of molecules (clonal populations) to be spatially discrete from other individual molecules or groups of molecules. Labeled nucleotides are then added in a manner that results in the addition of individual nucleotides to each individual molecule or group of molecules. After the nucleotide addition, the addition of the label is detected and identified.

[0132] In some cases, sequencing analysis utilizes the addition of a single type of nucleotide at a time, followed by a washing step. The added labeled nucleotides are then detected, their labels removed, and the process is repeated using different types of nucleotides. The sequence of each template sequence is determined by the order in which the labels appear at given positions on the substrate.

[0133] In other similar cases, the immobilized complex is contacted with all four types of labeled nucleotides, each type of nucleotide having a distinguishable fluorescent label and a terminator group that prevents the addition of more than one nucleotide in a given step.

[0134] After a single binding to each individual template sequence (or group of template sequences), unbound nucleotides are washed away, and the immobilized complex is scanned to identify which nucleotide was added at each position. This process is repeated to produce sequence information for each template sequence. In other cases, more than four labeled nucleotides are used.

[0135] In a particularly concise method, the labeled nucleotide is detected in real time by a separate molecular complex during the binding process itself. Such a method is described, for example, in U.S. Patent No. 7,056,661, which is incorporated herein by reference in its entirety for all purposes. In these processes, the nucleotide is labeled on the terminal phosphate group released during binding to avoid accumulation of the label on the extended product, thereby avoiding any label removal process that might be detrimental to the complex. The primer / template polymerase complex is observed during the polymerization process, and the added nucleotide is detected by its associated label. In one particular aspect, an optically confined structure such as a zero-mode waveguide (see, for example, U.S. Patent No. 6,917,726, which is incorporated herein by reference in its entirety for all purposes) is used to observe the labeled nucleotide, which restricts the volume of excitation radiation exposed to the individual primer / template polymerase complex. As a result, only the labeled nucleotide retained by the polymerase during binding is exposed to excitation irradiation for a period sufficient to produce fluorescence and thus identify the bound nucleotide. In another approach, the label on the nucleotide is configured to interact with a complementary group on or near the complex, for example, by attaching to a polymerase, where the interaction provides a unique signal. For example, a donor fluorophore that is excited at a first wavelength and emits at a second wavelength can be provided to the polymerase, while the nucleotide to be added is labeled with a fluorophore that is excited at a second wavelength but emits at a third wavelength (see, for example, U.S. Patent No. 7,056,661, previously incorporated herein). As a result, a unique signal is generated when the nucleotide and the polymerase are close enough to allow energy to be transferred from the donor fluorophore to the label on the nucleotide. Again, in these cases, various types of nucleotides are set with unique fluorescent labels that allow identification by the spectral or other optical characteristics of their label.

[0136] In the various exemplary processes described above, the detection of a signal event from the reaction region indicates that a reaction has occurred. Furthermore, for many of these processes, the nature of the reaction is identified by distinguishing the spectral characteristics of the signal event, such as which nucleotide or complementary nucleotide to a given position in the template molecule was added at a given time in the primer extension reaction.

[0137] The optical path of the entire system disclosed herein serves one or more of the following functions: transmitting excitation radiation to the reaction region, for example, to excite fluorescently labeled molecules that subsequently emit associated optical emission signals; transmitting the optical signals emitted from the reaction region to a detector; and, for multispectral signals (i.e., multiple signals that can be distinguished by their emission spectra), separating those signals so that differential detection can be performed, for example, by directing the different signals to different detectors or different regions on the same detector array. The differentially detected signals are then correlated with the occurrence of the reaction (e.g., the addition of a nucleotide at a given position) and the determination of the nature of the reaction (e.g., identifying the added nucleotide as a specific nucleotide type, such as adenosine).

[0138] In conventional, fully free-space analytical systems used for nucleic acid sequencing, optical sequences for delivering excitation light to the reaction region and for transmitting optical signals from the reaction region to the detector can preferably reduce the overall system size, complexity, and cost. For example, such optical sequences may include a collection of lenses, dispersive elements, beam splitters, beam expanders, collimators, spatial and spectral filters, and dichroic mirrors, all assembled to deliver a targeted and uniform illumination distribution to different reaction regions. In large-scale systems, these components must be fabricated, assembled, and tuned to ensure proper alignment, focusing, and isolation from other light sources and vibration sources to optimize the transmission of excitation light to the reaction region. Addressing these and other issues becomes more critical as the number of addressable reaction regions increases, or as the system becomes more sensitive to variations in excitation light intensity, and often involves incorporating additional components into the optical sequence, such as alignment and focusing mechanisms, isolation structures, etc.

[0139] Conventional systems for collecting and detecting optical emission signals typically employ the following optical sequence: the emitted optical signal is collected from the reaction region via an objective lens system; various different signals are transmitted through one or more filter stages, typically constructed from one or more dichroic mirrors that differentially transmit and reflect light of different wavelengths, thus directing spectrally different optical signals to different detectors or multiple regions on a given detector. These separated optical signals are then detected and used to identify the nature of the response that elicits such signals. It should be understood that the use of such differentially oriented optics, in the form of multiple detectors, multiple lenses, and filter systems, imposes significant space, size, and cost requirements on the entire system, as well as complex alignment and related problems in many cases. Many of these difficulties are further exacerbated when the optical sequence shares one or more subpaths with the excitation irradiation, as signal processing will include the further requirement of separating the background excitation irradiation from each detected signal.

[0140] Furthermore, similar to the excitation optical sequences described above, increased system sensitivity and multiplexing introduce more problems that must be addressed, thus increasing the complexity of the already intricate optical sequences. Moreover, the greater the number of optical components in the optical sequence, the greater the risk of introducing unwanted perturbations into the system and the greater the ability to detect the resulting signal. For example, optical aberrations in optical elements introduce additional difficulties for signal detection; optical elements can also inject a certain level of autofluorescence into the optical sequence, which must then be distinguished from the signal event.

[0141] In some embodiments, the system of this disclosure further includes a computer that receives at least one electronic signal from a detector or a region of the detector, such as the detected signal described above, and analyzes the at least one electronic signal. More specifically, as will be understood by those skilled in the art, the analysis performed by the computer may include obtaining nucleic acid sequencing information from the electronic signal.

[0142] Backpropagation waveguide irradiation

[0143] In addition to reducing LEV, the inventors have also usefully discovered that using a light source with multiple longitudinal modes enables the illumination of a target waveguide from both ends using a single light source without significant reduction in illumination due to optical interference. As background, in some cases it may be desirable to illuminate a waveguide extending across multiple reaction regions from both ends. This is done, for example, to improve the consistency of illumination across reaction regions or wells. Specifically, for a typical waveguide, some light intensity is lost as light propagates downwards along the waveguide. This intensity loss results in a lower illumination level in the reaction region at the distal end of the waveguide compared to the illumination level in the reaction region at the end of the waveguide from which the light is emitted. Intensity loss can be improved by illuminating the waveguide from both ends. However, when illuminating both ends of a waveguide using light from a laser with a single longitudinal mode, interference may occur between the two illumination sequences, which can adversely affect optical analysis due to temporal and spatial variations in illumination. The inventors of this invention have discovered that the light source described herein (e.g., a laser with multiple longitudinal modes over a relatively narrow wavelength range) can be used for such end-to-end illumination with significantly less interference. This method allows for illumination from both ends using a single light source (e.g., a single laser), instead of relying on a solution that requires two light sources with slightly different wavelengths, one at each end of the waveguide.

[0144] In some cases, the optimal number of longitudinal modes for reducing or eliminating interference in end-irradiation may differ from the optimal number of longitudinal modes for reducing or eliminating LEV. Specifically, to reduce or eliminate interference, it is generally desirable for the irradiating laser to have at least eight longitudinal modes. The longitudinal modes are at least 10 pm apart, and the intensity of the weakest mode is 20% or more of the intensity of the strongest mode. In some cases, the intensity of the weakest mode is 50% or more of the intensity of the strongest mode. The determination of the optimal number of longitudinal modes, the separation of modes, and the intensity of each mode are known to those skilled in the art. See, for example, Saleh and Teich (1991) Fundamentals of Photonics, ch. 10, JohnWiley & Sons, Inc., which describes the temporal coherence function used in such calculations.

[0145] To use the backpropagation method, light from a light source is typically split into multiple beams, each beam being sent to multiple corresponding couplers on a chip. Each coupler then directs the light into multiple corresponding waveguides. For a given pair of couplers, one coupler injects light into the waveguide routed to illuminate one end of the waveguide, while the other coupler injects light into the waveguide routed to illuminate the other end. See, for example, the following: Figure 8B In other cases, the illumination light from a single coupler is routed within the chip so that both ends of the waveguide are illuminated. This routing can be achieved, for example, using an optical splitter associated with the input coupler, splitting the light within that optical splitter into two or more outputs to be delivered to the respective ends of the waveguide. (See below for more details.)

[0146] Integrated device

[0147] Some of the apparatuses and methods disclosed herein are integrated into a small-scale analytical apparatus, which optionally also includes one or more reaction regions, fluid components, and excitation irradiation paths and optionally excitation irradiation sources. Integrating some or all of the above components into a single small analytical apparatus solves many problems faced by larger, non-integrated analytical systems, such as size, cost, weight, and inefficiencies associated with long-path or free-space optics. For example, highly multiplexed analytical systems including integrated waveguides for irradiating nanoscale samples are described in U.S. Patent Publications Nos. 2008 / 0128627, 2012 / 0085894, 2016 / 0334334, 2016 / 0363728, 2016 / 0273034, 2016 / 0061740, and 2017 / 0145498, all of which are incorporated herein by reference in their entirety. Other optical systems for analyzing nanoscale samples, including the illumination and detection of such samples, are described in U.S. Patent Publications Nos. 2012 / 0014837, 2012 / 0021525, and 2012 / 0019828, all of which are incorporated herein by reference in their entirety. Other nanoscale irradiation systems for highly multiplexed analysis are described in U.S. Patent Publications Nos. 2014 / 0199016 and 2014 / 0287964, all of which are incorporated herein by reference in their entirety.

[0148] Other examples of such integrated systems are described in, for example, U.S. Patent Application Publications 2012 / 0014837, 2012 / 0019828, and 2012 / 0021525, all of which are incorporated herein by reference in their entirety. By integrating the detection element directly with the reaction region or as a coupling component, the need for many of the various components required in free-space optical systems can be eliminated, such as numerous transmission optics, lenses, mirrors, etc. Since alignment is achieved by directly integrating the detection element with the reaction region, other optical components, such as various alignment functionalities, can also be eliminated. The systems, apparatuses, and methods of this disclosure further enhance the benefits offered by such apparatus by greatly simplifying the optical components of the analytical apparatus, further reducing the cost and complexity of such apparatus, and improving the signals available during the process.

[0149] The analysis system according to this disclosure employs one or more analysis devices. In an exemplary embodiment, the system includes an array of analysis devices formed as a single integrated device. An exemplary array is configured to function as a disposable consumable. In various embodiments, the integrated device includes other components, including but not limited to local fluidics, electrical connections, power supplies, irradiation elements, detectors, logic, and processing circuitry. Each analysis device or array is configured to perform the analysis operations described above.

[0150] Although the components and configurations of each device in the system can vary, each analytical device within the system can at least partially include Figure 3 The block diagram illustrates a general structure. As shown, the analytical apparatus 300 typically includes a reaction unit 302, in which reactants are arranged and from which optical emission signals are emitted. "Reaction unit" is understood to refer to the location where the reaction of interest occurs, as commonly used in analytical and chemical fields. Therefore, a "reaction unit" can include a completely self-contained reaction well, container, flow unit, chamber, etc., for example, enclosed by one or more structural barriers, walls, caps, etc., or a "reaction unit" can include a specific region on a substrate and / or within a given reaction well, container, flow unit, etc., for example, without structural constraints or enclosures between adjacent reaction units. The reaction unit can include structural elements that enhance the reaction or its analysis, such as optically confined structures, nanowells, pillars, surface treatments such as hydrophobic or hydrophilic regions, binding regions, etc.

[0151] In various respects, "analytical device" refers to a reaction unit and functionally connected associated components. In various respects, "analytical system" refers to one or more associated analytical devices and associated components. In various respects, "analytical system" refers to a larger system including the analytical system and other instruments for performing analytical operations. For example, in some cases, the analytical device of the present invention is part of an analytical instrument or analytical system. The analytical device can be removably coupled into the instrument. Reagents can be brought into contact with the analytical device before or after coupling the analytical device to the system. The system can provide electrical signals and / or illumination to the analytical device and can receive electrical signals from detectors in the analytical device. The instrument or system may have a computer to operate, store, and analyze data from the device. For example, the instrument may have the ability to identify the sequence of nucleotide analogs added in a nucleic acid sequencing reaction. For example, this can be identified as described in U.S. Patent No. 8,182,993, which is incorporated herein by reference for all purposes.

[0152] In some cases, one or more reactants involved in the reaction of interest can be immobilized, entrained, or otherwise positioned within a given reaction unit. Various techniques are available for positioning and / or immobilizing reactants, including surface immobilization via covalent or non-covalent linkages; bead- or particle-based immobilization followed by positioning of the beads or particles; entrainment in a matrix at a given location, etc. A reaction unit may comprise an aggregate of molecules, such as a solution or molecular plaque, or it may comprise individual molecular reaction complexes, for example, one molecule from each molecule involved in the reaction of interest forming a complex. Similarly, the entire device and system of the present invention may comprise individual reaction units, or may comprise an aggregate, array, or other group of reaction units in an integrated structure, such as a multi-walled or multi-unit plate, chip, substrate, or system. Some examples of such arrayed reaction units include nucleic acid array chips, such as GeneChip® arrays (Affymetrix, Inc.), zero-mode waveguide arrays (as described elsewhere herein), microwell plates and nanowell plates, multichannel microfluidic devices, such as LabChip® devices (Caliper Life Sciences, Inc.), and any of a variety of other reaction units. In all respects, the “reaction unit,” sequencing layer, and zero-mode waveguide are similar to those described in U.S. Patent No. 7,486,865, the entire contents of which are incorporated herein by reference for all purposes. In some cases, the devices of these arrays may share optical components within a single integrated monolithic device (e.g., a single waveguide layer) to deliver excitation light to each reaction region. Methods for illuminating an analysis apparatus with waveguides are provided in U.S. Patent Nos. 8,207,509 and 8,274,040, each of which is incorporated herein by reference for all purposes.

[0153] Although the analysis system may include an array of analysis devices having a single waveguide layer and a reaction unit layer, it is understood that multiple layer compositions may be used in the waveguide array substrate and the cladding / reaction unit layer while still achieving the objectives of the present invention (see, for example, U.S. Patent No. 7,820,983, which is incorporated herein by reference for all purposes).

[0154] Analysis systems typically include one or more such as Figure 3The analytical apparatus 300 shown has a detector element 320 arranged in optical communication with a reaction unit 302. The optical communication between the reaction unit 302 and the detector element 320 can be provided by an optical sequence 304, consisting of one or more optical elements, typically designated 306, 308, 310, and 312, to efficiently guide signals from the reaction unit 302 to the detector 320. Depending on the specific application, these optical elements can typically include any number of elements, such as lenses, filters, gratings, mirrors, prisms, refractive materials, etc., or various combinations thereof. In addition to the components for guiding optical emission signals from the reaction region to the detector, the chip may also have optical components for transmitting illumination light to the reaction region to perform fluorescence measurements.

[0155] In various embodiments, the reaction unit 302 and detector element 320 are arranged together with one or more optical elements in an integrated device structure. Integrating these elements into a single device architecture improves the optical coupling efficiency between the reaction unit and the detector. As used herein, when referring to different components of an analytical device, the term "integration" generally refers to two or more components coupled to each other so that they remain stationary relative to each other. Thus, integrated components can be integrated irreversibly or permanently, meaning that separation would damage or destroy one or both elements, or integrated components can be integrated movably, where one component can be detached from another, and when integrated together, they remain substantially stationary relative to each other. In some cases, these components are integrated in a single silicon chip, for example, as a single-fabricated device. In some cases, the detector portion is part of a separate instrument, and the reaction unit component is part of a removable device (e.g., a removable chip). Where the reaction unit component is located on a separate chip from the detector component, the optical element components for directing optical emission signals from the reaction unit to the detector can be located in the reaction unit component, in the detector component, or in a combination thereof, with some components located in the reaction unit component and others in the detector component.

[0156] For the apparatus, method, and system of this disclosure, even when the detector is part of the instrument and separate from the reaction unit component, the reaction unit component is typically placed directly on the detector, and there is a minimum open space between the reaction unit component and the detector component. In some cases, the space between the reaction unit component and the detector component will be less than 1 micrometer, less than 10 micrometers, less than 100 micrometers, or less than 1 millimeter.

[0157] The reaction unit components will typically have alignment structures to allow for precise alignment of the reaction unit with the corresponding detector section.

[0158] Where reaction unit components, optical components, and detectors are irreversibly or permanently integrated into a single integrated device, such a device can be manufactured as a single unit, or two or more components can be manufactured separately and joined together to form a single device. The connection between individual components can be achieved by any suitable method, including bonding and wafer bonding.

[0159] Depending on the application, one can choose to integrate all components into a single device or associate the detector components separately with the instrument. The advantage of the permanently integrated device approach is that the detector can be manufactured under controlled conditions in close contact with the reaction unit and other components to achieve precise registration. The approach where the detector is not integrated into the reaction unit chip but is part of the instrument has the advantage that the detector can be repeatedly used with different reaction unit components.

[0160] In conventional optical analysis systems, discrete reaction vessels are typically housed within optical instruments that utilize only free-space optics to transmit optical signals between the reaction vessels and the detector. These free-space optics often comprise higher-quality, larger components with free-space interfaces, which introduce numerous drawbacks to such systems. For example, such systems are more prone to light loss due to the introduction of undesirable leakage paths from these higher-quality components. They also typically introduce higher levels of autofluorescence. All these inherent weaknesses reduce the system's signal-to-noise ratio (SNR) and overall sensitivity, consequently impacting system speed and throughput. Furthermore, in multiplexed applications, signals from multiple reaction regions (i.e., multiple reaction units, or multiple reaction locations within a single unit) often traverse a common optical sequence or a common portion of an optical sequence that uses the full volume of the optical elements in that sequence to be imaged onto the detector plane. As a result, the presence of optical aberrations (e.g., diffraction, scattering, astigmatism, and coma) in these optical components degrades signal amplitude and across the field of view, leading to greater noise contributions and crosstalk between the detected signals.

[0161] The analytical systems and apparatus according to this disclosure typically include a reaction region, a container or area that is physically integrated with a detection component or sensor, or configured to be sufficiently close to and sensingly connected to the detection component or sensor to improve performance.

[0162] In some cases, the reaction region comprises a nanoscale well, for example, a nanoscale well with a linear dimension not greater than 500 nm. The nanoscale well in the systems and apparatus of this disclosure can, for example, be a cylinder having a base diameter between about 50 nm and 200 nm. The depth of the well can, for example, be from about 50 nm to about 400 nm. In some cases, the reaction region may comprise a zero-mode waveguide (ZMW). Zero-mode waveguides are described, for example, in U.S. Patent Nos. 7,170,050, 7,486,865, and 8,501,406, each of which is incorporated herein by reference in its entirety.

[0163] Such devices attempt to leverage the proximity of the reaction region or container in which signals are generated to the detector or detector element that senses those signals, in order to take advantage of the benefits derived from that proximity. As mentioned above, such benefits include reducing the size, weight, and complexity of the optical sequence, resulting in increased potential multiplexing of the system, for example, increasing the number of different reaction regions that can be integrated and detected in a single system. Furthermore, compared to systems utilizing large free-space optics and multiple cameras in signal collection and detection, this proximity offers the following advantages: reduced signal loss during transmission, reduced signal crosstalk from adjacent reaction regions, and reduced overall system cost when utilizing such integrated devices.

[0164] In the systems disclosed herein, there are many design criteria that can benefit from optimization. For example, in the case of integrated detection systems, the primary objective is to minimize the intervention of optical elements that may interfere with the effective transmission of optical emission signals from the reaction region to the detector, and that contribute to increased system cost and space requirements by increasing the complexity of the optical elements between the reaction region and the sensor.

[0165] Furthermore, given the increasing importance of single-molecule detection systems, maximizing the amount of detected optical emission signal for any given reaction event is also crucial. In particular, optical detection of single-molecule events typically relies on a relatively small number of photons corresponding to the event of interest in the measurement. Although high quantum yield labeling groups (e.g., fluorescent dyes) can improve detectability, such systems still operate at the lower end of the detectability range for optical systems. Useful fluorescent dyes found in the analytical reactions performed using this system are known. Any suitable fluorescent dye may be used, such as those described in PCT International Publication No. WO2013 / 173844A1 and U.S. Patent Application Publications Nos. 2009 / 0208957A1, 2010 / 0255488A1, 2012 / 0052506A1, 2012 / 0058469A1, 2012 / 0058473A1, 2012 / 0058482A1 and 2012 / 0077189A1.

[0166] In the cases of the systems, apparatuses, and methods disclosed herein, the size and complexity of the optical path length present greater challenges because the available space is limited in which the objectives of excitation and signal separation, or separating one signal from the next, are achieved. Therefore, the systems, apparatuses, and methods of the present invention utilize the advantage of a simplified optical path length associated with the analysis performed, in order to optimize those analyses for the integrated nature of those systems, apparatuses, and methods.

[0167] Figure 4 An example of a device architecture for performing optical analysis (e.g., nucleic acid sequencing procedures or single-molecule binding assays) is shown in more detail. As shown, the integrated device 400 includes a reaction region 402 defined on a first substrate layer 404. As shown, the reaction region 402 includes wells disposed in the substrate surface. Such wells can be formed by creating recesses on the substrate surface or by forming holes through attaching the substrate layer to an underlying transparent substrate (e.g., used in zero-mode waveguide arrays). See, for example, U.S. Patent Nos. 7,181,122 and 7,907,800. Figure 4 A portion of an apparatus having a single reaction region 402 is shown. Typically, an apparatus will have multiple reaction regions; for example, an apparatus may include an array having thousands to millions or more individual reaction regions.

[0168] Excitation irradiation is transmitted from an excitation source (not shown) to the reaction region, which may be separate from or integrated into the substrate. As shown, an optical waveguide (or waveguide layer) 406 is used to transmit excitation light (indicated by arrows) to the vicinity of the reaction region 402, wherein the dissipation field emitted from the waveguide 406 irradiates the reactants in the reaction region 402. Irradiation of reaction regions using optical waveguides is described, for example, in U.S. Patent Nos. 7,820,983, 8,207,509, and 8,274,040, which are incorporated herein by reference for all purposes.

[0169] The integrated device 400 optionally includes an optical channel component 408 to efficiently guide emitted light from the reaction region to a detector layer 412 disposed below the reaction region. This detector layer will typically include multiple detector elements, such as the four illustrated detector elements 412a-d optically coupled to a given reaction region 402. For DNA sequencing applications, it is generally desirable to monitor four different signals in real time, each corresponding to a nucleic acid base. Although pixels 412a-d are shown as linearly arranged, it should be understood that the detector elements can be arranged in a grid in an n×n square, a ring array, or any other convenient orientation or arrangement. In some cases, each detector element or channel will have a single pixel. In some cases, each detector element will each include multiple pixels. The detector elements are electrically connected to conductors extending from the chip to provide electrical signals to and from the detector elements, for example, to a connected processor, as described in more detail elsewhere herein. In some embodiments, the detector layer is a CMOS wafer, i.e., a wafer composed of a CMOS sensor or a CCD array. See, for example, CMOS Imagers From Phototransduction to Image Processing (2004) Yadid-Pecht and Etienne-Cummings, eds.; Springer; CMOS / CCD Sensors and CameraSystems (2007) Hoist and Lomheim; SPIE Press.

[0170] Then, the emission signals from impacts on these detector elements from reaction region 402 are detected and recorded. For example... Figure 4 As shown in the integrated device, this device can additionally include color filters above each detector element, such as those disposed in filter layer 410. As illustrated, "filter a" corresponds to the color filter associated with "channel a", "filter b" corresponds to the color filter associated with "channel b", and so on. This set of filters is selected to allow a high yield of captured photons; for example, each color filter has one or more blocking bands that block a portion of one or more different spectral signals emitted from the reaction occurring in reaction region 402. Specifically, the filters are designed to allow most of the emitted photons to pass through while still distinguishing the four bases.

[0171] In some cases, optical elements are configured to selectively direct light from a given group of wavelengths to a given detector element. Typically, nonspecific light redirection is used so that the light arriving at each region of the filter layer is substantially the same.

[0172] The detector layer is operatively coupled to appropriate circuitry, typically integrated into the substrate, for providing a signal response to a processor, which may be integrated into the same device structure or separated from the detector layer and associated circuitry but electrically coupled to the processor and associated circuitry. Examples of circuit types useful in such devices are described in U.S. Patent Application Publication No. 2012 / 0019828, which is previously incorporated herein by reference.

[0173] The integrated analysis apparatus array of the present invention, also referred to herein as a target waveguide apparatus or target device, typically has at least one optical coupler and an integrated waveguide optically coupled to the optical coupler. In some embodiments, the optical coupler of the apparatus of the present invention is a low numerical aperture coupler, and in some embodiments, the optical coupler is a diffraction grating coupler. In some cases, the light source is directed to a single coupler, while in other cases, the light source is directed to multiple couplers, for example, 2 to 16 couplers. In some cases, each coupler receives substantially the same power. In some cases, different power levels are directed to different couplers on the target device. Although this description may refer to a "coupler" on the device, it should be understood that in some cases there may be a single coupler, while in other cases there will be multiple couplers on the device. An analysis apparatus with multiple couplers is described, for example, in U.S. Patent Application Publication No. 2016 / 0363728, the entire contents of which are incorporated herein by reference.

[0174] Grating couplers and their use in coupling light (typically from optical fibers) to waveguide devices are known in the art. For example, U.S. Patent No. 3,674,335 discloses reflective and transmissive grating couplers suitable for routing light into thin-film waveguides. Additionally, U.S. Patent No. 7,245,803 discloses an improved grating coupler comprising multiple elongated scattering elements. The coupler preferably has a horn-shaped structure with a narrow end and a wide end. This structure is believed to provide enhanced efficiency when coupling optical signals inside and outside a planar waveguide structure. U.S. Patent No. 7,194,166 discloses a waveguide grating coupler suitable for coupling wavelength division multiplexed light back and forth between single-mode and multimode optical fibers. The disclosed device comprises a set of waveguide grating couplers arranged on a surface, all of which are illuminated by a spot of light from the optical fiber. At least one grating coupler in this set is tuned to each channel in the beam, thus demultiplexing the channels propagating in the optical fiber. Other examples of grating couplers are disclosed in U.S. Patent No. 7,792,402 and PCT International Publications Nos. WO 2011 / 126718 and WO 2013 / 037900. U.S. Patent No. 7,058,261 discloses the integration of prism coupling and grating coupling into an integrated waveguide device.

[0175] As described above, referring to the integrated device for four-color analysis, Figure 5 An exemplary structure is illustrated. As shown, the analytical device includes a reaction unit 502 coupled to a reagent container or fluid conduit 506 that delivers reactants to the reaction unit 502. The reaction unit may be a nanoscale well or a zero-mode waveguide. In some cases, biomolecules, such as polymerases, will be immobilized within the reaction unit. The fluid conduit may provide reagents across multiple reaction units. Below the reaction unit is a waveguide 514 for providing excitation irradiation to the reagents within the reaction unit. The irradiation light can be used to excite fluorescence emission from the reagents within the reaction unit. Light emitted from the reaction unit is directed downwards through a transport layer for transmitting light from the reaction unit to a detector. In some cases, the transport layer will have optical components to enhance light transmission or modulate the efficiency of light. Figure 5 In the analysis apparatus, a light tunnel or conduit 508 is configured to be optically connected to a reaction unit 502, which in turn is optically connected to a sensing element 510 in a detector, wherein light reaching each of the four sensing elements on the detector passes through a different color filter. As shown, each reaction unit is optically coupled to a detector or detector element comprising four regions or pixel subsets, each region or pixel subset including a filter layer, and each filter layer including at least one blocking band. Each filter layer sends an appropriate set of light wavelengths to an appropriate pixel subset. The pixel subset or sensor element is coupled to appropriate electronics 512 that make up the entire sensor or camera, such as buses and interconnects. The electronics may also include processing elements for processing signals from the detector.

[0176] The inventors have usefully discovered that, in the SiON / SiO2 waveguides of this integrated device, longer waveguides are more likely to exhibit LEVs compared to shorter waveguides. This observation, without being bound by theory, is consistent with the LEV mechanism proposed above. Therefore, in addition to using illumination light with multiple longitudinal modes, it has been found that dividing the initially illuminated waveguide on the waveguide device into multiple waveguides can reduce LEVs. As previously mentioned, it has also been found that LEV accumulation may depend on the power (or spatial power density) of the illumination light. Furthermore, dividing the initial waveguide into multiple branch waveguides over a relatively short distance on the waveguide device can reduce the level of LEV accumulation.

[0177] For the waveguide device of this disclosure, as described above, there may be a significant distance between the coupler and the region of interest (e.g., the reaction region). The distance light travels in the waveguide from the coupler to the region of interest can be, for example, a few centimeters, such as 1 cm to 10 cm. The distance referred to herein is the distance light travels within the waveguide, for example, the distance light routes through one or more waveguides. Typically, when light is routed from the coupler over a relatively long distance to the region of interest, a single waveguide is used to route the light from the coupler to a region close to the region of interest, where the routed waveguide may split into multiple waveguides. In cases where multiple waveguide branches are required within the region of interest, the splitting from the routed waveguide to the waveguide branch within the region of interest is typically performed near the region of interest rather than near the coupler. One routed waveguide per coupler is generally the most efficient method for routing over relatively long distances. Using a single routed waveguide involves fewer components and typically requires less space on the device compared to using multiple routed waveguides per coupler. In contrast to this typical architecture, the inventors have discovered that, for the waveguide device of this disclosure, it is advantageous to carry light from the coupler to the region of interest or a region near the region of interest by splitting the irradiating light into multiple routing waveguides close to the coupler, relative to more than one waveguide per coupler. By reducing the power carried in each routing waveguide, the tendency to establish LEV back reflections can be reduced. When the LEV dependence on power is greater than linear, the reduction in LEV can be more significant by splitting the light near the coupler rather than near the region of interest.

[0178] In some cases, the inventors have discovered that within a distance of 1 mm to 3 mm from the coupler, the waveguide can advantageously be divided into two or more waveguides, for example, 2 to 512 waveguides. In some cases, within a distance of 3 mm from the coupler, the waveguide can advantageously be divided into two or more waveguides. In some cases, within a distance of 3 mm from the coupler, the waveguide can be divided into four or more waveguides. In some cases, within a distance of 3 mm from the coupler, the waveguide can be divided into 2 to 512 waveguides. In some cases, within a distance of 3 mm from the coupler, the waveguide can be divided into 4 to 64 waveguides.

[0179] The inventors have also discovered that separation can advantageously occur within a small fraction of the distance the light travels from the coupler to the region of interest. For example, in some cases, separation can occur within less than 30% of the distance the light travels from the coupler to the region of interest. In some cases, separation occurs within 20% of the distance the light travels from the coupler to the active area. In some cases, separation occurs within 10% of the distance the light travels from the coupler to the region of interest. In some cases, the coupled light is split into 2 to 512 waveguides within 20% of the distance the light travels from the coupler to the region of interest. In some cases, the coupled light is split into more than 2 waveguides within 20% of the distance the light travels from the coupler to the region of interest. In some cases, the coupled light is split into more than 4 waveguides within 20% of the distance the light travels from the coupler to the region of interest. In some cases, the coupled light is split into 2 to 512 waveguides within 20% of the distance the light travels from the coupler to the region of interest. In some cases, the coupled light is split into 4 to 64 waveguides within 20% of the distance the light travels from the coupler to the region of interest. Typically, for a given coupler, the most relevant distance is the longest distance the light travels through the branches. As used herein, when describing the longest distance of light from the coupler to the active region, this usually refers to the distance the light travels in the longest route branch from the coupler to the region of interest. This distinction is made because, in many cases, the distance the light travels differs in each of the different route branches. Generally, minimizing the LEV across all branches is preferred.

[0180] The distance by which a waveguide is split into multiple waveguides can be the distance between the last linear portion of the waveguide before one or more splitters and the first linear portion of the multiple waveguides after passing through one or more splitters. Alternatively, this distance can be determined by identifying the distance at which a correlated power reduction occurs (e.g., for one or more splitters that split light into four waveguides, the distance at which approximately one-quarter of the power is reduced, taking into account losses from one or more splitters).

[0181] One or more splitters used to separate a waveguide from a coupler into multiple routed waveguides can be of any suitable type. In some cases, a set or more sets of binary (1:2) splitters can be used to split a waveguide from a coupler into multiple waveguides. For example, two sets of binary splitters can be used to split light into four routed waveguides, one set of binary splitters splits light from an initial waveguide into two waveguides, and then a set of binary couplers is used for each of these two waveguides, thus forming four routed waveguides. In some cases, one or more higher-order splitters (1:3, 1:4, 1:5, 1:6, or even higher) can be used. Those skilled in the art will understand that combinations of splitters of the same or different orders can be used to split illumination light into multiple routed waveguides, as provided herein. As mentioned above, in some cases, the waveguide device has multiple couplers, each coupler optically couples to a single waveguide, and each single waveguide is divided into multiple routed waveguides, as described herein.

[0182] In many cases, branched waveguides further subdivide into numerous waveguides near the region of interest to monitor many response regions. For example, the number of waveguide branches within the region of interest can be hundreds or thousands. Alternatively, the number of waveguides in the region of interest can be approximately one hundred to approximately ten thousand. In some cases, each of these waveguides can illuminate one million to ten thousand response regions, such as nanoscale wells or ZMWs. For example, the illumination light can be divided into approximately 1,000 waveguides, each illuminating one,000 response regions, allowing for the simultaneous analysis of one million response regions.

[0183] The number of waveguides and the number of analysis regions per waveguide can be varied to achieve the desired level of multiplexing and performance. For example, one to 100,000 waveguides, 100 to 10,000 waveguides, or 500 to 5,000 waveguides can be used on the waveguide device. The number of analysis regions (e.g., nanowells) per waveguide can be, for example, one to 100,000 analysis regions, 100 to 10,000 analysis regions, or 500 to 5,000 analysis regions on the waveguide device. Those skilled in the art will understand how these values ​​are set to achieve the desired performance and multiplexing level.

[0184] In some cases, light is routed such that each waveguide in the region of interest is illuminated from both ends (i.e., illuminated by backpropagation from the first and second ends of the waveguide). As described elsewhere in this document, a light source with multiple longitudinal modes can be used to provide backpropagating illumination to the waveguides in the region of interest without significant loss due to optical interference. In some cases, it may be advantageous to route light into the waveguide from only one end (e.g., from the first end of the waveguide).

[0185] In some cases, the waveguide will be referred to as a waveguide branch herein. As those skilled in the art will understand, a waveguide branch, as used herein, is still a waveguide. For clarity, the waveguide will be referred to as a branch when discussing the separation of light. For example, in this document, references to illuminating a waveguide will include illuminating a waveguide branch.

[0186] Therefore, in some aspects, the present invention provides an optical detection system that reduces the attenuation of the optical emission signal emitted from the reaction region and ultimately reaching the detector. This allows for detection and signal differentiation based on a large number of emitted and detected signals, thereby improving the signal-to-noise ratio.

[0187] light source

[0188] The light source used in the system of this disclosure preferably has multiple longitudinal modes spaced at least 10 pm apart. The longitudinal modes are typically spaced from 10 pm to 500 pm apart, and in some cases from 10 pm to 100 pm apart. Each longitudinal mode preferably has an intensity of 20% or greater of the intensity of the strongest longitudinal mode. In some cases, the intensity of each longitudinal mode is 30%, 50%, or 70% of the intensity of the strongest longitudinal mode.

[0189] The illumination spectrum, wavelength range, or bandwidth of the shortest to longest longitudinal modes is typically between 10 pm and 20 nm. In some cases, the wavelength range is between 30 pm and 10 nm. In some cases, the wavelength range is between 30 pm and 5 nm. In some cases, the wavelength range is greater than 200 pm; in some cases, the wavelength range is greater than 1 nm; in some cases, the wavelength range is greater than 3 nm; in some cases, the wavelength range is greater than 5 nm; and in some cases, the wavelength range is greater than 10 nm. The inventors have found that a relatively wide range of wavelengths with appropriate performance can be used for the excitation of fluorescent materials. For example, even a longitudinal mode with a wavelength range of 50 nm can be used. However, the inventors have also found that if the wavelength range is too wide, there will be significant losses at the coupler on the target waveguide device. Although this loss will depend on the coupler design, the inventors have found that in some cases, wavelength ranges less than 20 nm, less than 10 nm, or less than 5 nm are preferred.

[0190] The number of longitudinal modes can be selected to optimize performance and cost. For example, there may be situations where a light source with two longitudinal modes spaced 10 pm apart provides sufficient improvement in both LEV and other objectives described herein. However, in some cases, a light source with eight longitudinal modes spaced 30 pm apart may be required to reduce LEV to the desired level. In some cases, a light source with dozens of longitudinal modes, all within the 3 nm wavelength range, may be the optimal solution. Therefore, in some embodiments, the number of longitudinal modes is at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 30, at least 50, or even more.

[0191] As described elsewhere in this document, the amount of LEV and other waveguide optical interference in a given system can depend on the waveguide material and size, as well as the wavelength and power of the incident light. Those skilled in the art will understand from the teachings provided herein how to implement a system with the desired analytical parameters.

[0192] In some cases, determining the number and spacing of longitudinal modes for a given light source is straightforward. In other cases, especially when the number of modes is relatively large, the illumination light can be characterized more simply by measuring the width of the illumination spectrum or the wavelength range between the longitudinal mode with the shortest wavelength and the longitudinal mode with the longest wavelength. In some cases, a large number of modes exist such that multiple modes are close to the peak at the resolution of the wavelength scan taken. In this case, the wavelength range can be determined by measuring the peak width, for example, by determining the full width at half maximum (FWHM).

[0193] In other embodiments, broadband spectra can be synthesized by rapidly scanning the laser wavelength within a specific spectral range at a specific frequency, rather than using a laser with a broadband linear continuous-wave (CW) spectrum comprising different stationary or quasi-stationary modes to suppress spatial coherence. As described above, such a time-modulation method for suppressing LEV has previously been disclosed in U.S. Patent Application Publication No. 2016 / 0216538. As provided therein, the modulation frequency can be greater than about 0.001 Hz in some cases, and between about 1 kHz and 0.01 Hz in others (see paragraph 0008). As further disclosed therein, wavelength modulation can be performed at the laser, or it can be performed using optical elements between the laser and the target waveguide (see paragraph 0053). As also noted therein, the modulation frequency can depend on the time required to form a back reflection in the waveguide (ibid.).

[0194] The above method can also be extended to reduce spatial coherence during excitation of the waveguide device by backpropagation from a single light source. Specifically, the coherence function is the Fourier transform of the laser spectrum, while the time-averaged coherence function is the Fourier transform of the time-averaged spectrum of the laser. By modulating the laser wavelength to broaden the time-averaged spectrum, this will at least partially suppress the time-averaged interference effect.

[0195] If the laser wavelength scanning frequency is 10 times greater than the chip sensor frame rate, time synchronization between the laser and the sensor is generally not a concern because the optical signal will be averaged over many wavelength periods within the sensor frame. In some cases, even if the laser scanning frequency is less than 10 times greater than the sensor frame rate, such as only 3 times greater, synchronization is acceptable. However, once the laser wavelength scanning frequency begins to approach the sensor frequency, it becomes desirable to synchronize the laser and the sensor to ensure that each sensor frame appears to be exactly within the same spectral range of the wavelength.

[0196] In contrast to gradually changing the wavelength, changing the laser wavelength in a continuous manner is advantageous compared to a discrete set of wavelengths because continuous changes help suppress repeating fringes in the coherence function. For example, U.S. Patent No. 7,668,216 (the entire contents of which are incorporated herein by reference) demonstrates that a reduction in the time-averaged coherence of laser radiation can be achieved by modulating the laser drive current using a modulation function optimized to obtain a predetermined time-averaged spectral profile (ideally Gaussian-shaped) of the laser radiation. In particular, U.S. Patent No. 7,668,216… Figure 3 A illustrates how Gaussian-shaped spectra improve fringe contrast (or coherence) and significantly reduce fringe visibility compared to trapezoidal spectra. As further described in the patent, the slow decay of fringe contrast with increasing optical path difference (OPD) stems from the sharp edges of the spectrum (λ). min and λ max (See, for example, U.S. Patent No. 7,668,216) Figure 2 (B) To obtain a rapidly decreasing coherence function, these sharp discontinuities in the spectrum should be avoided. The spectral profile with the fastest decrease in Fourier transform is a Gaussian profile. Therefore, when using such a light source in the present application, it is preferable to modulate the laser wavelength in a manner that results in a target spectral distribution with a time-averaged spectrum that approximates a Gaussian distribution.

[0197] In other embodiments of this disclosure, the light source used in this system is a superluminescent light-emitting diode (SLED or SLD), which is a superluminescent edge-emission semiconductor light source. This type of light source is also called a superluminescent diode or superluminescent LED. They combine the high power and brightness of laser diodes with the low coherence of conventional light-emitting diodes. A typical superluminescent light-emitting diode has an emission band approximately 5-100 nm wide. If such a light source is designed with a smooth Gaussian spectral distribution, its coherence function will decrease on a spatial scale of tens of micrometers, and there will be no repetitive visible edge lobes.

[0198] Therefore, both of the preferred methods described above rely on creating a time-averaged or CW-shaped Gaussian laser spectrum. The advantage of generating such a laser spectrum is that only one distinct coherence peak is formed at the zero OPD point, and the coherence function drops rapidly from this point. Thus, by introducing a small delay between the self-reverse propagating beams on the chip, the zero OPD point can be shifted outside the effective region of all waveguides on the chip, and the impact of laser coherence on detection performance can be minimized.

[0199] In another embodiment, a CW or time-averaged CW source can be used for LEV suppression. In this case, different light sources can be used to illuminate one or both sides (i.e., backpropagation) of the waveguide in the active region of the target device.

[0200] As described above, the integrated target device of this disclosure typically includes one or more single-mode waveguides. For some analyses, such as single-molecule sequencing, a single-mode waveguide may be preferred. When using a single-mode waveguide, it is preferable that the light source, in addition to having multiple longitudinal modes, has a large portion of its energy in a single lateral or spatial mode. In some cases, the illumination light from the light source has greater than 80% of its intensity in a single lateral mode; in some cases, the illumination light from the light source has greater than 50% of its intensity in a single lateral mode; in some cases, the illumination light from the light source has greater than 20% of its intensity in a single lateral mode; and in some cases, the illumination light from the light source has greater than 10% of its intensity in a single lateral mode. In some cases, the beam quality described by the M² factor of the illumination light from the light source is less than 1.5; in some cases, the M² factor of the illumination light from the light source is less than 1.3. The single lateral mode is typically a Gaussian mode or a fundamental mode. In some cases, the single lateral mode is a TEM. 00 model.

[0201] Typically, the illumination light from a light source is linearly polarized. For example, in some cases, linear polarization of the illumination light from a light source is useful in order to more effectively couple it into the TE mode in the waveguide of the target device.

[0202] It will be understood that the characteristics of the irradiating light can change along the path from the emanating source to the irradiated reaction region. For example, the irradiating light can be described as, after leaving the source, being emitted into free space, passing through optional free-space optics, traveling down a waveguide along the target device, and striking the reaction region. Although irradiation from all these locations is described here as irradiating light, it should be understood that the characteristics of the light can differ at each of these points. For clarity, the point most often referred to is the light leaving the source and entering free space. This provides a convenient point for measuring the characteristics of the irradiating light. This point is chosen for clarity and ease of measurement; generally, the number of longitudinal modes does not change significantly at various points along the optical path. An alternative point for determining the characteristics of the irradiating light is a point after the optional free-space optics and before the light enters the target device. As described elsewhere in this document, in some cases, there are optical components connected to the source before the light is emitted into free space. In some cases, the source will have associated optical elements, such as coupled optical fibers or lenses.

[0203] In some cases, free-space optical elements are placed between the light source and the target device. These free-space optical elements can be used to modify, tune, focus, or shape the beam, for example, to provide light to the target device for more efficient coupling or more efficient transmission via waveguides. Free-space optical elements can be any suitable element or group of elements, including, for example, one or more lenses, polarizers, wedges, filters, prisms, mirrors, or gratings.

[0204] As those skilled in the art will understand, the light source used in this system can be any suitable light source. In particular, the light source possesses the properties described above regarding the minimization of LEV in the target waveguide device. In a preferred embodiment, the light source is a laser source. Particularly useful for the analysis system of this disclosure are lasers emitting in the visible wavelength range, for example, lasers emitting between 450 nm and 700 nm or between 500 nm and 650 nm. In other preferred embodiments, the light source is a superluminescent diode.

[0205] Any suitable type of laser can be used in this system. In some cases, solid-state lasers, such as III-V semiconductor lasers, are used. Recently, progress has been made in the production of solid-state lasers that emit the desired wavelength range. A particularly useful laser is the GalnN solid-state laser. For example, lasers suitable for the systems disclosed herein are described in Sizov et al., “Gallium Indium Nitride-Based Green Lasers,” Lightwave Technol., 30, 679-699 (Mar. 1, 2012); Nakamura et al., “Current Status and Future Prospects of InGaN-Based LaserDiodes,” JSAP Int. No. 1, January, 2000; Jeong et al., Nature, Scientific Reports, “Indium gallium nitride-based ultraviolet, blue, and green lightemitting diodes functionalized with shallow periodic hole patterns,” DOI:10.1038; and Tagaki et al., “High-Power and High-Efficiency True Green LaserDiodes,” SEI Tech Rev, No. 77, October 2013; the entire contents of these patents are incorporated herein by reference for all purposes.

[0206] Combining multiple light sources

[0207] Although in many cases it is most desirable for the illumination light of the present invention to be provided with a single light source, such as a single laser, in some cases it is desirable to use two or more light sources to provide multiple longitudinal modes, which have been found to provide significant advantages for optical analysis of the target waveguide device of the present invention.

[0208] The combined output of the illumination light into free space is the same as described elsewhere herein; however, in this embodiment, the outputs of two or more lasers are combined to generate multiple longitudinal modes released into free space and then emitted into a waveguide on the target device. Those skilled in the art will know how to use combined optical elements to generate multiple longitudinal modes of the desired illumination light.

[0209] Analytical Instruments and Systems

[0210] On the other hand, this disclosure provides analytical instruments for performing the disclosed methods and for use with the analytical apparatus described herein. For example, in some cases, the analytical apparatus of the present invention is part of an analytical instrument or analytical system. In some cases, the analytical apparatus may be removably coupled to the instrument or system. Reagents may be contacted with the analytical apparatus after and / or before coupling the analytical apparatus to the instrument or system. The system or instrument may provide electrical signals and / or illumination to the analytical apparatus and may receive electrical signals from detectors in the analytical apparatus. The instrument or system typically has a computer to operate, store, and analyze data from the apparatus. For example, for the purpose of nucleic acid sequencing, the instrument may have the ability to identify the sequence of added nucleotide analogues. For example, this identification may be performed as described in U.S. Patent Nos. 8,182,993 and U.S. Patent Application Publications Nos. 2010 / 0169026 and 2011 / 0183320, the entire contents of which are incorporated herein by reference.

[0211] For example, this disclosure provides an analytical instrument comprising: any suitable target device as described herein; a light source for providing illumination light to one or more waveguides of the target device; an electronic system for providing voltage and current to a detector and for receiving signals from the detector; and a computer system for analyzing the signals from the detector to monitor the analytical reaction, such as obtaining sequence information about the template nucleic acid.

[0212] As used herein, the terms sensor and detector are used interchangeably. In some cases, a sensor or detector may comprise a single pixel. In other cases, a sensor or detector may comprise multiple pixels, such as a pixel array. Those skilled in the art will understand the meaning of these terms in the context in which they are used.

[0213] Planar optical wave circuit (PLC)

[0214] In some aspects, the systems disclosed herein include optical transmission devices constructed with multiple integrated transmission waveguides for transmitting optical energy from one or more optical inputs to one or more optical outputs. Such integrated devices are sometimes referred to as planar optical wave circuits (PLCs). PLCs are most commonly used in telecommunications equipment for coupling and / or decoupling optical excitation signals from fiber cores for purposes such as multiplexing / demultiplexing, optical branching, and / or optical switching. These more specific PLCs are sometimes also referred to as fiber optic spacer concentrators (FSCs). PLCs typically comprise multiple components directly fabricated into the integrated device, with different components providing the desired functionality. For example, a PLC may include one or more waveguides, one or more splitters, one or more taps, one or more photodetectors, and one or more optical fibers, ideally polarization-maintaining fibers. Optical fibers and PLCs used in telecommunications applications typically transmit light in the infrared range, most commonly at wavelengths of about 1310 nm or about 1550 nm. In some cases, low-cost electronics, such as light-emitting diodes (LEDs) and vertical cavity surface-emitting lasers (VCSELs), may be used in devices that include PLCs (e.g., FSCs).

[0215] However, compared to PLCs used for transmitting optical communication signals, the optical devices of the present invention are generally designed for efficient transmission of shorter wavelengths of light. In particular, for example in DNA sequencing reactions using fluorescently labeled DNA reagents, optical irradiation for fluorescence analysis is typically in the visible light range, most typically in the range of about 450 nm to about 700 nm. Therefore, the waveguides and other components of the optical devices disclosed herein are preferably designed and proportionately determined and incorporated into the present system for DNA sequencing to efficiently transmit light energy in the visible light range. In some embodiments, the wavelength is in the range of about 450 nm to about 700 nm. In more specific embodiments, the wavelength is in the range of about 500 nm to 650 nm or even about 500 nm to about 600 nm. In some specific embodiments, the wavelength is, for example, about 520 nm to about 560 nm. In other specific embodiments, the wavelength is about 620 nm to about 660 nm, for example, about 635 nm or 650 nm. In some embodiments, combinations of visible light wavelengths can be transmitted within the device.

[0216] In this disclosure, a PLC is typically optically coupled to a single light source via fiber optics. The PLC is used to split the light emitted from the light source into multiple beams, all of which are directed to a target device including waveguides. As described above, this disclosure includes using a laser or other light source to provide light with specific characteristics, including light having multiple longitudinal modes. For the purposes of this application, the characteristics of light are described at the point where light enters free space. Therefore, in the case where the PLC is coupled to the light source via fiber optics, when the light leaves the PLC, it is considered that the light has reached free space.

[0217] PLC devices may include various functional components embedded within optical circuitry. For example, the device may include an optical splitter, allowing a single optical input to be split into multiple optical outputs as light energy passes through it. In the simplest case, for example... Figure 6 As shown in the PLC, the optical power emitted from each optical output of the device is the same or nearly the same. In this example, the optical device 600 may include various integrated waveguides optically coupled to each other to allow light transmission through the device. Specifically, the device may include an optical input 602, which is optically coupled to a 1x4 splitting element 608 via an optical input waveguide 604. The splitting element 608 splits the delivered input light energy into four optical outputs 620-623 of equal power intensity, which are optically coupled to the splitting element via four optical output waveguides 610-613. It should be understood that the same result can be achieved by combining a first 1x2 splitter and then further splitting the resulting optical outputs by two additional 1x2 splitters. For example, a PLC element suitable for this system is described in U.S. Patent Application Publication No. 2016 / 0273034, which is incorporated herein by reference for all purposes. Target waveguide device and system with a single light source.

[0218] On the other hand, this disclosure provides a system comprising a target waveguide device with a single input source that can transmit light from a light source to both ends of an optical waveguide (i.e., via backpropagation). As described above, backpropagation illumination is advantageous when using a single waveguide to provide illumination to multiple regions on a target device. For example, as light travels through the waveguide, propagation losses are inevitable, and therefore, regions facing opposite ends of the waveguide will receive less illumination than regions at the beginning of the waveguide. For optical analysis devices, a consistent amount of illumination is generally desired at each region, so this reduction in intensity is undesirable. Backpropagation illumination can mitigate this problem. While there is still a loss in illumination intensity of light entering from each end of the waveguide, the combination of intensities from two opposing beams tends to offset this effect and provide more consistent illumination across the entire analysis region. To minimize propagation losses within each waveguide, the need for backpropagation could be avoided by using shorter waveguides, but using shorter waveguides would require an increased total number of waveguides, thus reducing the efficiency of the device geometry (e.g., short waveguides would require a long and narrow device design). Therefore, using shorter waveguides limits the size of the active region of the device.

[0219] While backpropagation might be desirable for illumination uniformity, generating a backpropagating beam using a single longitudinal mode laser (e.g., by splitting a single input beam and directing the split beam to opposite ends of a waveguide on a target device) can present problems. Specifically, laser beams entering from opposite ends of the waveguide can cause standing waves to form, resulting in temporal and spatial intensity fluctuations along the length of the waveguide, especially over long coherence lengths. See, for example... Figure 7 (Adapted from https: / / en.wikipedia.org / wiki / Standing_wave), this figure illustrates the combination of two opposing waves to form a standing wave. For example, backpropagating beams originating from the same laser and emitted from two opposite ends into a waveguide can result in undesirable standing waves, leading to non-uniform illumination of the sample. Because the coherence length of single-mode lasers is typically very long (e.g., >10 m), it is not easy to introduce delay lines between the two beams to disrupt coherence, especially if separation is performed on the target device itself.

[0220] One approach to addressing this interference problem is to use two independent single-mode light sources, each with a different wavelength, to minimize or eliminate the interference. When using two light sources, one source illuminates one end of a waveguide or group of waveguides on the device, while the other source illuminates the other end of that waveguide or group of waveguides. Figure 8A An example of the construction of such a target waveguide device is shown, where the input optical coupler is located at the lower left corner of the device. In this example, a 532 nm input beam is directed around the left side of the device to enter the sample waveguide from the “top” end of the device, while a 530 nm input beam is directed to enter the same sample waveguide from the “bottom” end of the device. In this case, the standing wave will only persist for an approximate time scale as follows. The wavelength is very short compared to the fluorescence lifetime, so it has no impact on the detection system. Although using two single-mode light sources with different wavelengths can mitigate or resolve interference caused by backpropagation illumination, using two light sources is expensive and increases the complexity of the illumination system.

[0221] The inventors have discovered and demonstrated that by using a single broadband multimode light source to transmit light to a target waveguide device, one or more waveguides on the device can be illuminated in a backpropagation manner, which significantly reduces interference levels compared to using a light source with a single longitudinal mode. Figure 8BAn example of the construction of such a target device is shown, where the input optical coupler is located at the lower left corner of the device. In this example, a single 522 nm multi-mode laser provides optical input at the two input couplers shown. The two beams are oriented around the device such that they enter the same sample waveguide from opposite ends, thus providing back-propagating input light. Alternatively, a single coupler on the target device can receive input light energy from a single light source, and separate optical inputs can be created at both ends of the sample waveguide by splitting the light on the target device itself.

[0222] Therefore, one aspect of this disclosure relates to the illumination of a target waveguide device, wherein the waveguide is illuminated by splitting light from a single light source, such that the waveguide is illuminated from both ends. In some embodiments, the single light source emits the illuminating light into free space before coupling to the target device. In some embodiments, the emitted illuminating light comprises a plurality of longitudinal modes spaced at least 10 pm. In some embodiments, the intensity of each longitudinal mode is 20% or more of the intensity of the strongest longitudinal mode, wherein the wavelength range from the shortest to the longest longitudinal mode is between 10 pm and 20 nm. Characteristics of a broadband light source for backpropagation are provided throughout this application. Typically, the target device has multiple waveguides, and a single light source is used to illuminate each waveguide from each end of the waveguide.

[0223] In some cases, a single coupler on the target device is used to collect light from a single illumination source. The light from the source is routed on the device to both ends of a single waveguide or a group of waveguides. In some embodiments, the target waveguide device includes a plurality of optical waveguides, each optical waveguide including a first end and a second end, wherein the first end and the second end of each optical waveguide are configured to receive an optically excited beam from the source. More specifically, the target waveguide device may include at least 2, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 50000, or even more optical waveguides. Alternatively or additionally, the target waveguide device may include no more than 100,000, no more than 50,000, no more than 10,000, no more than 5,000, no more than 1,000, no more than 500, no more than 100, or even fewer optical waveguides. In a particular embodiment, the group of waveguides may be, for example, 1 to 100,000 waveguides, 100 to 10,000 waveguides, or 500 to 5,000 waveguides.

[0224] The ability to provide this backpropagating light using a single coupler is advantageous because it significantly reduces cost and the complexity of the illumination system, especially compared to systems that must simultaneously direct multiple beams to different couplers on the target device. As will be understood by those skilled in the art, on-device light routing can be achieved by including one or more embedded optical splitters within the device between the input coupler and the waveguide or a set of waveguides (which provide illumination to the reaction region). Optical alignment with the target device.

[0225] On the other hand, this disclosure provides systems and methods for using signals from the target optical device itself to perform source-to-device alignment, and in some cases, alignment with a single input coupler. Typically, the alignment of an illumination beam from a laser or other light source (where the light source is often part of an analytical instrument, such as a sequencing instrument) with an optical target device (which is typically detachably held in place within the instrument) makes it difficult to precisely direct the beam onto the coupler on the target device. On the one hand, the components within the instrument responsible for holding the movable target device in place may have geometric uncertainties; on the other hand, the position of the coupler on the target device may also be uncertain, for example, the edge of the chip may be + / - 300 μm or larger in the x and y directions, considering defects in the cell packaging process. To maximize coupling efficiency, the beam from the light source may also need to be guided at an angle to the coupler on the target device, and the optimal angle may vary depending on the target device. Additionally, the size of the coupler on the target device may be relatively small (e.g., 50 μm x 50 μm), and it may be difficult to remove and replace the target device within the tolerance required for beam-to-coupler alignment each time the target device is removed and reinserted. An exemplary method of such alignment is provided in U.S. Patent Application Publication No. 2016 / 0273034, which is incorporated herein by reference for all purposes. This prior method typically requires the inclusion of an alignment camera in the analysis system. See, for example... Figure 9 This illustrates an optical analysis system that includes an alignment camera. The requirement for an analysis system that includes an alignment camera increases the system's complexity and cost.

[0226] To address this problem, this disclosure also provides novel systems, apparatus, and processes for performing and optimizing alignment between a light source (particularly a laser source) and a target device (particularly a waveguide device including at least one input optical coupler, such as a DNA sequencing chip). The alignment process can include coarse alignment and fine alignment. Specifically, alignment can be performed by using signal feedback directly from sensors included in the target device (e.g., analyzing the sequencing chip) and adjusting the position of the light source relative to the target device to maximize that signal. For example, a raster scan can be performed on the region where the input coupler is intended to be located using a laser beam, and signals from one or more sensors optically coupled to an optical waveguide associated with the coupler can be monitored. Because alignment is typically performed over a short time (compared to the duration of sequencing analysis on a sequencing chip, for example) and at low power, relative optical damage to the sample in the reaction region is minimal upon completion of alignment.

[0227] In the alignment process according to some embodiments, in response to input light, a fluorescence signal is generated by one or more reaction regions (typically including at least one fluorescent reagent). In the simplest and most efficient embodiment, the same optical path and data path are used for both the alignment process and subsequent analytical measurements, where an "optical alignment signal" is obtained during alignment and an "optical emission signal" is obtained during analytical measurements. Furthermore, in some embodiments, signals from several sensor regions (e.g., from several pixels on a detector) can be combined, for example, using embedded software, to improve the signal-to-noise ratio of the feedback signal. However, it should also be understood that conventional data analysis workflows can be slow because they require processing signals from all sensor regions. Therefore, in alternative embodiments, signals from several reactions in the sensor board can be combined (e.g., by reprogramming the device using a field-programmable gate array (FPGA), and the resulting signal is fed into a computer to control alignment movement between the light source and the target device via a separate data path. This method is much faster than others, thus achieving faster alignment.

[0228] In another embodiment, a fluorophore or another reagent capable of generating an optical emission signal whose intensity varies with the input light energy can be deposited in a virtual region used solely for alignment purposes. The subsequent fine alignment step, which optimizes the position between the light source and the target device, should be easier to perform than a coarse alignment step because, after the coarse alignment step is completed, the signal emitted from the actual reaction region can be monitored and, as those skilled in the art will understand, optimized using methods such as the Nelder-Mead method to maximize that signal.

[0229] In other embodiments, specific features can be placed on the target device to further simplify the coarse alignment step. For example, the target device may have a small auxiliary alignment sensor, 2D, ID, or two orthogonally placed IDs, positioned near the input coupler at a distance precisely defined by photolithography steps during chip fabrication. The size of the auxiliary alignment sensor should be larger than the positioning uncertainty during packaging to ensure that the laser beam is recorded on the sensor immediately after the cell is placed in the instrument. Among these considerations, the geometric tolerance of the components responsible for the target device within the instrument is also important. For example, if the packaging uncertainty of the auxiliary alignment sensor on the target device is + / - 300 micrometers, the sensor size should preferably be larger than 600 micrometers. Because the auxiliary alignment sensor is positioned very precisely on the target device relative to the input coupler, once the input beam is recorded on the auxiliary alignment sensor, it is immediately known how much the beam and / or the target device needs to be moved to align the input beam with the input coupler. Submicron positioning accuracy is known within the art using photolithography processes. If a degree of rotational and cross-coupling between the x and y axes of the target device is expected, the beam can be scanned on the sensor first to determine the required rotation angle. The sensor can be part of a sensor responsible for detecting signals in the reaction region, or preferably a separate sensor, as the sensor may optionally include an intermediate filter layer to attenuate the laser beam to a level that does not cause saturation of the sensor output.

[0230] In another variation, during cell fabrication, a machine vision camera can be used to calibrate the positions of different alignment features on the chip (e.g., auxiliary alignment sensors, auxiliary alignment couplers, etc.), and these positions can be stored in an E2PROM (Electrically Erasable Programmable Read-Only Memory) placed on the cell or cell package. Specifically, the position of the input coupler relative to a known reference or the distance between the alignment feature and the input coupler can be precisely measured. If the offset associated with the cell's positioning in the instrument is also precisely calibrated, this information can significantly accelerate alignment time.

[0231] In another variation for aligning the light source and target device, instead of a sensor array with small pixels, a quad-cell structure, such as a square configuration, can be used, consisting of four larger detectors assembled together. In some embodiments, two detectors can be used to determine the linear position of the beam along one axis, and two detectors can be used along another axis. The beam is initially located in the middle of the quad-cell and then translated to the input coupler using a known separation distance, as described above.

[0232] In another embodiment of the alignment system and process, one or more auxiliary alignment couplers can be placed at a lithographically defined distance from the input coupler, which is optically connected to the sequencing region, and these auxiliary couplers can be used to assist coarse alignment. For example, one to six, one to four, one to three, or one to two auxiliary couplers can be arranged on the target device to provide coarse alignment. In one example, four auxiliary input couplers can be constructed in a rectangular, e.g., square configuration, packaged close to each other, with one of the four couplers being the actual input coupler. One advantage of having multiple input couplers is that the likelihood of the laser encountering one of the input couplers during alignment scanning is increased. For example, the auxiliary couplers are preferably placed sufficiently far from the surface of the optical device such that the input beam scanning the surface of the device is guaranteed to encounter at least one of the auxiliary couplers during the coarse alignment process, given the manufacturing tolerances of the optical device. Light entering the device from the auxiliary couplers can be routed to the active region sensor or to a sensor dedicated to the alignment process.

[0233] In additional or alternative methods for target device alignment, the system for coarse alignment may include an extension lens, including a cylindrical lens, within the laser beam path to widen the laser beam point on the coupler. Alternatively or additionally, the beam can be widened by inserting a diffractive optical element (DOE) into the beam path, thereby also reducing the time required for coarse alignment. After achieving coarse alignment using the widened beam, a fine alignment step is performed, for example, by maximizing the intensity of the sequencing signal passing through the sample input coupler. In a broad application of this approach, coarse alignment can be achieved using the extended beam via the sample input coupler without the need for an auxiliary input coupler on the target device. In more specific applications, coarse alignment can be achieved using the extended beam coupled with one or more auxiliary couplers. For example, fine alignment is typically achieved using the sample input coupler after removing the extension lens from the path of the light source. Ideally, the beam extension should be greater than the uncertainty in the coupler position to ensure that the extended beam encounters the input or auxiliary input coupler at that size. However, where laser power output is limited, it is possible that the laser power density is too low after beam extension, and it may become more difficult to detect the alignment signal. In this case, it is advantageous to extend the beam with a smaller factor to maintain the optical power density. Even a small increase in beam size will help with alignment, as the area for coarse searching will be reduced accordingly.

[0234] After coarse alignment, the additional lens is typically removed from the optical path for fine alignment. In some cases, if the lens is not perfectly orthogonal to the beam, removing the lens from the optical path can cause misalignment between the beam and the input coupler. This displacement caused by the lens can be calibrated during instrument manufacturing, and the beam position can be corrected after removing the additional lens.

[0235] In another embodiment, instead of placing auxiliary sensors such as quad-unit sensors or photodiodes on the target device to assist coarse alignment, auxiliary features, such as diffraction features, can be placed on the target device at a known, lithographically defined distance from the input coupler to assist the coarse alignment process. Alternatively, 2D or 1D sensors or photodiodes can be placed near the target device. The sensor position can be configured such that if a scanning laser is applied to the surface of the target device and hits a diffraction feature, the light will be deflected towards the sensor. This method helps to quickly locate the input coupler, thereby accelerating the coarse alignment search.

[0236] While the alignment process can be performed by holding the target device in a fixed position and scanning the output from the light source across the surface of the fixed device, it should be understood that alignment can optionally be achieved by holding the optical device in a fixed position and moving the target device relative to the output of the light source. Typically, in this system, the target waveguide device or the light source is movable relative to the other in at least two dimensions, and alignment is achieved by configuring the system to monitor the optical alignment signal and moving the target waveguide device or the light source relative to the other to increase the optical alignment signal. In a particular embodiment, the system does not include an alignment camera. In other particular embodiments, the system is configured to move the target waveguide device relative to the light source until the optical alignment signal increases to a maximum level.

[0237] In some embodiments, at least one detector region in the target waveguide device is configured to detect both optical alignment signals and optical emission signals. In some embodiments, the target waveguide device further includes an auxiliary alignment sensor, which is located on the surface of the target waveguide device at a distance from at least one optical coupler, a distance precisely defined using photolithography during the fabrication of the target waveguide device. In some embodiments, the waveguide of the target waveguide device is configured to transmit backpropagating optical signals, for example, through a single light source such as a broadband, multimode light source, as described in detail above. The target waveguide device used in the alignment systems and methods just described may alternatively or additionally include any of the target waveguide device features described above, but is not limited thereto.

[0238] In some alignment methods, the beam is configured or scanned in space to project at least a coarse illumination spot size and a fine illumination spot size, wherein the coarse illumination spot size is larger than the fine illumination spot size. More specifically, in these embodiments, the beam may be configured to project the coarse illumination spot size onto the target waveguide device while the target waveguide device or the beam moves relative to each other to increase the optical alignment signal.

[0239] Improved optical system with multiple translation lenses

[0240] On the other hand, this disclosure provides an improved optical system for delivering light energy to a target device in a controllable manner. Specifically, such an optical system includes at least two translation lenses located between the light source and the target device to control the transmission of light from a laser or other light source through the beam in multiple directions.

[0241] exist Figures 10A to 10D An exemplary schematic design of a single-beam optical system with two translational lenses (also known as "light pens") is shown. This design is significantly simpler and less costly compared to systems that include light sources designed to simultaneously illuminate multiple input couplers on a chip. See, for example, the "light brush" and other optical delivery devices described in U.S. Patent Application Publications Nos. 2016 / 0273034A1 and 2016 / 0363728A1. Figures 10A to 10D In the illustrated optical transmission system, the transmission of light from a light source, such as an optical fiber, is controlled by at least two translation lenses located between the light source (at the top of each figure) and a movable target waveguide device (at the bottom of each figure), with the beam shown in each figure as passing through the lenses. In the illustrated design, the light from the light source is first collimated by a translational collimating lens (1001), which is typically directly connected to the light source, allowing the collimating lens and the light source (e.g., an optical fiber) to move in series. The collimated light is then focused onto the input coupler of the target device (1003) by a translational objective lens (1002). It should be understood in this design that the target device preferably remains stationary during lens translation. Existing designs of the target waveguide device (also referred to as a "chip") primarily require adjustment of the input beam along four axes: x, y, pitch, and yaw (i.e., a first direction, a second direction, a first angle, and a second angle), such as... Figure 10A As shown. (Note that the yaw angle lies in an orthogonal plane relative to the page, therefore it is not shown in the figure. Yaw error is usually likely to be small, but in this case, control along the yaw axis may not be necessary.) By moving the collimating lens and its associated light source (such as...) relative to the objective lens... Figure 10A and Figure 10B As indicated by the arrows in the diagram, the incident (pitch or yaw) angle changes, but the spatial (x, y) position of the target device remains unchanged. Figure 10A and Figure 10B The dashed lines in the diagram are intended to indicate that the objective lens remains fixed in these examples. This is achieved by moving the light source, collimator, and objective lens together (e.g., ...). Figure 10C and 10D(As indicated by the arrows in the diagram), the beam can be translated along the x or y spatial direction as needed. Alternatively, if the movement along the x and y directions is relatively small, and if the coupling to the target device is not very sensitive to small changes in the angle of incidence, the beam can be translated along the x or y direction by moving only the objective lens. For example, if the focal length of the objective lens is 100 mm, and the required x or y movement is at most about + / - 300 μm, the cross-coupling between the angular and horizontal movements will result in an angle of incidence change of less than + / - 0.2 degrees.

[0242] It should be understood that the systems for optical analysis just described are generally applicable to coupling optical systems from various light sources to various target devices, wherein at least two translation lenses are used to control the transmission of optical signals from the light sources to the target device. In particular, these methods are usefully used in systems having target devices having at least one optical coupler configured to receive an optical excitation beam from a light source traveling through free space. In some embodiments, the target waveguide device advantageously includes a plurality of optical waveguides configured to receive an optical excitation beam from at least one optical coupler. For example, the target waveguide device may include at least 2, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10000, or at least 50000 optical waveguides. In some embodiments, the target waveguide device may include no more than 100,000, no more than 50,000, no more than 10,000, no more than 5,000, no more than 1,000, no more than 500, or no more than 100 optical waveguides. In other embodiments, the target waveguide device may include 1 to 100,000, 100 to 10,000, or 500 to 5,000 optical waveguides.

[0243] In some embodiments, the target waveguide device of the disclosed system includes at least one optical splitter, wherein the at least one optical splitter includes an optical input and a plurality of optical outputs, and wherein the optical input of the at least one optical splitter is configured to receive an optical excitation beam from an optical coupler. Such a device typically also includes a plurality of optical waveguides configured to receive optical excitation beams from the plurality of optical outputs of the at least one optical splitter.

[0244] In a particular embodiment, the target waveguide device of this system includes no more than one optical coupler for providing illumination light to the reaction region. In other particular embodiments, at least one optical splitter includes 2 to 512 optical outputs.

[0245] The system includes at least two translation lenses located between the light source and the target device to control the propagation of light from a laser or other light source in multiple directions. The system can also be advantageously combined with any other of the above-described systems and methods, including more specific embodiments of those systems and methods. For example, in some embodiments, the light source of this system emits a broadband multimodal optical excitation beam, such as including at least 3, 5, 10, 15, 20, 30, or 50 longitudinal modes. In some embodiments, the target waveguide device of this system is any of the target waveguide devices described above, including those configured to receive excitation light provided by a counter-propagating beam.

[0246] In this system, lens control is further improved by using eccentric cams to facilitate precise movement of each lens in every dimension. For example, Figure 11A Two views of a suitable cam are shown, in which the axis of rotation is offset from the cam's center of mass. Figure 11B The diagram illustrates the displacement (mm) of this eccentric cam as a function of the cam angle (degrees) when the cam rotates about its axis. The sinusoidal displacement curve clearly shows that this device provides highly controllable coarse and fine motion as needed. As those skilled in the art will understand, this eccentric cam can be driven by any suitable motor device. Preferably, an inexpensive stepper motor (e.g., a stepper motor with an integrated planetary gearbox) is used to drive this eccentric cam.

[0247] For applications where lenses should be controlled along three degrees of freedom (e.g., along-track (AT) spatial control, across-track (CT) spatial control, and pitch and rotation control), eccentric cam control can provide the following exemplary specifications:

[0248]

[0249]

[0250] However, it should be understood that, as those skilled in the art will appreciate, a wide variety of other specified values ​​can be achieved by changing the nature of the stepper motor and the size and eccentricity of the cam.

[0251] Figure 11C This demonstrates how an eccentric cam control mechanism can be mechanically coupled to a lens (e.g., a collimating lens) to provide desired fine motion control over the lens on a single dimension. Figure 11COther optional features and components of this control mechanism are also shown, such as a “linear guide” mechanism for maintaining and controlling the movement of the lens in a single direction; and a “spring” mechanism for providing tension between the lens holder and the eccentric cam when the cam is rotated by the stepper motor. As shown in the figure, a roller mechanism attached to the lens holder can also be used to provide a smooth connection between the eccentric cam and the lens holder. As those skilled in the art will understand, the “stepper motor” and “gearbox” are selected to provide the desired functionality. For example, a stepper motor with an integrated planetary gearbox can be selected to provide a 100:1 gear reduction, a step size of 0.018°, a maximum torque of 4 Nm, and non-reverse drive; however, any suitable stepper motor and gearbox can be selected and adapted for use in the aforementioned drive system, provided the specifications are suitable for the required application.

[0252] Figure 11D The use of two fixed stepper motors and their associated eccentric cams to drive the lens mount along two orthogonal axes is shown, thereby enabling control of the objective lens in two spatial dimensions (AT and CT). Figure 11E and Figure 11F Two different views of an assembled optical alignment system are provided, which controls the objective lens along two orthogonal directions (X and Y) and also controls the pitch angle of the collimating lens. Therefore, in this system, fine motion control is provided for the lens-holding optical stage along three separate axes (e.g., AT, CT, and pitch). It should be understood that in such a system, motion control along additional axes (e.g., yaw, roll, and out-of-plane space control) can be easily achieved by adding appropriate motors / cams / linear or rotary guide mechanisms for each additional axis of motion. Therefore, in some embodiments of this system, motion is controlled along four separate axes (e.g., AT, CT, pitch, and yaw). In other embodiments, motion is controlled along five separate axes (e.g., AT, CT, pitch, yaw, and roll) or even six separate axes to provide the desired relative positioning and alignment between the light source and the target waveguide device.

[0253] It should be understood that the above design offers significant advantages over conventional designs for controlling lens translation in two dimensions. While in conventional designs the motor controlling the lens movement along the first directional axis (e.g., the AT axis) can be fixed, the motor and guiding mechanism controlling the lens movement along the second directional axis (e.g., the CT axis) are typically cantilevered AT guiding mechanisms. Therefore, the weight of the motor and related mechanical components significantly increases the cost of the AT components, which must be overcome by addressing the inertia of the entire CT drive system.

[0254] As in Figure 11FAs is particularly evident in this design, all motors are preferably mounted on the system housing or other rigid components, allowing the lens to move along each axis without affecting its position or movement along any other axis. Because the stepper motors are fixed, the payload moved by the motors is very light, resulting in a significant performance improvement compared to previous optical control systems. Figure 11E and Figure 11F The system does not show a spring for preloading each cam follower, or a mechanism for moving the collimating lens along the "yaw" axis.

[0255] Other embodiments

[0256] In another respect, this disclosure provides alternative embodiments, as described in the following numbered paragraphs.

[0257] 1. A system for improved fluorescence analysis on a chip, the chip including an illumination waveguide, the system comprising:

[0258] A laser that emits illumination light into free space, the emitted illumination light comprising multiple longitudinal modes spaced at least 10 pm apart, each longitudinal mode having an intensity of 20% or more of the intensity of the strongest longitudinal mode, wherein the wavelength range of the shortest to longest longitudinal mode is between 10 pm and 20 nm; and

[0259] A chip that receives the illumination light from free space, the chip comprising:

[0260] Multiple reaction zones;

[0261] At least one waveguide that provides illumination light to multiple reaction regions, said reaction regions comprising a fluorescent material that absorbs the illumination light and emits fluorescence; and

[0262] A coupler that couples the illumination light to the at least one waveguide, and

[0263] A detector that detects fluorescence emitted from the reaction region.

[0264] 2. According to the system in paragraph 1, wherein the plurality of longitudinal patterns includes at least two longitudinal patterns spaced at least 10 pm apart, wherein the intensity of each pattern is 50% or more of the intensity of the strongest pattern.

[0265] 3. According to the system in paragraph 1, wherein the plurality of longitudinal patterns comprises at least four longitudinal patterns spaced at least 10 pm apart, wherein the intensity of each pattern is 50% or more of the intensity of the strongest pattern.

[0266] 4. According to the system in paragraph 1, wherein the plurality of longitudinal patterns comprises at least eight longitudinal patterns spaced at least 30 pm apart, wherein the intensity of each pattern is 50% or more of the intensity of the strongest pattern.

[0267] 5. According to the system in paragraph 1, the multiple longitudinal patterns include 2 to 100 longitudinal patterns, wherein the intensity of each pattern is 50% or more of the intensity of the strongest pattern.

[0268] 6. According to the system in paragraph 1, the wavelength range from the shortest longitudinal mode to the longest longitudinal mode is greater than 200 pm.

[0269] 7. According to the system in paragraph 1, the wavelength range from the shortest longitudinal mode to the longest longitudinal mode is greater than 1 nm.

[0270] 8. According to the system in paragraph 1, the wavelength range from the shortest longitudinal mode to the longest longitudinal mode is greater than 3 nm.

[0271] 9. According to the system in paragraph 1, the wavelength range from the shortest longitudinal mode to the longest longitudinal mode is between 200 pm and 10 nm.

[0272] 10. The system according to paragraph 1 further includes an optical element, wherein the optical element modulates the illumination light between the laser and the chip.

[0273] 11. According to the system in paragraph 1, the illumination light from the laser has more than 80% of its intensity in a single transverse mode.

[0274] 12. According to the system in paragraph 1, the illumination light from the laser has more than 50% of its intensity in a single transverse mode.

[0275] 13. According to the system in paragraph 1, the illumination light from the laser has greater than 10% of its intensity in a single transverse mode.

[0276] 14. According to the system in paragraph 11, the single horizontal mode is the Gaussian mode.

[0277] 15. The system according to paragraph 1 also includes a computer that receives and analyzes electronic signals from the detector.

[0278] 16. According to the system in paragraph 1, the analysis includes obtaining nucleic acid sequencing information.

[0279] 17. According to the system in paragraph 1, the wavelength of the shortest longitudinal mode is from about 450 nm to about 700 nm.

[0280] 18. According to the system in paragraph 1, the wavelength of the shortest longitudinal mode is about 500 nm to about 650 nm.

[0281] 19. According to the system in paragraph 1, the waveguide includes a SiON core.

[0282] 20. According to the system in paragraph 18, the SiON core has a refractive index greater than about 1.6.

[0283] 21. According to the system in paragraph 1, the waveguide includes a core surrounded by silicon dioxide.

[0284] 22. According to the system in paragraph 1, the dimensions of at least one waveguide on the chip are determined to carry a single lateral mode.

[0285] 23. According to the system in paragraph 1, the chip includes a silicon chip.

[0286] 24. According to the system in paragraph 1, the detector includes a CMOS sensor.

[0287] 25. According to the system in paragraph 1, the laser is fiber-coupled, and the light emitted from the laser into free space is emitted from the fiber.

[0288] 26. According to the system in paragraph 1, the laser is coupled to the PLC via an optical fiber, and light emitted from the laser into free space is emitted from the PLC.

[0289] 27. The system according to paragraph 1 also includes one or more optical elements arranged between the laser and the chip.

[0290] 28. The system according to paragraph 27, wherein one or more optical elements include one or more lenses, polarizers, wedge prisms, filters, prisms, mirrors, or gratings.

[0291] 29. The system according to paragraph 1 also includes a computer that receives and analyzes electronic information from the detector relating to the light detected by the detector.

[0292] 30. According to the system in paragraph 1, the reaction region contains nucleic acids, and the analysis performed by the computer includes determining nucleic acid sequence information.

[0293] 31. The system according to paragraph 1, wherein at least one waveguide providing illumination to multiple reaction regions is irradiated from both ends with illumination from the same laser, and the number of longitudinal modes is greater than 8.

[0294] 32. According to the system in paragraph 1, multiple reaction regions include nanoscale wells.

[0295] 33. According to the system in paragraph 1, multiple response regions include zero-mode waveguides.

[0296] 34. According to the system in paragraph 1, the chip includes multiple couplers.

[0297] 35. According to the system in paragraph 34, the chip has 2 to 16 couplers.

[0298] 36. According to the system in paragraph 1, the coupler couples light into a single waveguide, and the waveguide is divided into 2 to 512 waveguide branches within 3 mm of the coupler.

[0299] 37. According to the system in paragraph 36, the chip includes multiple couplers, and each coupler couples light into a single waveguide, and the waveguide is divided into 2 to 512 waveguide branches within 3 mm of the coupler.

[0300] 38. According to the system in paragraph 1, wherein the coupler couples light into a single waveguide, and the waveguide is divided into 2 to 512 waveguide branches within 20% of the longest distance the light travels from the coupler to the region of interest through the waveguide branches.

[0301] 39. According to the system in paragraph 38, the chip includes multiple couplers and each coupler couples light to a single waveguide, and the waveguide is divided into 2 to 512 waveguide branches within 20% of the longest distance the light travels from the coupler to the region of interest through the waveguide branches.

[0302] 40. A method for performing fluorescence analysis on a chip, comprising:

[0303] Provides a laser that emits illumination light into free space, the emitted illumination light comprising multiple longitudinal modes, each longitudinal mode having an intensity of 20% or more of the intensity of the strongest longitudinal mode, wherein the wavelength range from the shortest to the longest longitudinal mode is between 10 pm and 20 nm; and

[0304] The chip is illuminated by light from free space, and the chip includes:

[0305] Multiple reaction zones;

[0306] At least one waveguide that provides illumination light to multiple reaction regions, said reaction regions comprising a fluorescent material that absorbs the illumination light and emits fluorescence; and

[0307] A coupler that couples illumination light into at least one waveguide; and a detector that detects fluorescence emitted from the reaction region.

[0308] 41. According to the method of paragraph 40, wherein the plurality of longitudinal patterns includes at least two longitudinal patterns spaced at least 10 pm apart, wherein the intensity of each pattern is 50% or more of the intensity of the strongest pattern.

[0309] 42. According to the method in paragraph 40, wherein the plurality of longitudinal patterns comprises at least four patterns spaced at least 10 pm apart, wherein the intensity of each pattern is 50% or more of the intensity of the strongest pattern.

[0310] 43. According to the method in paragraph 40, wherein the plurality of longitudinal patterns comprises at least eight patterns spaced at least 30 pm apart, wherein the intensity of each pattern is 50% or more of the intensity of the strongest pattern.

[0311] 44. According to the method in paragraph 40, wherein the plurality of longitudinal patterns comprises 2 to 100 longitudinal patterns, wherein the intensity of each pattern is 50% or more of the intensity of the strongest pattern.

[0312] 45. According to the method in paragraph 40, the wavelength range from the shortest longitudinal mode to the longest longitudinal mode is greater than 200 pm.

[0313] 46. ​​According to the method in paragraph 40, the wavelength range from the shortest longitudinal mode to the longest longitudinal mode is greater than 1 nm.

[0314] 47. According to the method in paragraph 40, the wavelength range from the shortest longitudinal mode to the longest longitudinal mode is greater than 3 nm.

[0315] 48. According to the method in paragraph 40, the wavelength range from the shortest longitudinal mode to the longest longitudinal mode is between 200 pm and 10 nm.

[0316] 49. The method according to paragraph 40 also includes optical elements, wherein the optical elements modulate the illumination light between the laser and the chip.

[0317] 50. According to the method in paragraph 40, wherein the illumination light from the laser has greater than 80% of its intensity in a single transverse mode.

[0318] 51. According to the method in paragraph 40, wherein the illumination light from the laser has greater than 50% of its intensity in a single transverse mode.

[0319] 52. According to the method in paragraph 40, wherein the illumination light from the laser has greater than 10% of its intensity in a single transverse mode.

[0320] 53. According to the method in paragraph 52, the single horizontal mode is the Gaussian mode.

[0321] 54. The method according to paragraph 40 also includes a computer that receives and analyzes electronic signals from the detector.

[0322] 55. The method according to paragraph 40, wherein the analysis includes obtaining nucleic acid sequencing information.

[0323] 56. According to the method in paragraph 40, the wavelength of the shortest longitudinal mode is from about 450 nm to about 700 nm.

[0324] 57. According to the method in paragraph 40, the wavelength of the shortest longitudinal mode is about 500 nm to about 650 nm.

[0325] 58. According to the method in paragraph 40, the waveguide includes a SiON core.

[0326] 59. According to the method in paragraph 58, the SiON core has a refractive index greater than about 1.6.

[0327] 60. According to the method in paragraph 40, the waveguide includes a core surrounded by silicon dioxide.

[0328] 61. According to the method in paragraph 40, wherein the dimensions of at least one waveguide on the chip are determined to carry a single lateral mode.

[0329] 62. According to the method in paragraph 40, the chip includes a silicon chip.

[0330] 63. According to the method in paragraph 40, wherein the detector includes a CMOS sensor.

[0331] 64. According to the method in paragraph 40, wherein the laser is fiber-coupled and the light emitted from the laser into free space is emitted from the fiber.

[0332] 65. According to the method in paragraph 40, wherein the laser is coupled to the PLC via an optical fiber, and light emitted from the laser into free space is emitted from the PLC.

[0333] 66. The method according to paragraph 40 also includes one or more optical elements arranged between the laser and the chip.

[0334] 67. According to the method of paragraph 66, one or more optical elements include one or more lenses, polarizers, wedge prisms, filters, prisms, mirrors, or gratings.

[0335] 68. The method according to paragraph 40 also includes a computer that receives and analyzes electronic information from the detector relating to the light detected by the detector.

[0336] 69. According to the method in paragraph 40, the reaction region contains nucleic acids, and the analysis performed by a computer includes determining nucleic acid sequence information.

[0337] 70. According to the method in paragraph 40, the chip includes multiple couplers.

[0338] 71. According to the method in paragraph 40, the chip has 2 to 16 couplers.

[0339] 72. According to the method in paragraph 40, wherein the coupler couples light into a single waveguide, and the waveguide is divided into 2 to 512 waveguide branches within 3 mm of the coupler.

[0340] 73. According to the method in paragraph 72, the chip includes multiple couplers, and each coupler couples light into a single waveguide, and the waveguide is divided into 2 to 512 waveguide branches within 3 mm of the coupler.

[0341] 74. According to the method in paragraph 40, wherein the coupler couples light into a single waveguide, and the waveguide is divided into 2 to 512 waveguide branches within 20% of the longest distance the light travels from the coupler to the region of interest through the waveguide branches.

[0342] 75. According to the method in paragraph 74, the chip includes a plurality of couplers and each coupler couples light to a single waveguide, and the waveguide is divided into 2 to 512 waveguide branches within 20% of the longest distance the light travels from the coupler to the region of interest through the waveguide branches.

[0343] 76. A system for improved fluorescence analysis on a chip, the chip including an illumination waveguide, the system comprising:

[0344] Multiple lasers, each with an output illumination;

[0345] A combined optical element that combines output illumination from multiple lasers, wherein the combined optical element emits the combined output illumination into free space, the emitted illumination light comprising multiple longitudinal modes spaced at least 10 pm apart, each longitudinal mode having an intensity of 20% or more of the strongest longitudinal mode intensity, wherein the wavelength range from the shortest to the longest longitudinal mode is between 10 pm and 20 nm; and

[0346] A chip that receives light from free space, the chip comprising:

[0347] Multiple reaction zones;

[0348] At least one waveguide that provides illumination light to multiple reaction regions, each reaction region comprising a fluorescent material that absorbs the illumination light and emits fluorescence; and

[0349] A coupler that couples illumination light into at least one waveguide; and a detector that detects fluorescence emitted from the reaction region.

[0350] In some embodiments, the chip in any of the above systems and methods may include at least one coupler.

[0351] It should be understood that, unless the context clearly indicates otherwise, the terms “chip” and “target waveguide device” are generally used interchangeably in this document.

[0352] It will be apparent to those skilled in the art that other suitable modifications and adaptations can be made to the analytical apparatus and system described herein without departing from the scope of the invention or any of its embodiments. The invention has now been described in detail, and will be more clearly understood by referring to the following embodiments, which are included herein for illustrative purposes only and are not intended to limit the invention.

[0353] Example

[0354] Irradiation and measurement from waveguide

[0355] Figure 9 A typical setup is shown that can be used to send light into a target waveguide device and measure the amount of light transmitted and reflected over time. Light can be delivered to one side of the target device using one of the lasers, or two lasers can be used to deliver light to both sides of the target device. A camera is aligned to measure the light transmitted through the waveguide and then coupled to the device. The target device includes a zero-mode waveguide (ZMW) region for fluorescence analysis, as described herein for single-molecule real-time sequencing.

[0356] Time-dependent back reflection

[0357] Figure 12 A set of measurements is shown, illustrating time-dependent backreflection. Light is emitted into the waveguide at approximately 532 nm. The axis to the left of the top curve shows the reflected light as a function of time. The axis to the right of the bottom curve shows the transmitted light. It can be seen that over time, a cumulative effect of backreflection occurs at the expense of transmitted light.

[0358] Improved transmittance using lasers with multiple longitudinal modes

[0359] This experiment demonstrates the effectiveness of using a laser with multiple longitudinal modes to reduce or eliminate back reflections from waveguides. Specifically, Figure 13 The wavelength spectrum of the output of a 522 nm broadband direct bandgap diode laser is shown. This laser has a single transverse spatial mode structure and a beam quality of M. 2 ~1.2 (almost diffraction-limited). From Figure 13 As can be seen, the laser exhibits multiple longitudinal modes, resulting in a broadband spectral linewidth (wavelength range) of -1 to 2 nm. For this particular optical system, the maximum fiber-coupled power output is approximately 55 mW, with a time stability of + / -0.5%. Figure 14 It is shown that when a broadband laser with a maximum incident laser power of 54 mW is coupled to a target device with a SiON waveguide, no LEV is observed during a 24-hour exposure. Figure 15 A comparative experiment is shown on the same target device when the device is irradiated with a single longitudinal mode laser at a comparable laser power using a 532 nm laser. The experiment clearly shows that when using a single longitudinal mode laser, the transmitted power drops significantly after two hours. This drop in transmitted power is characteristic of LEVs.

[0360] Nucleic acid sequencing

[0361] As described above, the inventors have discovered that by using a broadband multimode laser input source, it is possible to overcome the poor performance typically observed in the use of backpropagating single-wavelength narrowband optical excitation in target waveguide devices. The practicality of this method has been demonstrated in exemplary DNA sequencing experiments using a PacBio Sequel chip irradiated with light from a single broadband multimode laser source. Specifically, in the demonstrated system, the laser output is separated and the separated beam is coupled to a DNA sequencing device (e.g., having a...) Figure 8B In the apparatus shown (the optical path shown), excitation power is provided from a single exciter source. In these experiments, the optical input is provided by a direct bandgap multimode InGaN semiconductor diode laser, which delivers fiber-coupled power up to 55 mW (77 mW laser head power), with a center wavelength of approximately 522 nm and a spectral bandwidth of approximately 2 nm. This laser source is well coupled to the target apparatus and, as shown... Figure 14 As shown, the generation of LEV in the target optical waveguide is eliminated.

[0362] exist Figure 16 The image shows a signal-to-noise ratio heatmap generated from this exemplary DNA sequencing reaction using the aforementioned laser source and target device, where the laser power at the head is 37 mW and the power per unit input coupler is 2.4 mW. In this experiment, a 30 kB E E. coli fragment was sequenced for 10 hours. Figure 16 The SNR heatmap shown was obtained from an image captured 5 minutes into the sequencing run. The results of this sequencing run showed 88.7% accuracy, 10.1 kB read length, and 2.0 GB sequencing yield on only half the performance of a standard Sequel® chip.

[0363] Figure 17 The effect of laser power on wavelength bandwidth is shown. This comparison reveals that as the power of the 522 nm multimode laser decreases below 20 mW, the laser's spectrum begins to narrow significantly. Figure 18 This illustrates the impact of laser coherence on the sequencing SNR heatmap and the corresponding sequencing accuracy as laser power decreases. As shown in this example (including the following for...), Figure 20 As can be seen in the described analysis, a significant decrease in sequencing accuracy was observed when the laser head power was reduced to below approximately 20 mW.

[0364] Figure 19 The relationship between laser coherence, optical mode, spectral width, and optical path difference (OPD) is described. Based on optical coherence theory, the laser coherence length in the active region of the chip can be estimated as follows: Where λ is the center wavelength of the laser, Δλ is the spectral width of the laser, and n 芯片 This is the refractive index of the active region waveguide device. The refractive index *n* can be estimated using typical laser spectra. 芯片 ,like Figure 19 As shown in the lower left part. The mode interval is Δλ1~55 pm or Δv1~0.61 pm. 10 11 At Hz, the spectrum shown has approximately 36 optical longitudinal modes spanning 2 nm. According to optical coherence theory, the mode spacing defines the spatial periodicity (Lo) of coherence in the active region of the target device. 芯片 ): Therefore, the spatial period of experimental measurements of a coherent spike of ~1.3 mm on the sensor (see, for example, Figure 19 (SNR thermal map in the upper left corner), refractive index n of the active region 芯片 The estimate is 1.9.

[0365] For a 2 nm wide, 522 nm laser spectrum and n ~ 1.9, use the formula cited above, and as... Figure 19 As shown in the upper left, the coherence length on the target device can be estimated to be approximately 31.6 μm. Since the coherence fringes repeat in the active region of the cell with a period of ~1.3 mm, less than 3% of the ZMW experienced higher coherence, which, even at a small percentage, leads to a significant yield loss. Under experimental uncertainty, no yield difference was observed when comparing the irradiation schemes experimentally using self-propelled propagation from one laser versus counter-propelled propagation from two lasers.

[0366] like Figure 19As shown in the upper right, laser coherence is a strong function of the number of modes, and theoretically, for two beams with the same amplitude and polarization, it can be described by the following correlation: , where x is the OPD between the self-propagating beam and the zero OPD point normalized by the period of the coherence function. (See, for example, RJ. Collier, CB Burckhardt, LH Lin, Optical Holography, Academic Press Inc, 1971, pp. 146-150.) It can be seen that when the number of longitudinal laser modes is greater than about 30, the coherence function has a very narrow spatial peak, but the spatial peak broadens as the number of modes decreases.

[0367] These coherent fringes were observed to repeat with a spatial period of 1.3 mm. Their amplitudes decayed over a characteristic spatial scale defined by the spectral width of a single laser mode. For a typical CW laser diode, the coherence function is expected to decrease exponentially with increasing distance, and the characteristic spatial scale is very long (>10 m). Therefore, no significant attenuation of the coherence peaks was observed over the entire active region, which is only 1 cm wide. It should be noted that the period of the spatially coherent fringes observed in the active region is directly related to the laser cavity length and its effective refractive index. 芯片 L 芯片 = n L L 腔 .

[0368] Figure 19 The lower right portion shows the appearance of the slowly decaying coherence function. (This figure is taken from Epworth (1982), “The Temporal Coherence of Various Semiconductor Light Sources Used in Optical Fibre Sensors,” Eds. S. Ezekiel and HJ. Arditty, pp. 237-244.)

[0369] From Figure 17 , Figure 18 and Figure 20 The data from the DNA sequencing experiments shown provide further support for the view that high coherence is undesirable for irradiation of active regions. This is due to the reduced number of longitudinal modes (or spectral bandwidths) at low laser power (e.g., as...). Figure 17As shown), the SNR heatmap therefore exhibits significantly greater spatial variation (see [reference]). Figure 18 ). Figure 20 It also showed that the accuracy of sequencing data decreased as the laser spectrum narrowed. Figure 20 The six data points correspond to the following conditions: (1) 7.7 mW laser input (~0.5 mW per coupler), (2) 10 mW laser input (~0.65 mW per coupler), (3) 15 mW laser input (~1.0 mW per coupler), (4) 25 mW laser input (-1.6 mW per coupler), (5) 37 mW laser input (~2.4 mW per coupler), or (6) 61.6 mW laser input (~4 mW per coupler).

[0370] Alignment of cameraless laser with chip

[0371] Figures 21A to 21C This illustrates the requirement for coarse alignment between a laser beam and a target waveguide device (particularly a DNA sequencing chip) in an optical system without an alignment camera. In this example, the optical signal emitted by a fluorescent reagent in the reaction region of the arrayed waveguide device is measured to indicate the coupling efficiency of the input light source, thereby indicating the alignment of the device with the light source. The fluorescence signal is collected on a + / - 40 micrometer grid with a resolution of 10 micrometers in the x and y directions, and a pitch angle resolution of 0.25° + / - 0.5° relative to the optimal alignment point. Figures 21A to 21C In this example, the same set of data is plotted against three different coordinate systems. These plots illustrate that the laser beam is preferably within approximately + / - 20 micrometers in the x (21 A) and y (21 B) coordinates (referred to as AT and CT on the plot), and the optimal alignment point spacing is within approximately + / - 0.5° to excite a fluorescence signal that can then be detected by the chip sensor. In this example, the fluorescence signal is the average of 3x3 camera pixels in the sequencing region, and this averaging can be performed quickly within the FPGA.

[0372] Under these requirements, assuming a tolerance uncertainty of + / -300 μm for the input coupler position relative to the laser beam in the x and y directions, and a pitch angle of + / -2°, then for blind coarse search alignment, it is necessary to iteratively traverse (300μm / 20μm)*(300μm / 20μm)*(2° / 0.5°) ~ 15*15*4 = 900 positions. If approximately 0.1 seconds of camera exposure time is required per position to detect the fluorescence signal, and the laser beam is redirected much faster and therefore its delay can be ignored, then blind coarse alignment search can be completed in ~90 seconds. The additional auxiliary alignment features described above can accelerate this coarse alignment, which is particularly useful if the tolerance is large or the optical redirection time is slower than in the example above.

[0373] All patents, patent publications and other publicly available references mentioned herein are incorporated herein by full citation as if each reference were individually and specifically incorporated herein by citation.

[0374] Although specific examples have been provided, the above description is illustrative and not restrictive. Any one or more features of the foregoing embodiments can be combined in any way with one or more features of any other embodiment of the invention. Furthermore, many variations of the invention will become apparent to those skilled in the art upon reading the specification. Therefore, the scope of the invention should be determined by reference to the full scope of the appended claims and their equivalents.

Claims

1. A system for optical analysis, the system comprising: A light source, configured to emit an optically excited beam into free space, and Movable target waveguide device, comprising: At least one optical coupler, the at least one optical coupler being configured to receive the optical excitation beam from the light source through free space; At least one optical waveguide, the at least one optical waveguide including a first end; wherein the first end of the optical waveguide is configured to receive an optical excitation signal from the at least one optical coupler; At least one reactive region, said at least one reactive region being optically coupled to said at least one waveguide; and At least one detector region, the at least one detector region being optically coupled to the at least one reaction region and configured to detect an optical alignment signal from the at least one reaction region; Wherein, the target waveguide device or the optical excitation beam is movable relative to each other; and The system is configured to monitor the optical alignment signal and move the target waveguide device or the optical excitation beam relative to each other to increase the optical alignment signal.

2. The system according to claim 1, wherein, The system does not include an alignment camera.

3. The system according to claim 1, wherein, The system is configured to move the target waveguide device or the optical excitation beam relative to each other to increase the optical alignment signal to a maximum level.

4. The system according to claim 1, wherein, The target waveguide device or the optical excitation beam can move in two dimensions.

5. The system according to claim 1, wherein, The at least one detector region is configured to detect both the optical alignment signal and the optical emission signal from the at least one reaction region.

6. The system according to claim 1, wherein, The optical excitation beam is configured to project at least a coarse illumination point and a fine illumination point onto the target waveguide device, wherein the coarse illumination point is larger than the fine illumination point.

7. The system according to claim 6, wherein, The optical excitation beam is configured to project the coarse irradiation point onto the target waveguide device as the target waveguide device or the light source moves.

8. The system according to claim 1, wherein, The target waveguide device further includes at least one auxiliary alignment sensor or at least one auxiliary alignment coupler positioned on the surface of the target waveguide device at a certain distance from the at least one optical coupler.

9. The system according to claim 8, wherein, The distance is defined using photolithography during the fabrication of the target waveguide device.

10. The system according to claim 8, wherein, The at least one auxiliary alignment sensor or the at least one auxiliary alignment coupler is configured for use in the coarse alignment process.

11. The system according to claim 8, wherein, The target waveguide device includes multiple auxiliary alignment sensors or multiple auxiliary alignment couplers.

12. The system according to claim 11, wherein, The plurality of auxiliary alignment sensors or the plurality of auxiliary alignment couplers are arranged in a geometric pattern.

13. The system according to claim 12, wherein, The geometric pattern includes at least one optical coupler optically coupled to the at least one reaction region.

14. The system according to claim 1, wherein, The target waveguide device includes at least one alignment feature at a defined location on the device, and the location of the alignment feature is stored on the device in a readable format.

15. The system according to claim 1, further comprising: At least one optical element is located between the light source and the target waveguide device, wherein the at least one optical element modulates the optical excitation beam between the light source and the target waveguide device.

16. The system according to claim 15, wherein, The at least one optical element is a lens, polarizer, wedge prism, filter, prism, mirror, or grating.

17. The system according to claim 1, wherein, The target waveguide device includes multiple optical waveguides, each configured to receive the optical excitation signal from the light source.

18. The system according to claim 17, wherein, The target waveguide device includes at least 2, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10000, or at least 50000 optical waveguides.

19. The system according to claim 17, wherein, The target waveguide device includes no more than 100,000, no more than 50,000, no more than 10,000, no more than 5,000, no more than 1,000, no more than 500, or no more than 100 optical waveguides.

20. The system according to claim 17, wherein, The target waveguide device includes 1 to 100,000, 100 to 10,000, or 500 to 5,000 optical waveguides.

21. The system of claim 1, further comprising a computer that receives and analyzes at least one electronic signal from the at least one detector region.

22. The system of claim 21, wherein the analysis includes acquiring nucleic acid sequencing information.

23. The system according to claim 1, wherein, The optical excitation beam has an excitation wavelength of about 450 nm to about 700 nm.

24. The system of claim 23, wherein the excitation wavelength is about 500 nm to about 650 nm.

25. The system according to claim 1, wherein, The at least one optical waveguide comprises a SiON core.

26. The system according to claim 25, wherein, The SiON core has a refractive index greater than about 1.

6.

27. The system according to claim 1, wherein, The at least one optical waveguide includes a core surrounded by silicon dioxide.

28. The system according to claim 1, wherein, The target waveguide device is manufactured on a silicon chip.

29. The system according to claim 1, wherein, The at least one detector region includes a CMOS sensor.

30. The system according to claim 1, wherein, The light source is coupled to an optical fiber, and the optical excitation beam is emitted from the optical fiber.

31. The system according to claim 1, wherein, The light source is coupled to a planar optical wave circuit via an optical fiber, and the optical excitation beam is emitted from the planar optical wave circuit.

32. The system according to claim 1, wherein, The at least one reaction region includes a nucleic acid sample.

33. The system according to claim 1, wherein, The at least one reaction region includes a nanoscale well.

34. The system according to claim 1, wherein, The at least one reaction region includes a zero-mode waveguide.

35. The system according to claim 1, wherein, The target waveguide device includes 2 to 16 couplers.

36. The system according to claim 1, wherein, The target waveguide device includes 2 to 16 optical splitters.

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