spectroscopy

Abstract

A spectrometer includes an optical input for receiving spectral light, a single-photon avalanche diode (133), an array (131) of multiphoton detector elements (134), and a spectral dispersive optics (143) arranged to disperse the spectral light into a spectrum. The spectrometer further includes means (145) arranged to: direct the spectrum to both the single-photon avalanche diode (133) and the array (131) of multiphoton detector elements (134); and / or selectively direct the spectrum to either the single-photon avalanche diode (133) or the array (131) of multiphoton detector elements (134).

Description

Technical Field

[0001] The present invention relates to spectroscopy, and more particularly to a spectroscopic mirror, spectroscopic apparatus, and method for generating cumulative spectral data representing multiphoton arrivals at a photodetector and for performing time separation of single-photon arrivals at a photodetector. Background Technology

[0002] The Raman effect is the phenomenon where a sample scatters excitation light of a given frequency into a spectrum with characteristic peaks caused by the interaction between the incident light and the molecules constituting the sample. Different molecular species have different characteristic Raman peaks, and therefore this effect can be used to analyze the types of molecules present.

[0003] For some samples, excitation radiation and Raman radiation together produce fluorescence radiation. Since the Raman signal is emitted from the sample in a much shorter time frame (less than a picosecond) compared to the fluorescence signal (thousands of picoseconds or even tens of milliseconds), a fast time-resolved detector can be used to distinguish the spectra from the two processes. Time-correlated single-photon counting (TCSPC) is known to be used to separate the Raman and fluorescence signals. Due to the statistical nature of these processes and the finite width of the excitation pulse, the Raman and fluorescence signals typically overlap.

[0004] US2012 / 0194815 A1 discloses an apparatus that includes a single-photon detector, which may be a single-photon avalanche diode (SPAD) array operating in "Gieg mode," wherein a single photon illuminating a photodetector causes the generation of an output pulse (similar to the "click" of a Geiger counter). Detecting Raman radiation in pulse mode effectively filters out background noise and fluorescence because no optical or electrical pulse is considered outside the detection or registration period.

[0005] US2013 / 0342835 A1 discloses a time-resolved Raman spectroscopy device that uses SPAD via optical or electrical triggering.

[0006] EP2956748 A1 discloses an apparatus for measuring Raman radiation from an object, comprising a detector having multiple single-photon avalanche diode (SPAD) elements. A counter measures the individual timing of each detection performed in the detector array relative to an excitation pulse. The counter counts the number of detections within a Raman time window and one or more fluorescence time windows. The counter forms the Raman detection count by eliminating estimates of the number of fluorescence photons detected in the Raman time window based on the number of detections in at least one fluorescence window. Summary of the Invention

[0007] According to a first aspect, a spectrometer is provided, which includes an optical input for receiving spectral light and a single-photon detector element.

[0008] The spectrometer may further include an array of multiphoton detector elements, such as a two-dimensional multiphoton detector element array. The spectrometer may further include spectral dispersive optics arranged to disperse spectral light into a spectrum.

[0009] The spectrometer may further include means arranged to perform the following:

[0010] i) Directing the spectrum to both single-photon detector elements and multi-photon detector element arrays; and / or

[0011] ii) Selectively direct the spectrum from the optical input to either the single-photon detector element or the multi-photon detector element array.

[0012] In this way, multiphoton detector arrays can be used to detect spectra where the capability of a multiphoton detector array is needed to replace or supplement the capability of a single-photon detector element. Specifically, measurements obtained from a single-photon detector element can have a lower signal-to-noise ratio than those from a multiphoton detector element because fewer photons in the spectral light are detected on the single-photon detector element. In particular, a single-photon detector element (such as a SPAD) may not be able to detect photons during the "off" period after detecting the initial single photon, resulting in a smaller proportion of the spectral light being detected compared to a multiphoton detector element. Furthermore, multiphoton detector elements can allow for collection methods that are not feasible with single-photon detector elements (such as SPADs). For example, various techniques require the use of the two-dimensionality of the array, the high fill factor provided by typical multiphoton detector element arrays (such as CCDs), and the charge accumulation on the multiphoton detector elements of the array and the transfer of charge between these multiphoton detector elements, such as the techniques disclosed in WO2008 / 090350, WO2009 / 093050, and WO2015 / 049494, all of which are incorporated herein by reference in their entirety. However, single-photon detector elements allow the device to be used for time-correlated single-photon counting in specific regions of the spectrum. In this way, the spectrometer can provide time-correlated single-photon counting while also having the capability of a multiphoton detector element array for spectral light arriving at the optical input from the same source / spectrometer setup (e.g., spectral light generated with the same excitation source, the same microscope, the same sample, etc.). This allows for cross-correlation between spectral measurements performed using single-photon detector elements and measurements performed using a multiphoton detector element array.

[0013] A spectrum is light separated in space according to its wavelength.

[0014] A spectrometer may include a processor.

[0015] The processor can be configured to correlate the output from a single-photon detector element with the output from a two-dimensional multiphoton detector element array.

[0016] In a cross-correlation technique, an estimate of the fluorescence signal from a sample can be determined based on detections performed by an array of multiphoton detector elements, and the Raman signal from the sample can be determined by eliminating the estimate of the fluorescence signal from detections performed by a single-photon detector. Elimination can include subtracting several detections from the single-photon detector detections based on the fluorescence signal estimate, or attributing a portion of the single-photon detector detections to fluorescence based on the fluorescence signal estimate, such as during a fitting process.

[0017] Multiphoton detector arrays can generate signals that are the cumulative result of photon detections over a period including the time when the sample will fluoresce. Therefore, the spectrum constructed from detections performed by the multiphoton detector array will be dominated by fluorescence from the sample (because the Raman effect is weaker than fluorescence). Thus, the fluorescence signal can be estimated based on the detections performed by the multiphoton detector elements. Single-photon detectors generate timed single-photon detections, some of which are generated by the Raman effect and some by fluorescence. The time periods for generating Raman and fluorescence photons overlap. Therefore, it may be desirable to eliminate detections due to fluorescence from other detections generated by the single-photon detector in order to determine the Raman signal.

[0018] In embodiments of the invention, detection performed by a multiphoton detector element array is used to estimate the spectral form of fluorescence from the sample, and the estimation of the spectral form of fluorescence is used to determine the number of counts eliminated from detection performed by a single-photon detector for a specific time (e.g., a time chamber of a histogram). The form of the fluorescence signal determined from the multiphoton detector element array can be used in a fitting process, wherein a reference spectrum of known form, including the fluorescence spectrum determined from the multiphoton detector element array, is fitted to the detection generated by the single-photon detector for a specific time to determine the components in the sample and / or the amount of each component. For example, the fitting process can be a direct classical least squares fitting process, such as the process disclosed in WO2012 / 156667. This assumes that the spectral form of the fluorescence spectrum from the sample remains the same even as the intensity of the fluorescence spectrum changes over time.

[0019] In an alternative embodiment, this correlation between the output from the single-photon detector element and the output from the two-dimensional multiphoton detector element array can be performed on a processor separate from and / or far from the spectrometer.

[0020] The term "single-photon detector element" is used herein to refer to a photodetector element arranged to generate an output signal when a photodetector detects a single photon, thereby enabling the differentiation of the detection of each photon. For example, a single-photon detector element may generate a signal pulse for each photon detected by the photodetector. The single-photon detector element may be arranged to generate an output signal when the photodetector detects a single photon, and during a "off" period following the detection of the single photon, the single-photon detector is unable to detect any further photons illuminating the photodetector. The "off" period may be at least ten nanoseconds. The single-photon detector may include a single-photon avalanche diode (SPAD).

[0021] The term "multiphoton detector element" is used herein to refer to a photodetector element arranged to generate an output signal that varies (e.g., accumulates) with the number of photons incident on a photodetector element. Each multiphoton detector element can generate a signal proportional to the number of photons absorbed by the detector element over a period of time. For example, an array of multiphoton detector elements can be a charge-coupled device (CCD), in which, as photons are absorbed, electronic charge accumulates at each detector element until the charge is transferred to another element and / or read out. In alternative embodiments, the array of multiphoton detector elements is a CMOS sensor.

[0022] The device may include optical elements, such as a spectral light steering mirror, operable to selectively direct spectral light from an optical input to either a single-photon detector element or an array of multi-photon detector elements. For example, the optical element may be movable to selectively direct spectral light from an optical input to either a single-photon detector element or an array of multi-photon detector elements, such as being movable from a position inserted into the optical path of the spectral light to a position outside the optical path.

[0023] The device can be a mechanism configured to move spectral light into and out of the optical path of a single-photon detector element and a multi-photon detector element array to selectively guide spectral light from the optical input to either the single-photon detector element or the multi-photon detector element array.

[0024] The device can be a beam splitter arranged to split the spectral light received by the optical input between a single-photon detector element and an array of multi-photon detector elements. In this way, both the single-photon detector element and the array of multi-photon detector elements can generate signals produced by the co-excitation of the sample.

[0025] In one embodiment, the spectrometer includes an array of single-photon detector elements. For example, the array of single-photon detector elements may include a one-dimensional or two-dimensional array of single-photon detector elements. Spectral dispersive optics may be arranged to disperse the spectral light into a spectrum guided across the array of single-photon detector elements as the device directs the spectrum to the array.

[0026] The spectrometer may include a two-dimensional single-photon detector element array; a spectral dispersive optics arranged to disperse spectral light into a spectrum in a spectral plane such that the spectrum is spread across the two-dimensional single-photon detector element array; and an optical arrangement located between the spectral dispersive optics and the two-dimensional single-photon detector element array in the optical path of the spectral light.

[0027] Optical arrangements can be configured such that spectral light converges to and / or diverges from a spatial focal point in a spatial plane perpendicular to the spectral plane, wherein the spectral light converges to a spectral focal point at a location different from the spatial focal point in the spectral plane. The spatial focal point may be outside the detector plane of the two-dimensional single-photon detector element array. The spectral focal point may be in the detector plane or closer to the detector plane than the spatial focal point. In alternative arrangements, the optical arrangement includes a structured diffuser configured to diffuse the spectral light in a spatial plane perpendicular to the spectral plane. The structured diffuser can be arranged to diffuse the light in only one dimension. In this way, the spectrometer can achieve high spectral resolution while dispersing the spectrum across multiple single-photon detector elements in a spatial direction / spatial plane. Therefore, if a single-photon detector element is unavailable, another single-photon detector element for that spectral location can be used to detect the spectral light.

[0028] The optical arrangement can be configured to focus the spectral light in the spectral plane.

[0029] The optical arrangement can be configured to position the focal point of the spectral light such that the spectral light is incident in a spatial direction on two or more single-photon detector elements of the array. The optical arrangement can be configured such that the spectral light received from the spectral dispersive optics is dispersed across the two-dimensional array of single-photon detector elements in a spatial direction perpendicular to the spectral plane in which the spectral light is dispersed by the spectral dispersive optics.

[0030] Optical arrangements may include diverging optics. As used herein, the term "diverging optics" means an optical device that refracts and / or reflects received light to disperse it more widely than if the optics were not in the optical path.

[0031] The optical arrangement can be a converging optics device arranged to focus the spectral light outside the detector plane of a two-dimensional single-photon detector element array. Converging optics can also focus the light in front of the detector plane of a two-dimensional single-photon detector element array. As used herein, the term "converging optics device" means an optical device that refracts and / or reflects light to focus it to a greater extent than if the optics were not in the path of the light.

[0032] Defocusing or converging optics can be arranged to prevent light from scattering in the spectral plane.

[0033] In this way, the spectral light is defocused in the spatial plane / direction on the two-dimensional single-photon detector element array.

[0034] Optical arrangements may include non-circular optics having optical surfaces that are non-circularly symmetrical about the optical axis of the spectral light. Optical arrangements may include lenses or mirrors, such as cylindrical lenses or mirrors, axonocones, or lens arrays. Non-circular optics may be oriented such that the longitudinal axis of the non-circular optics is aligned (i.e., parallel) with the spectral direction / plane of the spectral light incident on the non-circular optics. The terms "spectral plane" and "spectral direction" are used herein to refer to the plane or direction of dispersion of the spectrum at a specified point in the optical path of the spectral light (e.g., at the detector or at the non-circular optics). Non-circular optics may have a plane of symmetry having a normal orthogonal to the optical axis and longitudinal axis of the non-circular optics.

[0035] It should be understood that the orientation of the spectral plane or spectral direction between points along the optical path can be transformed by the optical devices present in the path. Therefore, the spectral plane or spectral direction at the detector may not have the same orientation as the spectral plane or spectral direction at a non-circular optical device. Other planes / directions of the spectral plane (such as spatial directions / planes) should be defined as orientations relative to the spectral plane at the same point along the optical path; for example, the orientation of the spatial plane at the detector relative to the spectral plane also at the detector, or the orientation of the spatial plane at a non-circular optical device relative to the spectral plane also at a non-circular optical device.

[0036] Optical arrangements can be made to avoid causing spectral dispersion of light.

[0037] Spectral dispersive optics and optical arrangements can be configured to guide spectral light onto an array of single-photon detector elements such that the width of the spectral light (at the detector) in a spatial direction orthogonal to the spectral plane is equal to or greater than the width of two or more single-photon detector elements in the spatial direction.

[0038] A two-dimensional single-photon detector element array can be divided into pixels, each pixel comprising multiple single-photon detector elements in a spatial direction orthogonal to the spectral plane. Dispersion devices and optical arrangements can be configured such that spectral light is spatially dispersed across two or more of the single-photon detector elements in each pixel. The optical arrangement can be configured to guide the spectral light onto the single-photon detector element array such that the width of the spectral light (at the detector) in a spatial direction orthogonal to the spectral plane (in which the spectrum is dispersed by spectral dispersive optics) is equal to or greater than the pixel width in that spatial direction.

[0039] The pixel width of each pixel in a two-dimensional single-photon detector element array can be greater in the spatial direction than the pixel width of a multiphoton detector element in the spectral plane.

[0040] Each pixel may include a set of single-photon detector elements, each of which is used to detect light within the same wavelength range.

[0041] For an array, the detection of photons by the single-photon detector elements is recorded per pixel; that is, there is no separate count of photons detected by different single-photon detector elements among the single-photon detector elements that make up a pixel.

[0042] A single-photon detector element array can be a line sensor (a one-dimensional pixel array). A line sensor can be oriented to have a single pixel width only in the spatial direction and multiple pixels in the spectral direction. For example, a line sensor can be described as follows: “A CMOS SPAD Line Sensor With Per-Pixel Histogramming TDC for Time-Resolved Multispectral Imaging,” IEEE Journal of Solid State Circuits, Vol. 54, No. 6, June 2019, which is incorporated herein by reference in its entirety.

[0043] In another embodiment, the single-photon detector element array includes a two-dimensional pixel array.

[0044] In a typical array of single-photon detector elements, different single-photon detector elements can have different responses. In particular, it has been found that single-photon detector elements in a typical array have varying dark counts, some of which produce so many dark counts that spectral (e.g., Raman) counts are overwhelmed. Therefore, typically not all single-photon detector elements in a pixel can be used for spectroscopy. This means that the single-photon detector elements available for spectroscopy are not located in the same position in each pixel. Consequently, the sensitivity of each pixel can vary significantly in the spatial direction. If the spatial range of the spectral light at the detector (in the detector plane) is on the order of the width of the single-photon detector element, this can lead to measurement inaccuracies because, for some pixels, the spectral light may fall on less sensitive single-photon detector elements. By configuring the optics of the spectrometer to guide spectral light onto an array of single-photon detector elements, such that the width of the spectral light in a spatial direction orthogonal to the spectral plane (in which the spectrum is dispersed by spectral dispersive optics) is equal to or greater than the pixel width or equal to or greater than two or more single-photon detector elements, this measurement inaccuracy caused by the sensitivity variation of the single-photon detectors in each pixel can be mitigated.

[0045] The optical arrangement can be positioned between the optical elements and / or beam splitters and the single-photon detector array. Spectroscopic dispersive optics can be located between the optical input and the optical elements and / or beam splitters. In this way, the spectrometer can guide the spectrum onto a two-dimensional multiphoton detector array, whose spatial dispersion differs from that guided onto the single-photon detector array.

[0046] Spectral dispersive optics can be diffraction gratings or prisms. A spectral dispersive optics device may include a first diffraction grating or prism for dispersing spectral light across an array of multiphoton detector elements and a second diffraction grating or prism for dispersing spectral light across an array of single-photon detector elements. The diffraction grating disperses light into a spectrum at orders other than zero, wherein light passes through or is reflected by the diffraction grating without wavelength separation.

[0047] The processor can be configured to record the spectrum from a subset of the total number of single-photon detector elements in each pixel. For each pixel, the subset can include the same number of single-photon detector elements. The subset of single-photon detector elements in each pixel can be selected based on a quality factor that measures the size of the dark count generated by each single-photon detector element. Therefore, the single-photon detector elements for each pixel can be located at different positions within the pixel. Thus, guiding the spectrum of the spectral light onto the array of single-photon detector elements such that the width of the spectral light in a spatial direction orthogonal to the spectral plane is equal to or greater than the pixel width in that spatial direction ensures that the spectrum falls on the selected subset of single-photon detector elements.

[0048] According to a second aspect of the present invention, a spectrometer is provided, the spectrometer comprising:

[0049] Spectrometer;

[0050] Detector; and

[0051] Non-circular optical devices, which have optical surfaces that are non-circularly symmetrical about the optical axis, such as cylindrical lenses or mirrors.

[0052] The spectrometer includes an optical input port for receiving spectral light, a spectral dispersive optics arranged to disperse the spectral light into a spectrum, and an optical output port to which the spectrum is delivered.

[0053] The detector comprises a pixel array, which is mounted to an optical output port such that the spectrum is dispersed across the pixel array in the spectral plane.

[0054] Among them, the non-circular optical device is located in the optical path between the spectral dispersive optical device and the detector.

[0055] Each pixel may include multiple single-photon detector elements in a spatial direction orthogonal to the spectral plane, and non-circular optics may be configured to spread spectral light across two or more of the single-photon detector elements of each pixel in a spatial direction.

[0056] The pixel array may include a one-dimensional pixel array (line array). The spectrometer may include additional optical output ports. The spectrometer may include additional detectors. These additional detectors may be one-dimensional or two-dimensional multiphoton detector element arrays. These additional detectors may include charge-coupled devices (CCDs).

[0057] A spectrometer may include means configured to perform the following:

[0058] i) Directing spectral light from the optical input to both the optical output port and another optical output port; and / or

[0059] ii) Selectively direct spectral light from the optical input to either the optical output port or another optical output port.

[0060] Non-circular optical components can be part of a spectral analyzer. For example, a non-circular optical component can be located between a spectral dispersive optical component and an optical output port.

[0061] According to a third aspect of the invention, a spectroscopic device is provided, comprising a spectrometer according to a first or second aspect of the invention and a spectral path from a sample to the spectrometer, for detecting spectral light emitted from a sample when irradiated with excitation light.

[0062] Spectroscopic devices may include an excitation source. The excitation source can be configured to generate excitation light pulses, such as a pulsed laser, like a pulsed laser diode. The source can emit pulses with frequencies between 1 kHz and 100 MHz. The pulse length can be on the order of tens or hundreds of picoseconds.

[0063] According to a fourth aspect of the present invention, a spectroscopic method is provided, the spectroscopic method comprising: detecting spectral light generated by exciting a sample with excitation light using a single-photon detector element; and detecting spectral light generated by exciting a sample with excitation light using a multiphoton detector element array, such as a two-dimensional multiphoton detector element array.

[0064] The spectral light can be detected sequentially or simultaneously by each detector. The output of the single-photon detector element can be used for time-correlated single-photon counting (TCSPC), for example, to separate the Raman signal and the fluorescence signal.

[0065] The method may include correlating the output from a single-photon detector element with the output from a two-dimensional multiphoton detector element array.

[0066] The spectroscopic method can use a spectrometer according to the first or second aspect of the present invention.

[0067] The method may include determining an estimate of the fluorescence signal from the sample based on detections performed by an array of multiphoton detector elements, and determining the Raman signal from the sample by eliminating the estimate of the fluorescence signal from detections performed by a single-photon detector. Elimination may include subtracting several detections from the single-photon detector detections based on the fluorescence signal estimate, or attributing a portion of the single-photon detector detections to fluorescence based on the fluorescence signal estimate, such as during a fitting process.

[0068] This method may include estimating the spectral form of fluorescence based on detections performed by an array of multiphoton detector elements, and estimating the number of Raman detections for a specific time or time period by eliminating detections performed by a single-photon detector for a specific time or time period (e.g., a time chamber of a histogram or Raman window) allocated to a specific time or time period from the time the sample is exposed to excitation radiation, based on the estimation of the spectral form of fluorescence. The form of fluorescence can be used in the fitting process, wherein a spectrum of known form, including the fluorescence spectrum determined from the array of multiphoton detector elements, is fitted to each spectrum generated by a single-photon detector for a specific time or time period to determine the components in the sample and / or the amount of each component. For example, the fitting process may be a direct classical least squares fitting process, such as the process disclosed in WO 2012 / 156667. This assumes that the spectral form of the fluorescence spectrum from the sample remains the same even as the intensity of the fluorescence spectrum changes over time.

[0069] According to a fifth aspect of the present invention, a method for analyzing spectral data collected by a spectrometer according to a first aspect of the present invention is provided, the method comprising receiving first spectral data of a sample, the first spectral data being detected by a single-photon detector element; receiving second spectral data of the sample, the second spectral data being detected by an array of multiphoton detector elements; and correlating the first spectral data with the second spectral data.

[0070] The step of correlating the first spectral data with the second spectral data can be performed according to the method described with respect to the fourth aspect of the invention.

[0071] The methods of the fourth and fifth aspects of the invention may include analyzing Raman signals to identify the properties of a sample, and controlling the process and / or performing further processing on the sample based on the identified properties. For example, the process may be a manufacturing process. The sample may be a sample of one or more manufactured products, and the identified properties may be used to determine whether the manufactured product meets required specifications. If the required specifications are not met, the process may need to be adjusted to manufacture the product according to the required specifications. The sample may be a tissue sample, and the process may be treatment of a patient providing the tissue sample. The method may be used as part of an inspection procedure (e.g., a safety procedure or quality control procedure) that includes generating an alarm based on the analysis of spectral data.

[0072] According to a sixth aspect of the invention, a data carrier is provided having instructions that, when executed by a processor, cause the processor to perform the method of the fifth aspect of the invention.

[0073] The data carrier can be a non-transient data carrier, such as volatile memory (e.g., RAM), non-volatile memory (e.g., ROM), flash memory, and data storage devices (e.g., hard disk, optical disk), or a transient data carrier (e.g., electronic or optical signals).

[0074] According to a seventh aspect of the present invention, an apparatus is provided, the apparatus comprising: a receiving device configured to receive first spectral data of a sample, the first spectral data including a first detection performed by a single-photon detector element, and to receive second spectral data of the sample, the second spectral data including a second detection performed by an array of multi-photon detector elements; and a correlation device configured to correlate the first spectral data with the second spectral data.

[0075] The relevant apparatus can be configured to determine an estimate of the fluorescence signal from the sample based on a second detection, and to determine the Raman signal from the sample by eliminating the estimate of the fluorescence signal from the first detection. Elimination may include subtracting several detections from the first detection based on the estimate of the fluorescence signal, or attributing a portion of the first detection to fluorescence based on the estimate of the fluorescence signal, such as during a fitting process.

[0076] The correlation device can be configured to estimate the spectral form of fluorescence based on a second detection and to estimate the number of Raman detections for a specific time or time period by eliminating a first detection assigned to a specific time or time period from the time the sample is exposed to excitation radiation based on the estimation of the spectral form of fluorescence. The correlation device can be configured to use a fitting process in which a spectrum of known form, including a fluorescence spectrum, is fitted to each spectrum generated by a single-photon detector for a specific time or time period to determine the components in the sample and / or the amount of each component. For example, the fitting process can be a direct classical least squares fitting process, such as the process disclosed in WO 2012 / 156667.

[0077] According to an eighth aspect, a spectrometer is provided, comprising: means for receiving spectral light; means for detecting photons and generating a signal per photon; means for detecting photons across an array of photodetector elements, each photodetector element generating a signal that varies (e.g., accumulates) with the number of photons irradiating the photodetector element; means for dispersing the spectral light across the array of photodetector elements into a spectrum; and means for performing the following:

[0078] i) Directing the spectrum to both the device for detecting individual photons and the array of photodetector elements; and / or

[0079] ii) Selectively direct the spectrum to either the device for detecting individual photons or the array of photodetector elements.

[0080] According to a ninth aspect of the invention, a spectral method is provided using a detector comprising a row of pixels, each pixel comprising at least two single-photon detector elements perpendicular to the row of pixels, the method comprising: -

[0081] Receive spectral light;

[0082] Disperse the spectral light across the pixels in that row into a spectrum;

[0083] The spectrum is moved along these columns to direct the spectrum to the different single-photon detector elements for each pixel;

[0084] And record the detections performed by these pixels for different positions of the spectrum on each pixel.

[0085] This method may include generating spectral data by using a subset of recorded detections performed by different single-photon detector elements for each pixel. The subset of recorded detections may be performed by single-photon detector elements for each pixel determined to have a desired performance level. The desired performance level may be a number of dark counts below a threshold. The desired performance level may be one of the pixels with the lowest number of dark counts or a preset number of single-photon detector elements. For example, the subset of recorded detections may be performed by four single-photon detector elements for the pixels with the lowest number of dark counts.

[0086] According to a tenth aspect of the present invention, a data carrier is provided having instructions that, when executed by a processor, cause the processor to perform the method of the ninth aspect of the present invention.

[0087] According to an eleventh aspect of the present invention, a spectrometer is provided, the spectrometer comprising: -

[0088] An optical input terminal, used to receive spectral light;

[0089] The detector includes a row of pixels, each pixel including a column of at least two single-photon detector elements perpendicular to the row of pixels;

[0090] Dispersive optics, used to disperse spectral light across the row of pixels into a spectrum; and

[0091] A movable optics device is used to move the spectrum along these columns to guide the spectrum to different single-photon detector elements for each pixel.

[0092] The spectrometer may include a confocal microscope comprising a confocal slit or pinhole along the optical path of the spectral light, wherein movable optics are disposed behind the confocal slit or pinhole.

[0093] Movable optics may include movable mirrors. Movable optics may be rotatable to change the angle of incidence of spectral light on the movable optics.

[0094] The spectrometer may include a controller arranged to control the spectrometer to perform the method of the ninth aspect of the present invention. Attached Figure Description

[0095] Figure 1 This is a schematic diagram of a spectroscopic device according to an embodiment of the present invention;

[0096] Figure 2a and Figure 2b This is a schematic diagram of the spectral analyzer and detector in a spectral device;

[0097] Figure 3 This is a schematic diagram of a detector that includes an array of single-photon detector elements;

[0098] Figure 4 yes Figure 3 The schematic diagram of the detector shown illustrates how the spectrum is dispersed in a spatial plane by a diverging cylindrical lens;

[0099] Figure 5 This is a schematic diagram of an optical arrangement according to another embodiment of the present invention;

[0100] Figure 6 This is a schematic diagram of the spectrometer and detector of a spectrometer device according to another embodiment of the present invention;

[0101] Figure 7 This is an example of a histogram showing the detection performed by a single-photon detector; and

[0102] Figure 8 This is a graph illustrating the form of detection performed by a multiphoton detector. Detailed Implementation

[0103] refer to Figure 1 The spectroscopic apparatus includes an excitation source 110, a confocal microscope 118 for delivering a laser beam to a sample 124 and delivering spectral light (e.g., Raman-shifted light) to a spectrometer 117, and a spectrometer 128. The spectrometer disperses the received spectral light spatially into a spectrum according to wavelength and delivers the spectrum to a selected detector of two detectors 130 and 131. Detector 130 includes an array of single-photon detector elements, such as a two-dimensional single-photon avalanche diode (SPAD) array. Detector 131 includes an array of two-dimensional multiphoton detector elements 134, such as a charge-coupled device (CCD).

[0104] In this embodiment, the light source 110 is a pulsed laser configured to generate laser pulses along a laser optical input path 113 to the optical device. The optical device includes a mirror 114 and a Rayleigh filter 116. In this embodiment, the Rayleigh filter 116 is a notch or edge filter that acts as a dichroic beam splitter. The mirror 114 and the Rayleigh filter 116 reflect the laser beam guided along the optical input path 113 onto the sample 124. The laser beam guided to the sample 124 enters the microscope 118, where it is deflected by optics 120, passes through the objective lens 122, and focused onto the sample 124. Raman scattering occurs at the sample, producing Raman-shifted light with a wavenumber different from the incident laser line.

[0105] The sample is mounted on a stage 123, which is movable in two dimensions (x, y) perpendicular to the laser beam. The movement of the stage 123 allows the laser beam to be positioned at different locations on the sample 124, making it possible to collect spectral data at each of these locations.

[0106] The Raman-shifted light is collected by objective lens 122 and transmitted back to Rayleigh filter 116 via optics 120 along spectral path 115. Rayleigh filter 116 reflects the laser wavelength but transmits the Raman-shifted wavenumber. In doing so, the Rayleigh filter filters out laser lines with much higher intensity. The Raman-shifted light then passes through Raman analyzer 128 and reaches selected detectors 130, 131.

[0107] refer to Figure 2a and Figure 2b The spectrometer 128 includes a housing 135 having an optical input port 137 and two optical output ports 139 and 141. In this embodiment, the optical input port 137 is an entrance slit that serves as a confocal slit 129. However, in other embodiments, the confocal slit or pinhole 129 may be located elsewhere in the system and separate from the optical input port 137. The housing 135 includes a collimating lens 142, a spectral dispersive optics (in this embodiment, a diffraction grating 143), a focusing optics 144, a spectral light steering mirror 145, and an optical arrangement configured to diverge the spectral light from a virtual spatial focus 147 in a spatial plane perpendicular to the spectral plane. In this embodiment, the optical arrangement is a non-circular diverging optics having a plane of symmetry 155 in the form of a cylindrical lens 146. The diffraction grating 143 disperses the spectrum in the spectral direction / plane λ. The spatial plane (e.g., Figure 4(As shown) Perpendicular to the spectral direction / plane λ. A CCD 131 is mounted to a spectrometer 128 to receive spectral light from the optical output port 141, and a single-photon avalanche diode array 133 130 is mounted to the spectrometer 128 to receive spectral light from the optical output port 139 when the mirror 145 moves into the path of light dispersed by the diffraction grating 143 (as shown). Figure 2b (As shown). As can be understood, the spectrum is rotated by mirror 145 along the spectral direction in which it spreads (although the spectral plane remains the same). However, mirror 145 can be angled to guide the spectral plane outwards. Figure 2b The plane shown (i.e., the plane guiding the paper). When the reflector 145 leaves the optical path, the light dispersed by the diffraction grating 143 is guided to the multiphoton detector 131 (e.g., Figure 2a (As shown).

[0108] Cylindrical lens 146 causes spectral light to diverge from virtual spatial focus 147 in the spatial plane. This virtual focus 147 is located at a different position from the spatial focus 149, and the spectral light is converged to this spatial focus in the spectral plane by focusing optics 144.

[0109] A focusing optics (in this embodiment, a focusing lens 144) is arranged to focus the spectrum onto the CCD at the desired resolution. In this embodiment, the focusing lens 144 is arranged to produce a spectral line on the CCD 131 that is about one pixel wide in the spatial direction (for the CCD) (e.g., because it focuses the image of the confocal slit through which the Raman light passes to a width of about one pixel).

[0110] The spectral light steering mirror 145 can be moved into the optical path of the spectral light dispersed by the dispersive optics 143 to redirect the dispersed spectrum of the light to the detector 130. The diverging cylindrical lens 146 is located in the optical path between the dispersive optics 143 (and in this embodiment, the spectral light steering mirror 145) and the detector 130, and is oriented to disperse the spectrum across the detector 130 in the spatial direction S. Figure 4 Solid line 152 illustrates the light diverged by the cylindrical lens, and dashed line 154 illustrates the converging path the light would take in the absence of the cylindrical diverging lens 146. The cylindrical lens 146 diverges the light in a spatial plane perpendicular to its longitudinal axis BB and keeps it unchanged in a direction parallel to the longitudinal axis BB. Accordingly, the cylindrical lens 146 is oriented such that its longitudinal axis is parallel to the spectral direction λ. When the cylindrical lens diverges the spectral light, the (virtual) focal point 147 of the diverging light is outside the detector plane 148 of the detector 130.

[0111] refer to Figure 3The detector 130 includes single-photon detector elements 133 arranged in a two-dimensional array. The single-photon detector elements 133 are grouped into pixels P1 to P2. n Each pixel includes multiple single-photon detector elements 133, including multiple single-photon detector elements 133 in a spatial direction S orthogonal to the spectral direction λ. (The dashed box encloses the single-photon detector elements 133 constituting pixel P1. Each other pixel P1 to P...) n This includes a corresponding set of single-photon detector elements 133. In this embodiment, each pixel includes one single-photon detector element 133 in the spatial direction and two single-photon detector elements 133 in the spectral direction. The size of each single-photon detector element 133 is similar to the size of the detector element 134 of the CCD. Accordingly, without the cylindrical diverging lens 146, the spectrum will be focused to less than P1 to P2 per pixel. n On all single-photon detector elements 133. However, as Figure 4 As shown, using a cylindrical diverging lens 146, spectral light is dispersed to each pixel P1 to P2. n On all single-photon detector elements 133. A cylindrical lens 146 with a focal length to achieve this purpose is selected.

[0112] For a typical single-photon detector array 133, several single-photon detectors 133' may operate in a manner unsuitable for detecting Raman signals. For example, some of the single-photon detectors 133' may generate dark signals that overwhelm any Raman signals that could be detected. However, since the spectral light spans each pixel P1 to P2... n All single-photon detectors 133 are scattered, thus making it easier for the spectrum to be incident on each pixel P1 to P2. n The device is equipped with at least one single-photon detector 133 having the required sensitivity / sufficiently low dark current for Raman light. The device can be operated to use only each pixel P1 to P... n A single-photon detector 133 is considered to have satisfactory operation (e.g., sufficiently small dark current). The device can be operated to use only each pixel P1 to P2. n A single-photon detector 133 is considered to have a set number (e.g., four) with the minimum dark current. Because the spectral light spans each pixel P1 to P... n All single-photon detectors 133 are scattered, so they can be distributed from pixel P1 to P2. n Select a set number of single-photon detectors 133 from any of the single-photon detectors 133.

[0113] exist Figure 5In the alternative optical arrangement shown, a converging cylindrical lens 246 is used instead of a diverging cylindrical lens 146. The converging cylindrical lens 246 converges the spectral light in the spatial plane to a spatial focal point 247 outside the detector plane 148. Accordingly, in the spatial plane, the spectral light spans each pixel P1 to P2. n The single-photon detector elements 133 are distributed in an array. In the spectral plane, the spectral focus (typically on the detector plane 148) remains essentially constant.

[0114] Detector 130 is connected to laser 110 via trigger line 132, which is used to synchronize the operation of detector 130 with the pulses of laser 110. Further details on the operation and timing of detector 130 can be found in the following literature: “ACMOS SPAD Line Sensor With Per-Pixel Histogramming TDC for Time-Resolved Multispectral Imaging”, IEEE Journal of Solid State Circuits, Vol. 54, No. 6, June 2019. In particular, after detecting a photon, each single-photon detector element is not able to detect another photon until at least the next laser pulse.

[0115] Detector 130 stores a histogram of photon detections per pixel detected on multiple pulses (exposures) of the laser beam to the sample. Detector 130 assigns the detections performed by each pixel to the appropriate bin in the corresponding histogram based on the photon arrival time. Thus, the resulting histogram for each pixel plots the arrival time against the number of detections (counts). Each pixel corresponds to a wavenumber based on the spectral dispersion at detector 130. Figure 7 An example of the resulting histogram is shown (only one histogram is shown, but it should be understood that a histogram per pixel is generated, as illustrated by the wavenumber axis). The Raman and / or fluorescence spectra can then be determined from the histogram, for example, by the controller 150 based on the arrival time of the photons after the excitation pulse. In one embodiment, the controller 150 sends the spectral data to a separate computer from the spectrometer 117, and the Raman and / or fluorescence spectra are determined by this separate computer.

[0116] In use, the user can choose to use detector 130 or detector 131 to perform detection. Specifically, the user can use detector 130 to perform time-resolved spectral imaging of the sample, while detector 131 can be used to perform non-time-resolved spectral imaging of the sample. The user may choose to use detector 131 because the increased signal-to-noise ratio achievable with a charge-coupled device (CCD) may be desirable for certain applications, or in some spectral collection techniques, the ability to synchronize the migration of charge between CCD pixels with the movement of the laser spot on the sample may be desirable. However, for other applications or in addition to using detector 131, time-resolved spectral imaging may be desirable. The user can input an instruction to the controller 150 indicating which detector 130 or 131 they wish to use for detection, and the controller 150 generates a command that causes the spectrometer 128 to move the spectral light steering mirror 145 into or out of the dispersed spectral path.

[0117] Users can use both detectors 130 and 131 to detect Raman and / or fluorescence spectra from the same sample and analyze the spectra from both detectors 130 and 131 to determine the characteristics of the sample. For example, the spectra collected by each detector 130 and 131 can be analyzed together to find correlations with the characteristics of the sample.

[0118] In one embodiment, the results from one detector 130, 131 can be used to modify the results from another detector 130, 131 and / or in the spectral model of the results from another detector. For samples that produce Raman light and fluorescence, the detection performed by each detector 130, 131 will be a combination of photons from both effects.

[0119] Photons generated by the Raman effect tend to arrive earlier than photons generated by the sample's fluorescence. Therefore, for the single-photon detector 130, this can lead to... Figure 7 The pixel detection distribution is shown, where a first counting peak corresponding to Raman light is followed by a second counting peak corresponding to fluorescence. These two peaks can overlap, as illustrated by the dashed line 160 showing the Raman peak (which typically has a temporal form similar to that of the excitation light) and the dashed double-dotted line 161 showing the fluorescence peak. For clarity, the following is shown... Figure 7 The histogram shown is noise-free, but in practical applications, significant noise, such as noise from dark counts, can obscure the peaks. To analyze samples based on either Raman or fluorescence peaks, the data is post-processed to remove or otherwise eliminate either the fluorescence or Raman peak from the analysis.

[0120] For the multiphoton detector 131, there is no time division for counting. The count is accumulated by the multiphoton detector over a time period that includes both the Raman peak and the fluorescence peak. Therefore, the count for each wavenumber recorded by the multiphoton detector will be the sum of the photons detected by the Raman effect and the photons detected by the fluorescence. The Raman effect is typically a weak effect relative to fluorescence, and therefore, the number of counts due to the fluorescence of the sample will be much greater than the number of counts due to the Raman effect. Therefore, the form of the spectrum recorded by detector 131 (an example of which is shown in...) Figure 8 (As shown in the diagram) This can be attributed to fluorescence because the effect of Raman photons on the spectrum is negligible. Furthermore, compared to a single-photon detector, the multiphoton detector 131 will likely record a greater number of total counts during the collection period and suffer from less noise, such as dark counts. Therefore, the error in determining the form of the fluorescence spectrum based on detection by detector 131 is likely smaller than the error in determining the form of the fluorescence spectrum based on detection by single-photon detector 130. However, to determine the Raman spectrum of the sample, the output from single-photon detector 130 is analyzed.

[0121] In one embodiment, the fluorescence spectrum determined from the multiphoton detector is used to determine the number of counts to be removed from the detection performed by the single-photon detector 130 at each wavenumber. Specifically, the form of the fluorescence spectrum, such as that determined from the multiphoton detector 131, can be used as a component (reference spectrum) in a fitting process (e.g., a direct classical least squares fitting process), where several reference spectra of known form are fitted to the measured spectral data to determine the components that replicate the measured spectral data and their amplitudes. The fitting process can scale the selected reference spectra (including the fluorescence spectrum) to produce an optimal fit to the spectral data based on a quality factor (e.g., least squares, Bayesian Information Criterion (BIC), etc.). This process at least partially eliminates the contribution of fluorescence to the number of counts recorded by the single-photon detector, thereby enabling a more accurate determination of the components present in the sample and / or the amount of those components based on the Raman effect.

[0122] The fitting process can be applied to each time slot or to blocks of counts across multiple time slots. For example, counts within a Raman time window of 162 can be aggregated, and the fitting process can be performed on the aggregated counts derived from the Raman time window.

[0123] It should be understood that the analysis of the detections performed by detectors 130 and 131 is not limited to the techniques described above, but other analytical techniques may be used, such as those described in EP2956748 A1.

[0124] exist Figure 6In a further embodiment shown, a means for moving the spectrum across detector 130 in the spatial direction S is provided instead of a cylindrical lens. For example, an optical device (such as a mirror, e.g., mirror 145) can be mounted to rotate about an axis AA aligned with the spectral direction λ. A motor 151 is configured to rotate mirror 145 about axis AA such that the spectrum (e.g., having a spatial focus at the detector plane) spans each pixel P1 to P2. n Multiple single-photon detector elements 133 are moved. Motor 151 can rotate mirror 145 at a certain speed, such that for a given point of the laser beam spot on the sample, each pixel P1 to P2... n Multiple single-photon detector elements 133 receive photons generated by excitation at a given point on the sample. A laser pulse can excite a given region for a duration on the order of milliseconds, and then the laser is directed to another given region of the sample. Therefore, a motor 151 can rotate a mirror 145 at a certain speed, such that the spectrum is directed to each pixel P1 to P2 within milliseconds, tens of milliseconds, or hundreds of milliseconds. n All single-photon detector elements 133. Thus, from each pixel P1 to P... n The selected single-photon detector element 133, which is considered to have a sufficiently low dark count, forms a spectrum.

[0125] It should be understood that other optical components can be motorized to shift the spectrum across detector 130. In confocal microscopy, the motorized optics are located behind the slit.

[0126] Modifications and alterations can be made to the above embodiments without departing from the invention as defined herein. In a further embodiment, the positions of detectors 130 and 131 are reversed, such that a spectral light steering mirror 145 is inserted into the optical path of light dispersed by dispersive optics 143 to guide the dispersed spectrum of light to detector 130. The movable spectral light steering mirror 145 can be moved into and out of the optical path of the spectral light by linear or rotational movement. The spectral light steering mirror 145 can be moved in the optical path to change the deflection angle, thereby selectively guiding light to different ports 139, 141, rather than moving the spectral light steering mirror 145 into and out of the optical path of the spectral light. Alternatively, detectors 130 and 131 can be moved to selectively position one of detectors 130 and 131 at a port (e.g., port 141), instead of moving the movable spectral light steering mirror 145. In this embodiment, detector 130 and cylindrical lens 145 can be configured to move together as a single unit, and the movable spectral light steering mirror 145 (as well as the second port) can be omitted. In another embodiment, the movable spectral light steering mirror 145 can be replaced by a beam splitter, so that detectors 130 and 131 can be used simultaneously to detect spectral light during a sample exposure to a series of pulses.

[0127] In a further embodiment, another non-circular lens or mirror other than a cylindrical lens or mirror can be used. For example, a non-circular lens or mirror can cause spectral light to converge or diverge in the spectral plane, but to a different degree than the convergence or divergence of spectral light in the spatial plane, so as to position the spatial focus and the spectral focus at different locations (e.g., a spatial focus located outside the detector plane and a spectral focus located closer to the detector plane).

Claims

1. A spectrometer comprising an optical input for receiving spectral light, a single-photon avalanche diode, an array of multiphoton detector elements, a spectral dispersive optics arranged to disperse the spectral light into a spectrum, and means arranged to perform the following: i) directing the spectrum to both the single-photon avalanche diode and the multiphoton detector element array; and / or ii) Selectively direct the spectrum to either the single-photon avalanche diode or the multiphoton detector element array.

2. The spectrometer according to claim 1, wherein, The multiphoton detector element array is a charge-coupled device.

3. The spectrometer according to any one of the preceding claims, wherein, The device includes optical elements operable to selectively direct the spectral light from the optical input to either the single-photon avalanche diode or the multiphoton detector element array.

4. The spectrometer according to claim 3, wherein, The optical element is a spectral light steering mirror.

5. The spectrometer according to claim 3 or 4, wherein, The optical element is movable to selectively direct the spectral light from the optical input to either the single-photon avalanche diode or the multiphoton detector element array.

6. The spectrometer according to claim 5, wherein, The optical element can be moved from a position inserted into the optical path of the spectral light to a position outside the optical path.

7. The spectrometer according to claim 1 or 2, wherein, The device includes a beam splitter arranged to split the spectral light received by the optical input between the single-photon avalanche diode and the multiphoton detector element array.

8. The spectrometer according to any one of the preceding claims, comprising a single-photon avalanche diode array.

9. The spectrometer according to claim 8, wherein, The spectral dispersive optics are arranged to disperse the spectral light into a spectrum guided across the single-photon avalanche diode array when the device directs the spectrum to the single-photon avalanche diode array.

10. The spectrometer of claim 9, comprising a two-dimensional single-photon avalanche diode array; a dispersive optics device arranged to disperse the spectral light across the two-dimensional single-photon avalanche diode array in a spectral direction; and a diverging optics device located between the dispersive optics device and the two-dimensional single-photon avalanche diode array.

11. The spectrometer according to claim 10, wherein, The two-dimensional single-photon avalanche diode array is divided into pixels, each pixel including a plurality of single-photon avalanche diodes in a spatial direction orthogonal to the spectral direction, and the dispersion device and the diverging optics are configured such that the spectrum is dispersed across two or more of the single-photon avalanche diodes in each pixel in the spatial direction.

12. The spectrometer according to claim 11, wherein, The pixel width of each pixel in the array of the two-dimensional single-photon avalanche diode array is greater than the pixel width of the multiphoton detector element in the spatial direction.

13. The spectrometer of claim 11 or 12, further comprising a processor configured to record the spectrum from a subset of the total number of single-photon avalanche diodes in each pixel.

14. The spectrometer according to claim 13, wherein, Each subset of pixels comprises the same number of single-photon avalanche diodes.

15. A spectroscopic apparatus comprising a spectrometer according to any one of the preceding claims and a spectral path from a sample to the spectrometer for detecting spectral light emitted from the sample when irradiated with excitation light.

16. A spectroscopic method, comprising: Detection of spectral light generated by a sample excited by excitation light using a single-photon avalanche diode; And the spectral light generated by the sample excited by the excitation light is detected using a multiphoton detector element array.

17. The spectroscopic method according to claim 16, wherein, The spectral light is detected sequentially or simultaneously by each detector.

18. The spectroscopic method of claim 16 or 17, comprising correlating the output from the single-photon avalanche diode with the output from the two-dimensional multiphoton detector element array.

19. The spectroscopic method according to any one of claims 16 to 18, wherein, The spectrometer according to any one of claims 1 to 14 is used to detect the spectral light.

20. A method for analyzing spectral data collected by a spectrometer according to any one of claims 1 to 14, the method comprising receiving first spectral data of a sample, the first spectral data being detected by the single-photon avalanche diode; receiving second spectral data of the sample, the second spectral data being detected by the multiphoton detector element array; and correlating the first spectral data with the second spectral data.

Images