Multi-track raman analyser

Abstract

A spectroscopy system acquires Raman measurements from multiple samples or multiple regions of a liquid or solid sample simultaneously. At least two Raman probes acquire spectra from the sample simultaneously using an imaging spectrometer with a single shared two-dimensional scientific grade CMOS sensor. Each probe is coupled into a laser, which can be integrated into the probe, and a spectrometer, and includes a lens that focuses the laser light within or on the sample and collects light from the sample for the spectrometer. The spectrometer simultaneously images light from the multiple probes onto the scientific grade CMOS sensor, spaced apart from one another to reduce cross-talk. Sample positioning apparatus and probe positioning mechanisms can provide relative movement between the sample or sample regions and the probes to acquire data from different subsets of the sample or sample regions, and can also adjust the probe distance from the sample for desired laser focus size and location.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Application No. 19 / 037,340, filed January 27, 2025, which is a partial continuation-in-part of co-owned and equally pending U.S. Application No. 18 / 087,341, filed December 22, 2022, which claims the benefit of U.S. Application No. 63 / 293,733, filed December 24, 2021, pursuant to 35 U.S.SC §119(e). This application also claims the benefit of U.S. Application No. 63 / 625,237, filed January 25, 2024, pursuant to 35 U.S.SC §119(e). The disclosure thereof is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure generally relates to a multi-orbit analyzer that provides Raman measurements alone or in combination with measurements of fluorescence, absorbance and / or other spectral-based liquid samples, which may be contained in well plates, vials, flow cells or solid samples (such as wafers or disks). Background Technology

[0003] Raman measurements can be used alone or in combination with fluorescence, absorbance, and / or other spectral-based measurements for quality control and analysis of a variety of substances during manufacturing. For example, liquid samples can be contained in well plates, individual vials, flow cells, or moved through tubing. Solid samples on disks or wafers can also be analyzed.

[0004] Microplate readers, or well plate readers, are commonly used in life sciences and pharmaceutical applications for the quantification of biological and chemical assays in microplates that can have hundreds or thousands of wells (lumens, tubes, channels, etc.). The optical properties of a sample can change as a biological, chemical, biochemical, or physical reaction occurs (typically at a controlled temperature). Absorption, fluorescence intensity, and luminescence are typical detection methods. For example, automated readers for well plates (or similarly, vial arrays) can be used to provide high-throughput (high-speed) testing for typical well plates containing 8, 48, 96, or 384 wells. While fluorescence, luminescence, and absorption methods are used by many commercially available plate readers with well plates in various forms, including transparent, opaque, translucent, and black, Raman plate readers are rare and slow due to their single-well readout mode using low-power lasers and low-sensitivity CCDs. Depending on the type of reading (fluorescence, Raman, absorption, etc.), the well plate can exhibit a variety of background contributions from the selected material. Therefore, the characteristics (material, color, etc.) of the aperture plate should be evaluated and selected specifically for Raman applications at the chosen laser wavelength.

[0005] Raman microscopy has been used as a microprobe for well plate reading. Another strategy involves scanning 96 to 384 wells with a single Raman probe (using an XY platform) moving relative to the well plate. This solution involves 96 translational movements of a 96-well plate, which reduces the overall throughput, i.e., the data acquisition time for the entire well plate. This strategy typically sacrifices sensor exposure time per well to increase throughput (in terms of wells processed per minute) and is generally a low-sensitivity solution using a CCD sensor and a low-power laser. In this case, a linear (or two-dimensional (2D), vertically merged) CCD is sufficient for single-spectral acquisition when recording signals from 96 wells one by one using a single probe.

[0006] Another strategy for high-speed Raman well plate readout proposes a lens array optics setup (8 × 24 matrices with 4.5 mm spacing) combined with a non-polarizing beamsplitter cube that splits the output of a single laser into each of the multiple wells, resulting in a laser power of only about 7.5 mW per channel. This method provides static simultaneous acquisition of 192 wells. However, compared to other strategies using individual standard Raman probes, where these probes are carefully designed to eliminate crosstalk between the laser and Raman paths, stray light / crosstalk between adjacent wells increases. Laser splitting in free space can also present stability issues, leading to low sensitivity per channel and a low signal-to-noise ratio (SNR) associated with low laser power, especially for low-concentration sample solutions. Another drawback of this method is that it does not allow simultaneous acquisition of fluorescence or other spectroscopic methods on the well plate array. Furthermore, laser power cannot be adjusted up or down for individual probes or sub-sections of the 192-well array.

[0007] For example, various other methods are described in published patent applications US2017 / 0010153 A1, US8520204, and US6266140.

[0008] Various life science applications, such as pharmaceutical drug development or manufacturing, may include product vials moving on conveyor belts or liquids flowing through pipes or flow tanks in the shape of squares, circles or hexagons.

[0009] From development to manufacturing, semiconductors can include wafer process monitoring and control, and materials analysis. Triaxial positioning mechanisms can be used to fully scan the wafer using Raman, photoluminescence, or elliptic polarization methods, but this requires significant processing time when using a single probe due to crosstalk and signal-to-noise ratio limitations. Slurries and liquids, and plasmas, can again be analyzed via flow cells, pipes, or through one or more windows.

[0010] Another application in the energy sector, particularly hydrogen monitoring, can utilize Raman signals. Raman spectroscopy can be acquired through chambers, flow cells, windows in pipes, or using immersion probes, although contamination is a problem in many applications. The challenge in all these applications is achieving the optimal signal-to-noise ratio while keeping the cost of system components reasonable. Summary of the Invention

[0011] As described in more detail herein, multi-track Raman analyzers and methods include at least two Raman probes that deliver light from corresponding points of a solid, liquid, or gaseous sample to the input of an imaging spectrometer with a shared two-dimensional sensor to simultaneously acquire Raman data from the corresponding points and generate correlated Raman spectra. Various types of sample holders and sample / probe positioning devices or mechanisms can be used to analyze several points or regions of single or multiple samples. This system and method can be used to analyze liquid samples ranging from clarified solutions to slurries, solid / powder, gaseous or gas-liquid samples, and plasmas used in wafer fabrication. Solid samples can include semiconductor products or wafers, food, packaging, recycled items, energy production (H2), etc. Applications can include pharmaceutical manufacturing with conveyor belts having two or more rows of vials, semiconductor process liquid flow cells containing wafer cleaning solutions or slurries, semiconductor wafers, etc., for example, analysis from the side, top, or bottom. Liquids flowing in pipes or flow cells can be analyzed with different samples flowing in parallel or with a single flow cell having two or more probes aligned for cross-collection / excitation. Sample / probe positioning can be provided for rotational and / or translational relative motion between the probe and the sample, based on signals from distance sensors that detect the distance to a desired point or surface of the sample, or based on Raman signal optimization or maximization, using optional probe autofocus.

[0012] In various embodiments, a system for obtaining Raman spectra from a sample includes: at least one laser; at least two Raman probes, each Raman probe being positioned to irradiate and receive light from a corresponding point on the sample to simultaneously acquire the Raman spectrum of the sample, each Raman probe including an input end optically coupled to the at least one laser and a sample lens configured to receive light from the input end, focus the light from the input end onto the corresponding point on the sample, collect light from the corresponding point on the sample, and guide the collected light to an output end; and an imaging spectrometer optically coupled to the output ends of the at least two Raman probes, the imaging spectrometer including a diffraction grating (reflective or transmissive) configured to receive light from the at least two lasers. The light collected by the Raman probes is guided to a single two-dimensional scientific-grade CMOS sensor; a sample positioning device configured to move the sample relative to the at least two Raman probes; a probe positioning mechanism configured to translate the at least two Raman probes along at least one axis; and at least one controller communicating with the at least one laser, the imaging spectrometer, the sample positioning device, and the probe positioning mechanism, the at least one controller being programmed to simultaneously acquire the Raman spectra of the corresponding points of the sample based on signals from the two-dimensional scientific-grade CMOS sensor of the spectrometer, and to control at least one of the sample positioning device and the probe positioning mechanism to change the position of the at least two Raman probes relative to the sample. Each of the at least two Raman probes may include: an achromatic collimating lens positioned to receive light from the input; a reflector positioned to receive light from the achromatic collimating lens; a first dichroic long-pass filter configured to reflect light from the reflector having a wavelength below a first cutoff wavelength to the sample lens and transmit light from the sample lens having a wavelength above the first cutoff wavelength; a second dichroic long-pass filter configured to transmit light received from the first dichroic long-pass filter having a wavelength above a second cutoff wavelength and reflect light having a wavelength below the second cutoff wavelength; and an achromatic collecting lens configured to focus light transmitted through the second dichroic long-pass filter onto the output. The Raman probes may be fixed in position relative to each other and move together with the sample in response to a signal from at least one controller. The sample positioning device may include a rotary stage configured to rotate the sample in response to a corresponding signal from at least one controller. The probe positioning mechanism may be configured to translate the Raman probe relative to the sample positioning device along orthogonal X, Y, and Z axes in response to a corresponding signal from at least one controller.In various implementations, the probe positioning mechanism is configured to translate at least two Raman probes in response to a corresponding signal from at least one controller to control the distance between the probes and the sample.

[0013] In one or more embodiments, the output of each Raman probe is optically coupled to the input of the spectrometer via at least one fiber optic cable. The fibers from the Raman probes may be arranged linearly at the input of the spectrometer, wherein the fibers from each Raman probe are spaced apart from the fibers of another Raman probe by at least one fiber diameter. In one embodiment, the output of each Raman probe is optically coupled to the input of the spectrometer via multiple fibers, wherein the fibers from each Raman probe are spaced apart from the fibers of another Raman probe by multiple inactive fibers. In one embodiment, seven (7) active fibers for a first Raman probe are arranged linearly adjacent to each other, with five (5) inactive fibers between the active fibers of the first Raman probe and the active fibers of the second Raman probe.

[0014] In one or more embodiments, the system includes a plurality of lasers, wherein each of the plurality of lasers is optically coupled to the input end of a single, different Raman probe. In another embodiment, the system includes a plurality of lasers, each laser integrated with a corresponding Raman probe. The integrated laser may be contained within the housing of a corresponding Raman probe. In yet another embodiment, the system includes a plurality of lasers, wherein at least two of the plurality of lasers are coupled to the input end of a single Raman probe. In still another embodiment, a single laser is coupled to the input ends of at least two Raman probes.

[0015] The implementation may also include a system with at least two Raman probes positioned to have corresponding probe outputs aligned along a common axis, wherein the system is configured to allow sample movement between the corresponding probe outputs. The sample positioning device may be implemented by a tube, cuvette, or flow cell positioned between the corresponding probe outputs and configured to allow sample flow through the tube, cuvette, or flow cell. The controller may be programmed to sum or otherwise combine scientific-grade CMOS signals from the Raman probes to generate a combined Raman spectrum, wherein lasers output from at least two probes are focused on the same point on the sample. The sample positioning device may include a conveyor or other material handling system configured to move a vial containing the sample between opposing probe outputs. The integrated electronics of the scientific-grade CMOS sensor may also be designed and programmed to combine Raman spectra as needed in each channel, ignoring inter-channel readout regions.

[0016] Various embodiments may include a system for obtaining Raman spectra of a moving liquid sample, the system comprising: at least one laser; at least two Raman probes, each Raman probe positioned along a common axis on opposite sides of the moving liquid sample to illuminate and receive light from corresponding points on the moving liquid sample, thereby simultaneously acquiring the Raman spectrum of the sample, each Raman probe including an input end optically coupled to the at least one laser and a sample lens configured to receive light from the input end, focus the light from the input end onto the corresponding point on the sample, collect the light from the corresponding point on the sample, and [further details on the sample]. The collected light is guided to an output; an imaging spectrometer optically coupled to the output of the at least two Raman probes via multiple fiber optics, the fibers of the at least two Raman probes being linearly arranged at the input of the imaging spectrometer, the imaging spectrometer including a diffraction grating configured to guide the light collected by the at least two Raman probes to a single two-dimensional sensor; and at least one controller communicating with the at least one laser and the imaging spectrometer, the at least one controller being programmed to simultaneously acquire the Raman spectra of the corresponding points of the sample based on signals from the two-dimensional sensor of the spectrometer.

[0017] In various embodiments, moving the liquid sample involves moving the liquid through a tube (circular, square, or other shape) positioned between the output ends of Raman probes, wherein each Raman probe focuses its corresponding output laser onto the same point of the moving liquid sample moving through the tube. The controller can be programmed to sum the signals from the two-dimensional sensor corresponding to the output ends of at least two Raman probes to generate a combined Raman spectrum. In one embodiment, the two-dimensional sensor is a shutterless CCD sensor. In another embodiment, the two-dimensional sensor is a scientific-grade CMOS sensor.

[0018] Various embodiments of the system include multiple lasers, each of which is optically coupled to the input end of a single Raman probe within a Raman probe. In other embodiments, each Raman probe includes an integrated laser that may be contained within a housing of the probe. In one embodiment, the system includes multiple lasers, with at least two of the multiple lasers coupled to the input end of a single Raman probe within a Raman probe. In another embodiment, the system includes lasers coupled to the input ends of at least two Raman probes. In other embodiments, the lasers are integrated within the Raman probes. In various embodiments, moving the liquid sample includes vials containing the liquid sample, wherein the vials are moved between the output ends of opposing Raman probes via a material handling system, wherein the opposing Raman probes cause corresponding output lasers to be focused at points within different vials. In these embodiments, the two-dimensional sensor includes a scientific-grade CMOS sensor, optical fibers associated with each Raman probe are positioned adjacent to each other and spaced apart from optical fibers associated with other Raman probes by a distance corresponding to at least one fiber diameter, and a controller is programmed to generate individual Raman spectra for each Raman probe by simultaneously and independently reading signals from the scientific-grade CMOS sensor. The system may include a separate laser coupled to the input of each Raman probe. In various embodiments, each Raman probe includes: an integrated laser; an achromatic collimating lens positioned to receive light from the laser; a mirror positioned to receive light from the achromatic collimating lens; a first dichroic long-pass filter configured to reflect light from the mirror having a wavelength below a first cutoff wavelength to a sample lens and transmit light from the sample lens having a wavelength above the first cutoff wavelength; a second dichroic long-pass filter configured to transmit light received from the first dichroic long-pass filter having a wavelength above a second cutoff wavelength and reflect light having a wavelength below the second cutoff wavelength; and an achromatic collecting lens configured to focus light transmitted through the second dichroic long-pass filter to an output.

[0019] The implementation may further include a system for obtaining Raman spectra of a flowing liquid sample, the system comprising at least two lasers; at least two Raman probes, each Raman probe positioned along a path of the flowing liquid sample to illuminate and receive light from a corresponding point on the flowing liquid sample, thereby simultaneously acquiring the Raman spectrum of the sample, each Raman probe including an input end optically coupled to a corresponding laser of the at least two lasers and a sample lens, the sample lens being configured to receive light from the input end, focus the light from the input end onto the corresponding point on the sample, collect light from the corresponding point on the sample, and guide the collected light to an output end; and an imaging spectrometer. The imaging spectrometer is optically coupled to the outputs of at least two Raman probes via multiple fiber optics, the fibers of which are linearly arranged at the input of the imaging spectrometer, wherein the fibers associated with each probe are positioned adjacent to each other. The imaging spectrometer includes a diffraction grating configured to guide light collected by the at least two Raman probes to a single two-dimensional scientific-grade CMOS sensor; and at least one controller communicating with the at least one laser and the imaging spectrometer, the controller being programmed to simultaneously acquire Raman spectra of the corresponding points of the sample based on signals from the two-dimensional CMOS sensor of the spectrometer. In one embodiment, the Raman probes have outputs aligned in opposite directions along a common axis, wherein flowing liquid passes between the outputs, and the controller is programmed to sum the signals from the two-dimensional CMOS sensor to generate a combined Raman spectrum corresponding to the at least two Raman probes. In various embodiments, the fibers associated with each probe and the fibers associated with other Raman probes coupled to the imaging spectrometer are spaced apart by a distance corresponding to at least two fiber diameters, and the controller is programmed to generate individual Raman spectra associated with each Raman probe.

[0020] The implementation may also include a method comprising: simultaneously focusing a laser onto at least two points on a solid sample; simultaneously directing light from the at least two points to an imaging spectrometer having a diffraction grating (reflective or transmissive) that redirects the light to a shared two-dimensional scientific-grade CMOS sensor at a wavelength-dependent angle; acquiring Raman spectral data associated with the at least two points from the shared two-dimensional scientific-grade CMOS sensor; and controlling the rotation of the solid sample and the translation of the focused laser by at least one controller to simultaneously acquire Raman spectral data from at least two different points on the solid sample. Simultaneously focusing the laser may include simultaneously focusing light from different lasers associated with each of the at least two points. Simultaneously directing light from the at least two points may include coupling the light from each of the points to at least one corresponding optical fiber and arranging the optical fibers in a linear array at the input of the spectrometer, with the at least one optical fiber associated with each point and the optical fiber associated with the other point separated by a distance corresponding to at least one optical fiber diameter. In various embodiments, the rotation of the solid sample is controlled independently of the translation of the focused laser. Translation of a focused laser can include translation relative to a solid sample in orthogonal X, Y, and / or Z directions.

[0021] A system and method for simultaneously obtaining spectroscopic measurements from at least two liquid samples contained in an array of sample holders (such as microplates or well plates) may include an imaging spectrometer with a two-dimensional sensor that receives light emitted by at least two samples during illumination by a focused laser, the focused laser being directed to the input of the spectrometer and diffracted by a diffraction grating of the spectrometer. The spectroscopic measurements may include Raman and fluorescence measurements, as well as various other measurements obtained with the same or different spectrometers. To reduce inter-sample crosstalk and improve the signal-to-noise ratio (SNR), the at least two samples may be separated or spaced by at least one sample holder or well of the array that is not illuminated by the focused laser during the measurement. To reduce inter-channel crosstalk and further improve the SNR, the light imaged on the spectrometer sensor from each sample is spatially separated from the light from any other sample corresponding to the Raman and / or fluorescence measurements. A spectral probe may be fiber-coupled to the spectrometer and an illumination laser or other light source to facilitate probe positioning within the filling and reaction chamber of an automated liquid handling system, wherein the laser, other light source, one or more controllers, and the spectrometer are positioned outside the reaction chamber. Calibration standards can be integrated into the orifice plate holder for automatic periodic calibration and adjustment of laser wavelength, intensity, and / or focus.

[0022] In one embodiment, a system for obtaining Raman spectra from liquid samples within a sample holder array includes: at least one laser; at least two Raman probes, each Raman probe positioned perpendicular to the sample holder array and aligned to simultaneously acquire Raman spectra of liquid samples in non-adjacent sample holders, each Raman probe including an input end optically coupled to the at least one laser and a sample lens configured to receive light from the input end, focus the light from the input end onto an associated liquid sample, collect light from the associated liquid sample, and guide the light collected from the associated liquid sample to an output end; and a spectrometer optically coupled to the output ends of the at least two Raman probes, the spectrometer... The instrument includes a diffraction grating configured to guide light collected by the Raman probes to a sensor; a mechanical positioning mechanism configured to adjust the relative position between the sample holder array and the at least two Raman probes; and at least one controller communicating with the at least one laser, the spectrometer, and the mechanical positioning mechanism, the at least one controller being programmed to generate Raman spectra for the liquid samples in the at least two non-adjacent sample holders based on signals from the sensor of the spectrometer, and to control the positioning mechanism to change the position of the at least two Raman probes relative to the sample holder array to simultaneously acquire Raman spectra of the liquid samples in different non-adjacent sample holders of the array.

[0023] In various embodiments, each Raman probe includes: an achromatic collimating lens positioned to receive light from an input end; a reflector positioned to receive light from the achromatic collimating lens; a first dichroic long-pass filter configured to reflect light from the reflector having a wavelength below a first cutoff wavelength to a sample lens and transmit light from the sample lens having a wavelength above the first cutoff wavelength; a second dichroic long-pass filter configured to transmit light received from the first dichroic long-pass filter having a wavelength above a second cutoff wavelength and reflect light having a wavelength below the second cutoff wavelength; and an achromatic collecting lens configured to focus light transmitted through the second dichroic long-pass filter to an output end. The Raman probe may also include at least one bandpass filter in the optical path located between the achromatic collimating lens and the reflector. In various implementations, the output of each Raman probe is optically coupled to the input of the spectrometer via at least one fiber optic cable, wherein the fibers from the Raman probes are linearly arranged at the input of the spectrometer, and the fibers from each Raman probe are spaced apart from the fibers of another Raman probe by a distance of at least one fiber diameter. This spacing can be provided by one or more non-light-carrying, inactive or dead-zone fibers located between the fibers associated with the Raman or fluorescence probes in the linear fiber array. The fibers from the Raman probes can be arranged closer to the center of the linear array than the output fibers from the fluorescence probes, such that the light from the Raman probes is guided closer to the optical axis of the spectrometer's sensor than the light from the fluorescence probes.

[0024] In one or more embodiments, the spectrometer includes a diffraction grating comprising a reflective aberration-corrected holographic concave grating, and the sensor includes a two-dimensional imaging sensor, such as a scientific-grade CMOS sensor or a CCD sensor with a shutter. In one embodiment, all Raman and fluorescence probes are optically coupled to a single spectrometer having a single two-dimensional sensor. Other embodiments include spectrometers with separate diffraction gratings for each connected probe, wherein a linear CCD sensor is shared by two or more probes. Embodiments may also include separate spectrometers optimized for fluorescence / photoluminescence or Raman data and optically coupled to associated probes. The spectrometer may include a slit located in the optical path between the fiber from each Raman probe and the diffraction grating of the spectrometer. The slit width may be selected from a plurality of fixed-width slits by at least one controller, or may have a variable width controlled by at least one controller.

[0025] The mechanical positioning mechanism can be configured to move a sample holder array relative to at least two Raman probes (and a fluorescence probe, if present) to orthogonal X and Y positions corresponding to the sample holders in the array. The Z-axis position can also be controlled to change the position of the focused laser within the sample. The positioning mechanism may include a well plate holder with one or more integrated calibration standard samples (such as diamond samples). Furthermore, the well plate holder can be configured to hold a calibration plate having one or more calibration standard samples (e.g., diamond, silicon), and / or containing orifices of a custom liquid calibration sample for a specific application. At least one controller can be programmed to change the relative position between the Raman probes and the sample holder array to alter the position of the focused laser within the liquid sample while acquiring data from spectrometer sensors to generate Raman spectra, thereby reducing or eliminating localized heating and / or photobleaching of the sample and improving sample homogeneity and / or averaging of measurements from different locations within the sample.

[0026] Various implementations may include at least one laser associated with each Raman probe or with a small number of Raman probes (e.g., two to four probes). The laser may be a single-mode or multi-mode laser. Separate light sources may be provided for the fluorescence / photoluminescence probes, with multiple fluorescence / photoluminescence probes connected to a single light source. Raman and fluorescence / photoluminescence probes may be implemented as non-contact or immersion probes. Raman and fluorescence / photoluminescence probes may be located above or below the sample and may be positioned perpendicular to the sample array. In one implementation, the laser source focused on or within the sample is spatially offset from the Raman probes, at an angle or perpendicular to the sample array. The illumination source may be positioned opposite the probe's collection source to provide transmission Raman or fluorescence measurements.

[0027] In one embodiment, an additional laser is optically coupled to the input of an additional Raman probe, which has an output coupled to the spectrometer and is positioned to acquire the Raman spectrum of a reference sample not included in the sample holder array.

[0028] In one or more embodiments, a system for obtaining Raman spectra from a liquid sample contained within a well plate includes: at least one laser optically coupled to at least two optical fibers to selectively focus the laser light into a sample contained within at least two wells of the well plate, the at least two wells being separated by at least one well not irradiated by the focused laser; at least two Raman probes, each Raman probe aligned with a corresponding well among the at least two wells of the well plate irradiated by the focused laser, each Raman probe including a lens configured to focus light from the corresponding sample onto the output of the Raman probe; and a spectrometer optically coupled to the output of the at least two Raman probes. The spectrometer includes a diffraction grating configured to guide light collected by the at least two probes to a shared sensor; a mechanical positioning mechanism configured to adjust the relative position between the well plate and the at least two Raman probes; and at least one controller communicating with the spectrometer and the mechanical positioning mechanism, the at least one controller being programmed to generate Raman spectra for the liquid sample in the at least two wells based on signals from the shared sensor of the spectrometer, and to control the positioning mechanism to change the relative position between the at least two probes and the well plate to simultaneously acquire Raman spectra of the liquid sample in at least two different wells of the well plate. The relative position may include three-dimensional positioning of the well plate relative to the probes. Each of the at least two optical fibers can focus a laser at an angle relative to an associated Raman probe among the at least two Raman probes that collect light emitted from the associated sample into an associated well among the at least two wells. The optical fibers may be positioned to focus the laser into the sample from the opposite direction relative to the Raman probes. The system may include a separate laser associated with each optical fiber. The spectrometer may include a two-dimensional sensor, wherein the output of a Raman probe is connected to the input of the spectrometer via corresponding optical fibers arranged in a linear array at the input of the spectrometer, wherein the optical fibers associated with each probe are positioned adjacent to each other and spaced apart from the optical fibers associated with other probes by a distance corresponding to at least one optical fiber diameter.

[0029] In various embodiments, the system includes a plurality of fluorescent probes corresponding in number to a plurality of Raman probes, each fluorescent probe being fixed to move together with its associated Raman probe, the fluorescent probes being aligned with associated wells of a well plate, the associated wells being spaced apart by at least one well not aligned with any other fluorescent probe or Raman probe. The fluorescent probes may be optically coupled to an input of a spectrometer, and at least one controller may also be programmed to simultaneously acquire fluorescence data from a shared sensor. In some embodiments, the fluorescent probes and Raman probes may be aligned with the same wells for simultaneous Raman exposure, followed by simultaneous fluorescence exposure of each subset or group of wells associated with the probes. The shared sensor may include a two-dimensional scientific-grade CMOS sensor or a CCD sensor with a shutter.

[0030] In one or more embodiments, the outputs of at least two Raman probes are connected to the input of the spectrometer via corresponding optical fibers. Each of a plurality of fluorescence probes is coupled to the input of the spectrometer via at least one optical fiber. The optical fibers of each Raman probe are linearly arranged and grouped together, and the optical fibers of each fluorescence probe are linearly arranged and grouped together. The grouped optical fibers of each Raman probe are spaced apart from the grouped optical fibers of any other Raman probe or any fluorescence probe by a distance corresponding to at least one optical fiber diameter, and the grouped optical fibers of each fluorescence probe are spaced apart from the grouped optical fibers of any other fluorescence probe or any Raman probe by a distance corresponding to at least one optical fiber diameter. The grouped optical fibers of each Raman probe may be positioned closer to the center of the linear arrangement than the grouped optical fibers of each fluorescence probe. The Raman probes, fluorescence probes, at least two optical fibers coupled to at least one laser, and a mechanical positioning mechanism may be positioned within the reaction chamber of an automated liquid handling system, wherein the spectrometer and at least one laser are positioned outside the reaction chamber. The mechanical positioning mechanism may include an orifice holder having an integrated calibration standard sample capable of being positioned by at least one controller for alignment with one or more of the probes. The wavelength and / or intensity of the laser associated with the corresponding probe may be adjusted in response to measurements obtained from the calibration standard sample. In some implementations, the system may include a second spectrometer optimized for fluorescence measurements, which has an input that is optically coupled to the output of a plurality of fluorescent probes.

[0031] The implementation may also include a method comprising: simultaneously focusing a laser into at least two samples contained in corresponding wells of a well plate, the at least two samples being contained in wells spaced apart by at least one well not illuminated by the focused laser; simultaneously directing light emitted from the at least two samples to a spectrometer having a diffraction grating that redirects the light to a shared sensor at a wavelength-dependent angle; acquiring Raman spectral data associated with the at least two samples from the shared sensor; and controlling the positioning of the well plate relative to the focused laser by at least one controller to simultaneously acquire Raman spectral data from at least two different samples contained in previously unexamined wells of the well plate. Simultaneous focusing of the laser may include simultaneously focusing light from different lasers associated with each of the at least two samples. The method may further include: simultaneously directing light from a source into at least two samples contained in corresponding wells of the well plate, each well being spaced apart by at least one well illuminated by an unfocused laser or light from the source; simultaneously directing light emitted from samples illuminated by light from the source to a shared sensor of the spectrometer; and acquiring fluorescence data associated with the at least two samples illuminated by light from the source from the shared sensor of the spectrometer.

[0032] In at least one embodiment, simultaneously guiding light emitted by at least two samples includes coupling the emitted light to at least one optical fiber for each of the at least two samples, and arranging the optical fibers in a linear array at the input of the spectrometer, wherein the optical fibers associated with each sample are grouped together and separated from the optical fibers associated with the other sample by a distance corresponding to at least one optical fiber diameter.

[0033] Various implementations may also include positioning a calibration standard integrated within an orifice plate holder for irradiation by a focused laser via at least one controller; acquiring Raman spectral data from the calibration standard; and adjusting the wavelength of the focused laser based on a comparison of the Raman spectral data of the calibration standard with a previously stored peak wavelength of the calibration standard.

[0034] One or more embodiments according to this disclosure can provide associated advantages. For example, various embodiments provide high-throughput Raman measurements using Raman spectroscopy and spatially shifted Raman spectroscopy (SORS), as well as various combinations of Raman spectroscopy with absorbance measurements, fluorescence intensity, luminescence, time-resolved fluorescence, alpha screen detection, and many other types of spectral and non-spectral measurements of samples contained in the well plate. Embodiments can use various types of two-dimensional (2D) sensors, such as one or more CCD, CMOS, or scientific-grade CMOS sensors, to provide full-well imaging. Using a laser in its full-power / full-well mode (any typical UV, VIS, and NIR laser, typically 532 nm and 785 m), or laser output split across several wells, maintains a reasonably high laser output / channel and facilitates tuning the laser wavelength and / or intensity of the associated probes. Assigning Raman probes to each channel ensures optimal sensitivity, low stray light, and maximized SNR. The system can provide automated calibration using one or more calibration standards embedded in a plate holder or a calibration well plate containing one or more calibration standards and / or wells configured to contain liquid calibration samples. Fiber-coupled Raman probes can be integrated with automated plate processor systems positioned within the filling and reaction chambers and coupled to remotely positioned lasers and spectrometers to enable real-time or near-real-time spectral acquisition for real-time monitoring of reactions within the well plates without prior quenching or dilution before analysis. Various implementations may include integration with an automated plate processor system that automatically transfers the well plate from the filling and reaction chambers to the analysis chamber for Raman data acquisition via a multi-probe system. Attached Figure Description

[0035] Figure 1 A representative scanning pattern of a well plate with four Raman probes is shown.

[0036] Figure 2 Existing technology strategies for full-frame readout of CMOS sensors are illustrated.

[0037] Figure 3A The readout of the 2D CCD sensor is shown.

[0038] Figure 3B The readout of a shutterless 2D CMOS or scientific-grade CMOS sensor is shown.

[0039] Figure 3C A plan view of a representative 2D scientific-grade CMOS sensor is shown.

[0040] Figure 4 The components of a multi-probe Raman plate reader are shown.

[0041] Figure 5 Components of another embodiment of the multi-probe Raman plate reader are shown.

[0042] Figure 6 A system or method for simultaneously acquiring Raman and fluorescence data from a well plate is shown.

[0043] Figure 7A The diagram shows a linearly arranged optical fiber, with each channel having a single active fiber and an optional slit for input to the spectrometer.

[0044] Figure 7B Another configuration of linearly arranged optical fibers is shown, with three active fibers per channel and an optional slit at the spectrometer input, the opposite ends of the fibers being grouped into triangular clusters.

[0045] Figure 7C Another configuration of linearly arranged optical fibers is shown, with seven active fibers per channel and an optional slit at the spectrometer input, the opposite ends of the fibers being grouped into hexagonal clusters.

[0046] Figure 7D Another configuration of a dual-row linear arrangement of optical fibers is shown, wherein each channel has three active fibers and an optional slit array at the spectrometer input, wherein the resulting spectrum is laterally shifted on the spectrometer sensor.

[0047] Figure 8 This is a simplified diagram illustrating a perforated plate reader with combined Raman and full-hole imaging.

[0048] Figure 9A This is a perspective view showing the components inside a representative Raman probe, including the input and output fiber optic connectors.

[0049] Figure 9B yes Figure 9A The optical layout of a representative probe.

[0050] Figure 10 A representative fluorescent probe is shown.

[0051] Figure 11 A representative configuration of spatially shifted Raman spectroscopy (SORS) is shown.

[0052] Figure 12 An SORS system according to this disclosure is shown, which can be used to obtain simultaneous Raman measurements from multiple orifices of an orifice plate.

[0053] Figure 13 A representative imaging spectrometer is shown for use in a perforated plate reader according to this disclosure.

[0054] Figure 14AA representative non-imaging spectrometer with a shared CCD sensor for use in an orifice plate reader is shown according to this disclosure.

[0055] Figure 14B This is a simplified functional block diagram illustrating sensor control for a non-imaging spectrometer with a shared CCD sensor.

[0056] Figure 14C This is a cross-sectional view showing the lower left / lower right optical chamber of a non-imaging spectrometer with a shared CCD sensor.

[0057] Figure 15 This is a partial perspective view showing the various components of a representative perforated plate reader.

[0058] Figure 16 A representative positioning mechanism with an orifice plate holder having an integrated calibration standard sample is shown according to this disclosure.

[0059] Figure 17 A representative calibration plate with one or more probes for a well plate reader is shown according to this disclosure.

[0060] Figure 18 This is a block diagram illustrating the operation of a system or method for simultaneously acquiring spectral measurements.

[0061] Figure 19 This is a block diagram illustrating the integration of a multi-probe spectroscopy system with an automated liquid handling system according to the present disclosure.

[0062] Figure 20 This is a block diagram illustrating an embodiment of a Raman analyzer according to the present disclosure, wherein opposing Raman probes are focused on the same point on a moving or flowing liquid sample.

[0063] Figure 21 This is a block diagram illustrating an embodiment of a Raman analyzer according to the present disclosure, wherein a Raman probe is focused on different points or regions of a moving or flowing liquid sample.

[0064] Figure 22 This is a block diagram illustrating an embodiment of a Raman analyzer according to the present disclosure, wherein opposing Raman probes are focused on different points of a moving or flowing sample.

[0065] Figure 23 A representative embodiment of a Raman analyzer according to the present disclosure is shown, which includes a sample positioning device and a probe positioning mechanism for analyzing solid samples.

[0066] Figure 24 A representative embodiment of a sample positioning device including a controllable rotary stage according to the present disclosure is shown.

[0067] Figure 25 This is a diagram illustrating the operation of a system or method for simultaneously acquiring Raman spectral data from at least two Raman probes using a rotating sample positioning device and a translational probe positioning mechanism.

[0068] Figures 26A to 26C Representative locations of sample points for simultaneously acquiring Raman spectra of solid samples are shown according to this disclosure.

[0069] Figure 27 The results of software simulations of a representative Raman analyzer embodiment according to this disclosure are shown, demonstrating that there is no crosstalk between Raman probe channels sharing a single two-dimensional scientific-grade CMOS sensor.

[0070] Figure 28 This is a diagram illustrating the operation of a Raman analyzer or method according to the present disclosure for simultaneously acquiring Raman spectra from two or more points on a sample.

[0071] Figure 29 Another representative implementation of a Raman analyzer with multiple probes focused at different points within a liquid sample is shown.

[0072] Figure 30 Another representative implementation of a Raman analyzer is shown, which has two or more Raman probes focused at the same point on a solid sample, thereby improving signal or time gain through cross-collection. Detailed Implementation

[0073] This document describes embodiments of the present disclosure. However, it should be understood that the disclosed embodiments are merely examples, and other embodiments may take various and alternative forms. The drawings are not necessarily drawn to scale; some features may be enlarged or minimized to show detail of specific components. Therefore, the specific structural and functional details disclosed herein should not be construed as limiting, but only as a representative basis for teaching those skilled in the art to employ the claimed subject matter in various ways. As will be understood by those skilled in the art, the various features shown and described with reference to any of the drawings may be combined with features shown in one or more other drawings to produce embodiments not explicitly shown or described. The combinations of features shown provide representative embodiments for typical applications. However, for a particular application or implementation, various combinations and modifications of features consistent with the teachings of this disclosure may be required.

[0074] As described in this disclosure, a well plate refers to a flat plate or tray having multiple wells spaced apart from each other and configured to accommodate liquid samples typically in the nanoliter to milliliter volume range. Well plates typically contain 96,384 or 1,536 wells, although they can be customized for specific applications. Well plates may also be referred to as multiwell plates, microplates, microtiter plates, microplates, or similar variations. Well plates may be made of molded plastics such as polystyrene, polypropylene, or polycarbonate, or may include more complex constructions to provide desired properties for a particular type of measurement. Well plates may be transparent or colored with white or black pigments, for example, to provide desired optical properties suitable for a particular desired measurement. Similarly, well plate measurements can be obtained with open cavities, or a sealing cap or cover plate can be placed over the cavities after sample loading.

[0075] One or more configurations of the well plate reader according to this disclosure use multiple Raman probes arranged in an array to provide rapid readout of the well plate, wherein a laser is associated with each well to be read, thereby enabling full-power laser operation. Commercially available UV, VIS, or NIR lasers can be used. Many applications use lasers operating at 532 nm or 785 nm. This configuration allows for individual laser power adjustment for each of the corresponding mapped / associated portions of the individual wells scanned by the laser. For example, a well plate with 96 wells including four probes (each probe having an associated laser) would allow for individual adjustment of the laser power for each of four regions comprising 24 wells. This is advantageous for handling wells containing varying sample concentrations while reducing or eliminating the risk of localized heating and / or photobleaching of the sample material during readout. In some configurations, one or more lasers may include outputs separated between a small number of wells (e.g., using associated optical fibers) to maintain the desired laser output / channel using a fully optically sealed Raman probe associated with each channel, ensuring optimal sensitivity, low stray light, and the desired SNR. The probe array can consist of Raman probes or a combination of Raman and fluorescent probes, or various other types of probes.

[0076] Two-dimensional (2D) CCD sensors can be used in known configurations that utilize commercially available spectrometer and CCD camera arrangements to simultaneously acquire spectra from a large number of apertures. For example, Isoplane... TM (Roper Princeton Instruments) The spectrometer can be configured with Pixis Isoplane. TMBoth the CCD camera (Roper Princeton Instruments) and the camera are available from Teledyne Princeton Instruments, Inc. Conventional CMOS sensors suffer from excessively high dark current and insufficient thermoelectric (TE) cooling performance, making them unsuitable for high-speed Raman detection systems. According to one or more embodiments of this disclosure, a compact scientific-grade CMOS sensor / camera, such as the Sylent-Blue from HORIBA Instruments Incorporated, can be used to achieve low readout noise in a camera integrated at -25°C to -40°C with air or water TE vacuum cooling, ranging from less than 1e to 2e and low dark current (less than 1e / pixelsec). This allows for the simultaneous acquisition of virtually noise-free Raman spectra in the described embodiments. Such scientific-grade CMOS sensors are typically back-side-illuminated (BSI) (also known as back-illuminated (BI) or back-thin (BT)) sensors to achieve high quantum efficiency (QE) approaching 95% in the visible range.

[0077] Scientific-grade CMOS sensors eliminate the need for a shutter when using 2D CCDs (a problem carefully avoided in Isoplane / Pixis configurations), as this issue exists in full-frame CCDs and is eliminated using only interline CCDs (no shutter), which unfortunately is characterized by very low quantum efficiency (QE). They are generally not used due to the prohibitive cost and size of frame-transfer CCDs, but can be used for specialized applications. Imaging spectrometers, such as many compact imaging spectrometers available from Horiba Instruments Co., Ltd., combine high spectral resolution (required for Raman) with multi-fiber input (arranged along a slit) capability. The CiCi-Raman-532 HORIBA spectrometer according to one or more embodiments described herein, wherein the CCD is replaced by a SylentTE-cooled CMOS camera, is characterized by having 4 to 12 channels, each with high sensitivity if properly configured with custom fiber bundles (see possible arrays, each channel having 1 to 7 active fibers separated by 1, 2, 3 or more dead-zone fibers for spacing and crosstalk minimization, well below 1%) and optimized slit widths (optional for high-resolution requirements). The active fiber channels can be arranged linearly in two or more columns shifted or offset from each other.

[0078] As the inventors of this disclosure recognize, the need for a high-throughput well plate reader can be addressed by combining Raman probes / lasers and fluorescence (FLUO) probes / sources into a hybrid array, allowing for the simultaneous acquisition of Raman and fluorescence measurements from different wells. This avoids crosstalk and reduces the verbose processing time otherwise associated with sequential acquisition. Furthermore, all spectra can be collected on the same imaging spectrometer with a scientific-grade CMOS sensor, which can optionally be cooled. Fluorescence typically covers a broad spectral region, while Raman is used for a narrow range (3,000–4,000 1 / cm). Compared to a Raman-only configuration, the hybrid system may require translation between the probes and the well plate, allowing the two combined arrays of Raman and fluorescence probes to be positioned to cover all wells. Because the Raman and fluorescence probes are not positioned to be simultaneously focused into the same wells, there is no significant stray light or crosstalk (such as crosstalk from the laser to fluorescence measurements or from fluorescence measurements to Raman), and there is no need to continuously turn the light source on / off for Raman and fluorescence measurements. Therefore, the total acquisition time for the entire well plate is significantly reduced. All probes can be positioned above the well plate, or some probes can be positioned below the well plate, depending on the specific configuration. Two probe arrays (Raman and fluorescence) can be provided that move on the same platform; these two arrays can be non-adjacent, or if one array is on top of the well plate and the other is below, then the two probe arrays move on two separate platforms. The latter configuration may require wells with flat, transparent bottoms for optimal performance.

[0079] In some configurations, the hybrid system may include Raman and fluorescence probes positioned to read the same well. For example, the Raman and fluorescence probes may be aimed at the same well at a small angle (away from vertical), where Raman and fluorescence readings are performed sequentially on the well before the platform (or probe, depending on the application) is adjusted to read the next well.

[0080] Figure 1A representative array of sample holders implemented as a multiwell plate 100 and a related scanning pattern for a multiprobe plate reader are shown. The well plate 100 is shown in a top / bottom view or plan view, where individual wells 102 are arranged in a rectangular array of twelve columns and eight rows, providing a total of 96 wells extending into or beyond the plane of the drawing, which can be filled with a liquid sample. As previously mentioned, well plates or microplates are commercially available in various sizes, with common configurations including arrays of 48, 96, 192, 384, or 1,536 wells. While rectangular arrays are shown, other configurations are possible, such as arrays with offset alternating rows, or two different arrays of the same or different shapes / configurations separated by space between them. Those skilled in the art will recognize that the claimed subject matter is generally independent of the specific configuration or number of wells or other sample holders within the array. Similarly, well plates can be made of various types of materials having properties suitable for the specific types of liquid samples described above. Well plate or other sample holder materials can be selected to provide the desired optical properties for spectroscopic analysis. For example, black or other optically opaque or absorbing apertures can be used to reduce or eliminate stray light and inter-aperture crosstalk during exposure readouts that acquire spectral measurements from multiple apertures simultaneously. Various system optics can also be selected or adjusted to optimize one or more desired system parameters, such as sensitivity, exposure time, and associated throughput, based on the optical properties of the selected aperture plate material.

[0081] The well plate 100 may include a cap (not shown) placed over the wells to contain liquid samples. Depending on the specific application and implementation, the cap may remain in place or may be removed during spectral analysis. In this example, the reader includes four (4) Raman probes configured to simultaneously read four wells with initial positions as shown by solid dots 110, 112, 114, and 116. In this example, the well plate is translated in the xy plane in a pattern indicated by arrow 120 via an associated mechanical positioning mechanism including a well plate holder, such that each probe will read one of its associated 24 wells, with four probes simultaneously reading the corresponding four wells of the well plate. Alternatively, the probes may be translated relative to the well plate. Using multiple probes to simultaneously capture spectra from multiple wells reduces the number of xy movements compared to applications with fewer probes or a single probe, thereby increasing system throughput.

[0082] like Figure 1As shown, four probes are aligned with non-adjacent wells to reduce stray light from other probes and associated inter-well crosstalk. In the representative configuration shown, probe position 110 is separated from probe position 112 by five wells and from probe position 114 by four wells. The probe spacing can vary depending on the specific requirements of each application, which may include the optical properties of the liquid sample, the well plate material, the laser wavelength, the sample concentration, etc. Figure 1 In the example, the adjacent holes of probe position 110 will include the holes at positions A2, B1, and B2, where all other holes are not adjacent to probe position 110 (A1). Similarly, the adjacent holes of probe position 116 will include the holes at positions D6, D7, D8, E6, E8, F6, F7, and F8, where all other holes are not adjacent to position 116.

[0083] Figure 2 This illustrates the trailing that can occur when reading a full-frame CCD (vertical transfer) without using a shutter. As previously described, frame-transfer CCDs are generally not used due to their prohibitive cost and size, but can be used for specialized applications.

[0084] Figure 3A The reading of a scientific CCD sensor with a shutter (not shown) that can be used in various applications according to this disclosure is illustrated. The shutter can be closed during sensor readout to reduce or eliminate motion blur. Figures 3B to 3C The following are illustrated examples of shutterless scientific-grade CMOS (sCMOS) used in various embodiments according to this disclosure. TM The sensor provides fast and direct readouts without trailing. (As in...) Figure 3B Generally represented herein, a 2D CMOS or scientific-grade CMOS sensor includes a 2D array 200 of pixels 210, each pixel having associated readout circuitry 212 to provide fast and direct readout without using a shutter and without trailing. The 2D scientific-grade CMOS sensor allows for multiple spectral readouts along its height within the image-free defocus plane of an imaging spectrometer, as described in more detail herein.

[0085] Figure 4 The components of a representative multi-probe plate reader 400 are shown, which include four Raman probes 410 to provide simultaneous reading of samples contained in four non-adjacent wells, the initial positions of which are at A1, A7, E1 and E7 of the well plate 420. Figure 4The quaternary configuration comprises four fiber-coupled 532 nm, 150 mW, single-mode lasers 430 and probes 410, fixed relative to each other and vertically positioned above an aperture plate 420, as shown in the solid circles. Each probe 410 is optically coupled via at least one fiber 432 to provide excitation light from a corresponding laser 430 perpendicular to a plane of the aperture plate 420, wherein the laser is focused within a liquid sample contained in a corresponding aperture extending into the plane of the drawing. In various applications, the laser is focused directly below the surface of the liquid sample to minimize the laser's influence on the sample and to minimize absorption to improve the SNR of Raman measurements. Other applications may focus the laser deeper within the liquid sample and / or obtain measurements from more than one depth within the sample.

[0086] The Raman signal shares the same lens 434 with the excitation light from laser 430. The Raman signal is collected through this common (identical) lens 434. The output from the probes is provided to a single 4-channel imaging spectrometer 440 by at least one corresponding optical fiber 480 associated with each probe. This spectrometer includes a 2D imaging cooled camera 450 with a scientific CCD (in combination with a shutter) or a 2D CMOS sensor 460 (without a shutter). As shown, the output light from the probes can be provided to the input of a single spectrometer by a linear array 470 of optical fibers 480, 482, 484, and 486, spaced at least one fiber diameter apart to reduce inter-channel crosstalk. The spacing between the active fibers can be provided by one or more dead-zone or inactive fibers 492, which do not carry light and may extend partially or completely within the fiber bundle to the corresponding probe 410, or may not extend partially or completely within the fiber bundle to the corresponding probe 410. Other mechanical spacers can be used instead of dead-zone fibers. In the representative example shown, each active fiber 480, 482, 484, and 486 is spaced apart by three fiber diameters from the inactive or dead fiber indicated at 492. The active fibers are linearly arranged to form a line entering the spectrometer and may optionally be coupled to the entrance slit (in... Figures 7A to 7D (Best illustrated in the diagram). The entrance slit can be a fixed slit. In other embodiments, the slit is selected from one of a plurality of fixed slits and is moved into place by an associated controller, such as by translation or rotation of a multi-slit wheel or device. Alternatively, a variable-width slit can be controlled by a corresponding controller to provide the desired slit width.

[0087] Each valid fiber 480, 482, 484, and 486 generates corresponding lines 480′, 482′, 484′, and 486′ on sensor 460, where the unread lines of sensor 492′ are matched with invalid spaced fibers 492. Raman signals 496 correspond to selected merged lines of sensor pixels, which can be merged by associated software or hardware (FPGA / FW). For example, each line 480′, 482′, 484′, and 486′ may correspond to multiple rows of sensor pixels. Reads associated with a specific line or channel are merged or grouped together to generate the corresponding Raman spectrum 496, thereby simultaneously acquiring Raman spectral data to generate four corresponding Raman spectra. After acquiring Raman measurements of the samples in orifices A1, A7, E1, and E7, at least one controller moves the associated mechanical positioning mechanism (such as a 2D or 3D platform with a plate holder 420) to the next position to simultaneously acquire data from the samples in orifices A2, A8, E2, and E8. This process is repeated, as shown in the representative scanning pattern, to obtain data for all samples contained in the well plate 420. A reset or reset signal returns the well plate 420 to the indicated initial position. As previously described, the relative position between the well plate 420 and the Raman probe 410 can be controlled by one or more controllers by moving the probe 410, the well plate 420, or both.

[0088] The spectrometer 440 can be implemented using a near-1:1 imaging spectrometer including a reflective concave aberration-corrected holographic grating 442 or various other concentric spectrometer configurations (Dyson, Offner, toroidal, Schmidt-Czerny-Turner, grating imaging spectrometers, etc.), all characterized by low to astigmatism. The number of fiber optic channels used in this multi-track configuration is limited by the spectrometer's aberrations (affecting crosstalk and resolution) and the height of the sensor 460 of the cooled camera 450. Furthermore, the number of fiber optic channels is also limited by the size of the physical array of probes that can be mounted on the top or bottom of the aperture plate and the desired cost of the system.

[0089] Figure 5 It shows something similar to about Figure 4A representative embodiment 500 of the illustrated and described scheme includes twelve Raman probes 510 for simultaneously acquiring data from liquid samples contained in corresponding wells of a well plate 520 having 96 wells. The system may include twelve lasers 530 (e.g., tailstocks) coupled to the probes 510 via optical fibers 532. Alternatively, fewer lasers may be used, each with an associated beam splitter to deliver an excitation beam to two or more probes 510. In this embodiment, the twelve non-adjacent probes 510 are arranged perpendicular to the plane of the well plate 520 to read associated spaced-apart wells, which are spaced apart from each other by at least one well in each direction, and which at least one well does not receive focused laser light from the probes 510 during the current measurement.

[0090] A representative scanning pattern 502 is shown, such that each of the twelve probes 510 acquires Raman data from one of the six wells of the plate during each measurement cycle. For example, in Figure 5 The initial position is shown, and measurement results are simultaneously obtained for samples contained in wells A1, A5, A9, C1, C5, C9, E1, E5, E9, G1, G5, and G9. After each measurement, the relative position between the probe and the well plate is changed by moving the well plate, moving the probe, or redirecting the excitation light from the probe. In one example, after obtaining simultaneous measurement results from a first subset of the wells, at least one controller operates at least one mechanical positioning mechanism to change the relative position between the well plate 520 and the probe 534, wherein measurement results are simultaneously obtained from the next subset of twelve wells corresponding to wells A2, A6, A10, C2, C6, C10, E2, E6, E10, G2, G6, and G10 in this example. This process is repeated until measurement results are obtained from samples in all wells of the well plate 520. Of course, the positioning / arrangement of the probe 510 and the reading or scanning pattern 502 can vary depending on the application and implementation.

[0091] Figure 5 Examples are similar to Figure 4In this example, the output of probe 510 is optically coupled to a multichannel spectrometer 540 via corresponding optical fibers (each probe may include one or more optical fibers), which has an imaging camera 550 with a 2D CCD or CMOS sensor 560. One end of the output optical fibers 580, 581, ..., 590, 591 from probe 510 is linearly arranged at the input of spectrometer 540 as shown in 570, spaced apart from each other by a distance of at least one fiber diameter (three fiber diameters in this example). The spacing between fibers 580-591 may be provided by one or more invalid or non-functional optical fibers 592. Light from each fiber forms corresponding lines 580′, 581′, ..., 590′, 591′ on the 2D sensor 560. Each line may correspond to multiple rows of sensor pixels, where each column in the multiple rows is merged or combined to generate a corresponding Raman spectrum 596. Each line is separated from multiple rows of non-read sensor pixels by non-functional optical fibers 592′ to reduce inter-channel crosstalk and increase SNR. The position of the fiber corresponding to a particular Raman probe relative to the fiber corresponding to another probe is chosen for convenience and can vary depending on the application. For example, fiber 580 can be positioned anywhere within a linear arrangement, rather than at the top or bottom. For applications with more than one fiber associated with the output from one probe, the fibers from each probe are grouped together and positioned adjacent to each other, spaced apart from the fibers of other probes by a distance of one or more fiber diameters, as shown and described in more detail with respect to Figure 7.

[0092] Figure 6 Operation of a system or method 600 for simultaneously acquiring Raman and fluorescence measurements from samples contained in different wells of a well plate 620 is shown. Figure 6 The dual quaternary configuration comprises four (4) Raman probes 610 (A~D) and four (4) fluorescence probes 612 (A~D), wherein each probe 610, 612 is positioned to obtain a corresponding measurement from a sample contained in different non-adjacent wells. In this example, four (4) lasers 630 provide excitation light to the corresponding Raman probes 610, i.e., each laser 630 provides excitation light to one of the Raman probes 610. Four different illumination sources 636 provide illumination for the corresponding wells for fluorescence measurements. For example, the fluorescence illumination source 636 may be an LED, a laser diode, or filtered white light to provide the desired wavelength (UV-VIS-NIR) for illumination.

[0093] Figure 6The illustrated configuration requires additional positioning of the well plate 620 in the xy plane to allow the fluorescent probes 612 (A-D) to read the holes between the initial positions of the fluorescent probes (at A3, A9, E3, and E9) and the initial positions of the Raman probes (at A1, A7, E1, and E7) for which only fluorescence measurements are obtained. However, it can provide an overall speed gain of 75-80% while simultaneously performing fluorescence and Raman measurements on the other holes. In the representative configuration illustrated, the fluorescent probes 612 require additional relative motion between the well plate and the probes to scan the holes in columns 1, 2, 7, and 8. Those skilled in the art will recognize similar requirements for different spacings or other orientations of the Raman probes 610 and the fluorescent probes 612. As a non-limiting example, the configuration may include a fluorescent probe 612 offset by one or more positions in the x-axis (row) direction and / or y-axis (column) direction relative to the Raman probe 610 in the xy array, i.e., the initial positioning of the Raman probes 610 (A~D) measurement holes A1, A7, E1, and E7 relative to the fluorescent probes 612 (A~D) measurement holes B2, F2, B8, and F8. The probe positioning may vary based on the requirements of a specific application and implementation.

[0094] Figure 6The configuration utilizes an optical fiber bundle 680 connecting the output of Raman probe 610 to the input of imaging spectrometer 640, and an optical fiber bundle 682 connecting the output of fluorescence probe 612 to the input of imaging spectrometer 640. One end of optical fibers 680 and 682 is linearly arranged at the input of spectrometer 640. The optical fiber associated with each probe (whether Raman or fluorescence) and the optical fiber associated with another probe are separated by a distance of at least one optical fiber diameter 692 to reduce inter-channel crosstalk. The spacing between fluorescence probes may differ from the spacing between Raman probes. Furthermore, the optical fiber associated with Raman probe 610 (AD) is located in the middle of the linear arrangement, and the optical fiber associated with fluorescence probe 612 (AD) is located on either side of the Raman probe fiber, because the spectral resolution along the height of the scientific-grade CMOS sensor 660 decreases with distance from the optical center. In other words, the fiber associated with Raman probe 610 is positioned closer to the center of the linear arrangement than the fiber associated with fluorescence probe 612, resulting in the corresponding imaging rays 612A', 612B', etc., being farther from the optical axis of sensor 660 than imaging rays 610A', 610B', etc. Therefore, the angle of incidence (AOI) for reading out fluorescence (low resolution requirement) is furthest from the optical center of the sensor. This configuration works well for hybrid configurations including both Raman and fluorescence probes because the resolution required for fluorescence is typically significantly lower than that required for Raman (on the order of approximately 5X to 15X). Similar to the previously described example, each imaging ray can correspond to multiple rows of sensor pixels grouped or merged in columns to generate corresponding Raman spectra 696 and fluorescence spectra 698. The number of pixel rows associated with each imaging fluorescence line can differ from the number of pixel rows associated with each imaging Raman line. Figure 6 As shown in the example, a single spectrometer with a single diffraction grating and a 2D sensor can be used to simultaneously capture Raman and fluorescence measurements from samples in multiple wells.

[0095] While the inventors have recognized that positioning or aligning the probe on non-adjacent holes reduces inter-hole crosstalk when acquiring measurement results, various applications may impair crosstalk and SNR by acquiring fluorescence measurements from the same holes as Raman measurements in order to achieve desired throughput or other overall system performance.

[0096] Figures 7A to 7D Various linear arrangements of optical fibers are shown, which couple light emitted by a sample contained in an aperture plate in response to excitation light to the input of a spectrometer. For example... Figure 7AAs shown, the first configuration includes four probes, with one active fiber 702, 704, 706, and 708 used for simultaneous acquisition of each of the four corresponding channels from the aperture. Each active fiber 702, 704, 706, and 708 is separated by multiple inactive fibers 710. As previously mentioned, the inactive fibers are used for convenience and can be replaced by any suitable spacer. However, the use of inactive fibers provides low cost and precise diameter, which facilitates the formation of a linear arrangement of fibers, and the positioning of such fiber arrays is a collaborative effort of fiber optic component providers. Other mechanical spacers can be used when the desired spacing between active fibers is not an integer multiple of the fiber diameter.

[0097] exist Figure 7A In the example of the first configuration, three invalid fibers 710 are positioned between adjacent valid fibers, namely fibers 702-704; 704-706; and 706-70, and are positioned in a linear arrangement at the input of the spectrometer. However, other configurations may include more or fewer invalid fibers or other spacers. An optional slit 720 can be used to provide the desired spectral resolution for any of the configurations shown. As previously mentioned, the slit 720 can be of a fixed width, selectable by a controller from a plurality of slits with different widths, or it can be a variable-width slit controlled by an associated controller. The number of invalid spacer fibers can be selected to reduce crosstalk below a desired threshold for a particular selected imaging spectrometer, where greater spacing between fibers generally corresponds to lower inter-channel crosstalk.

[0098] Figure 7B An example of two or more active fibers per aperture / channel separated by one or more inactive fibers is shown. In the representative example shown, three active fibers 730, 734, 738, and 742 are provided for the corresponding channels 1-4, wherein two inactive fibers 732, 736, and 740 separate each group or set of three active fibers. Active fiber 742 is arranged in a triangular cluster 750 at one end to capture the Raman output signal and in a linear array at the opposite end at the input of the spectrometer. As previously mentioned, an optional slit 746 may be provided.

[0099] Figure 7C Each channel comprises seven active optical fibers 760, with four inactive optical fibers 762 separating each group of active fibers. The fibers are linearly arranged at the input of the spectrometer. The active fibers 760 are clustered at one end 764 to capture the Raman output signal from the probe, and arranged in a linear array at the opposite ends of the fibers, which are linearly arranged at the input of the spectrometer together with additional inactive optical fibers 762 or similar spacers. As previously described, an optional slit 766 may be provided.

[0100] Figure 7DThis includes three active optical fibers 770 for each channel, separated by one or more inactive fibers 772. The fibers are arranged in two parallel columns 780, 782 at the input of the spectrometer and can be used without slits, or optionally with patterned slits or an array of slits as shown. This results in a shift of the spectrum on the 2D sensor, as represented by spectrum 786 relative to spectrum 788, but with the advantage that strong peaks, such as those represented at 790 and 792, do not cross-contaminate each other; otherwise, these strong peaks might align and potentially overlap on the same sensor pixel.

[0101] As mentioned earlier, high-throughput orifice plate readers can combine Raman spectroscopy with other types of measurements. Figures 7A-7D The fiber optic arrangements shown can be used to couple various types of probes to their respective spectrometers. For example, applications with fluorescence probes can combine Raman probes as described above, using any fiber optic arrangement shown for the fluorescence probe. The number of fibers per fluorescence channel can vary.

[0102] Figure 8 This is a simplified diagram illustrating a well plate reader with combined Raman and full-hole imaging. As shown, a Raman probe 800 is used to read one hole 802 of a well plate 804, while an LED 810 or a filtered broadband source is used to simultaneously read a second non-adjacent hole 812. Light from the probe is guided by a first reflector 830 and a second dichroic mirror or beam splitter 832 to a camera 820 with a 2D scientific-grade CMOS sensor, which can be cooled or uncooled, to provide full-hole imaging. Figure 8 As illustrated in the example, a Raman probe 800 illuminates a liquid sample in a corresponding well 802 from one side of a well plate 804 (the top side in this example), where light emitted or transmitted by the sample is collected from the opposite side of the well plate 804 (the bottom side in this example). Similarly, an additional source, such as an LED 810, illuminates a non-adjacent well 812 from the top of the well plate 804, while emitted and transmitted light are collected from the bottom of the well plate 804. Depending on the specific application and implementation, optical fibers may be used to transmit illumination light from one or both probes to the associated wells 802, 812, and / or collect light and transmit the collected light to a camera 820.

[0103] Figure 9A and Figure 9BComponents of a representative Raman probe 900, including an input fiber optic connector 910 and an output fiber optic connector 912, are shown. The input 910 is connected to a laser source (not shown). The probe 900 can be positioned above or below the well plate, as previously described, depending on the specific application and implementation. Similarly, in some applications, the illumination / exposure components configured to deliver laser light to the sample may be separate from the components used to collect light emitted by or transmitted through the sample, as previously described.

[0104] exist Figure 9A and Figure 9B In the example shown, various optical components are contained within housing 902 (in Figure 9A (The image shows the cover removed). Light from the laser source passes through input 910, achromatic collimating lens 914, and one or more optional bandpass filters 916, 918 to reach reflector 920. The bandpass filters can be positioned at an angle relative to the optical axis of bandpass filter 916 to reduce or eliminate back reflection of the laser light to the laser input, which could otherwise have an unstable effect on the laser. The laser light is reflected by reflector 920 to dichroic long-pass filter 930, which reflects the wavelength of the laser light to plano-convex sample lens 932, which focuses the light onto the liquid sample contained within the various orifices. Light emitted from the liquid sample is collected by the same plano-convex sample lens 932 and guided to a dichroic long-pass filter 930. This filter passes the light (containing an offset wavelength relative to the reflected laser) to a second dichroic long-pass filter 934 (or notch filter), which then passes the filtered light to an achromatic collecting lens 940. This lens focuses the light onto one or more optical fibers coupled to an output 912. The optical fibers couple the Raman signal light from each probe to the input of an associated spectrometer (not shown). Various fiber arrangements can be used to collect the signal light and couple the Raman probe to the spectrometer input, such as a reference... Figures 7A-7D The arrangements shown and described are not identical. Other Raman probe configurations may be used, as are known to those skilled in the art. For example, an immersion probe may be used, which is submerged in a sample contained within the well being measured; however, self-cleaning between measurements may be necessary to avoid cross-contamination of the sample.

[0105] Figure 10A representative fluorescent probe 1000 is shown. A light source 1010 (such as an LED, a filtered broadband light source, etc.) is coupled to a conduit of the probe 1010 via a bundle of optical fibers 1012 and illuminates a liquid sample 1004 contained in one well of a well plate from above or below, depending on the specific application and implementation. The fluorescence emitted by the liquid sample is then coupled in a linear arrangement to the input of an associated imaging spectrometer 1016 via one or more optical fibers 1014 to one or more other fluorescent and / or Raman probes as described above. The optical fibers at the tip 1020 of the probe 1000 can be arranged in a cluster, wherein at least one central illumination fiber 1022 coupled to the bundle of optical fibers 1012 and the light source 1010 is surrounded by a group of collecting fibers of the bundle of optical fibers 1014 coupled to the spectrometer 1016, or vice versa. The fluorescent probe 1000 can be configured as a remote sensing (as shown) or an immersion / impregnation probe. One or more optical fibers within the probe can be angled relative to the sample surface to reduce backscattering of the illumination light.

[0106] Figure 11 and Figure 12 A representative configuration for spatially shifted Raman spectroscopy (SORS) of an orifice plate reader according to this disclosure is shown. The SORS configuration can be used to eliminate background from the orifice plate material (bottom, sides, etc.) and any contributions from the cap / cover that can be applied to the orifice plate. SORS allows for the detection of liquid samples at different levels within the orifice plate. Figure 11 The relative positioning of the excitation laser beam 1110 and the collection probe 1120, positioned above the orifice and spatially offset by a distance Δs, is shown. The SORS implementation can be used to obtain measurements within the upper layer 1130 and / or lower layer 1140 of a liquid sample. Alternatively, the upper layer 1130 may correspond to the orifice cap, wherein measurements are obtained only below the orifice cap at a selected depth of the liquid sample.

[0107] Figure 12A configuration suitable for line scanning to determine the desired spatial offset is shown. The use of an imaging spectrometer with a large number of pixels along the vertical (spatial) axis facilitates the line scanning configuration, which may be particularly suitable for applications involving a aperture cover / cap placed on top of the aperture, or, for example, in configurations where Raman signals are obtained through the bottom of the aperture. The SORS system 1200 includes a laser 1210 coupled via an optical fiber to a laser focusing unit 1214. The laser focusing unit 1214 may include a collimating lens 1216, a laser line filter 1218, and a focusing lens 1220 with an associated adapter 1222. The laser focusing unit 1214 is positioned at an angle above the aperture plate and offset from the collecting optics. The laser focusing unit 1214 generates a scan line 1230 on or within the aperture plate sample 1232. Raman signals 1234 emitted by the sample at various spatial offsets 1236 from the center of the scan line 1230 are directed to the imaging spectrometer 1250. One or more optical elements (such as lens 1252, long-pass filters 1252, 1254 and input slit 1256) may be used to provide Raman signal 1234 to the spectrometer input to generate scattering image 1270 for subsurface evaluation using associated signals / data provided to one or more controllers (such as computer 1280).

[0108] Figure 13 A representative spectrometer for a perforated plate reader according to this disclosure is shown. The spectrometer 1300 includes a reflection concave aberration-corrected holographic grating (ACHG) 1310 and a camera 1320 with a cooled 2D sensor 1322. The camera can be implemented by a Sylent-Blue scientific-grade CMOS camera available from Horiba Instruments Co., Ltd., which includes a scientific-grade CMOS sensor with 2048 x 2048 effective pixels and an effective photosensitive area of ​​22.528 mm × 22.528 mm, and may include a Peltier-cooled vacuum head, or alternatively, a scientific-grade CMOS sensor filled with nitrogen and purged with argon. The imaging spectrometer 1300 includes an optical fiber input 1312, which can accommodate multiple linearly arranged optical fibers from a correlated probe having optional slits as described above. Light from two or more coupled fiber optic probes 1316 is reflected by mirrors 1314 onto a diffraction grating 1310. The diffraction grating 1310 images the diffracted light from the two or more probes onto a 2D sensor 1322 of camera 1320 to simultaneously acquire multiple Raman spectra from samples within relevant apertures of a well plate. Imaging spectrometer 1300 and camera 1320 can communicate with one or more controllers or computers (not shown) to acquire associated data from sensor 1322 and control various features of spectrometer 1300 (such as selectable or variable slits) and camera 1320.

[0109] Figures 14A to 14CA representative non-imaging spectrometer 1400 for use in an orifice plate reader according to this disclosure is shown. The non-imaging spectrometer 1400 can be implemented, for example, by a PoliSpectra® quaternary multichannel grating spectrometer available from Horiba Instruments Co., Ltd. The PoliSpectra® quaternary spectrometer integrates four VS70 spectrometers, each with a planar aberration-corrected holographic concave grating. This high-throughput spectrometer 1400 includes four fiber optic inputs 1402, 1404, 1406, and 1408, four concave aberration-corrected gratings 1410, 1412, 1414, and 1416, and two non-imaging linear CCD sensors 1420 and 1422 to provide high-speed simultaneous acquisition of four channels with minimal crosstalk. The spectrometer 1400 includes four optical chambers, each including an associated fiber optic input and a grating. Light from the top fiber optic inputs 1402 and 1404 is guided to the corresponding gratings 1410 and 1412, and then to the associated portion of the shared linear CCD sensor 1420. Similarly, as... Figure 14C As best described, light from the bottom fiber optic inputs 1406 and 1408 is guided to the corresponding gratings 1414 and 1416, wherein light from each grating 1414 and 1416 is guided to the corresponding portions 1422A and 1422B of the shared linear CCD sensor 1422.

[0110] like Figure 14B As shown in the simplified functional block diagram, the main electronic board 1450 is designed to simultaneously read linear CCD sensors 1420 and 1422 and provide 1-8X merging capability to acquire data from all four channels simultaneously. Signals from CCD sensors 1420 and 1422 are provided to analog conditioning circuits 1460 and 1462, respectively. The conditioned sensor signals are provided to the corresponding channels of a 16-bit analog-to-digital converter (ADC) 1470, where the resulting digital data is captured in a temporary computer-readable storage device 1480 for subsequent reading, analysis, and storage by one or more connected controllers or computers (not shown).

[0111] Figure 15This is a partial perspective view showing various components of a representative multi-well plate reader. In this example, system sub-assembly 1500 includes Raman probes 1510, 1512, 1514, and 1516 fixed relative to each other and aligned with corresponding non-adjacent wells of well plate 1520. A mechanical positioning mechanism 1530 can be positioned by controlling a plurality of motors 1540 to control the positioning of an associated well plate holder 1560 in orthogonal X, Y, and Z directions in response to corresponding control signals from at least one controller. As previously described, at least one controller can control the positioning of well plate 1520 relative to focused laser light from Raman probes 1510, 1512, 1514, and 1516 to simultaneously acquire Raman spectral data from different samples contained in corresponding wells of well plate 1520. In some configurations, during the acquisition of Raman measurement results, the positioning mechanism 1530 is controlled to change the relative position between the well plate 1520 and the Raman probes 1510, 1512, 1514, 1516 in at least one of the X, Y, and Z directions, while maintaining the focused laser from each probe within its associated orifice to mix the liquid sample and / or obtain data from multiple points within the liquid sample. The positioning mechanism 1530 is then controlled to align previously unchecked orifices of the well plate with the probes in a predetermined pattern as described above.

[0112] Figure 16 A representative positioning mechanism 1600 according to this disclosure is shown, having an orifice plate holder 1606 with at least one integrated calibration standard sample 1610, 1612. The positioning mechanism 1600 includes at least one motor 1620 for moving the orifice plate holder 1606 in at least two orthogonal directions and an optional third orthogonal direction. One or more associated controllers can control the positioning mechanism 1600 to periodically align the integrated calibration standard samples 1610, 1612 with corresponding probes positioned above or below the orifice plate holder 1606 during the calibration process. Measurements obtained by the system from the calibration standards can be compared with Raman data of previously established and stored standard materials. Detected deviations in peak wavelength or intensity can be used to adjust the wavelength / intensity of the corresponding laser or other light source associated with the probe. In addition to the maximum available travel of the positioning mechanism relative to the standard for positioning the probes and the specified time for completing the calibration of all probes, the number and material of the standard samples can vary based on the number and type of probes being calibrated. Different standard samples can be provided for different types of probes, and / or probes can be calibrated for a desired specified wavelength. In a representative example, diamond calibration samples 1610 and 1612 are fitted into corresponding arms / legs of the orifice plate holder 1606, wherein each of the diamond calibration samples is used during the calibration of the two probes.

[0113] One or more controllers in a typical multi-well plate reader system can be programmed to periodically (e.g., once / day) automatically calibrate the probes. The controllers can be programmed to align associated calibration standards with the probes, collect measurement data, compare the measurement data with previously stored data for the standard materials, and control the corresponding light source wavelength and / or intensity based on the comparison. For example, when the calibration process detects a wavelength (X-axis) drift between the measured calibration sample material and the known sample material spectrum for one or more peak wavelengths, the controller will automatically correct the drift by shifting the laser wavelength to reduce the difference between the measured peak wavelength and the standard peak wavelength of the material. Similarly, the calibration process can detect intensity drift and adjust the light source intensity accordingly. Alternatively, the system can suggest different calibration processes using a calibration plate with multiple standard samples held by a well plate holder 1606. The calibration plate can be used to calibrate the intensities of various light sources relative to each other for throughput matching between different probes.

[0114] Figure 17 A representative calibration well plate 1700 according to this disclosure is shown, having at least one calibration standard sample with one or more probes for a well plate reader. The dimensions of the calibration well plate 1700 are based on a corresponding well plate that can be secured in a well plate holder as described herein in a well plate reader system. In the example shown, the calibration well plate 1700 is sized similarly to a 96-well plate and is configured to be positioned within a corresponding well plate holder. Furthermore, the calibration well plate 1700 provides a representative example of a well plate reader system with four Raman probes. Therefore, the number and positioning of various calibration samples are selected based on application requirements and can vary depending on the specific application and implementation.

[0115] exist Figure 17 In the example, calibration plate 1700 includes symmetrically positioned calibration standard samples and calibration wells, each group associated with two probes of a four-probe system. Each group of calibration standard samples includes a diamond calibration sample 1710, a silicon calibration sample 1720, and six wells 1730, which can be filled with any desired liquid calibration sample for a specific application. The dimensions of the calibration standard samples can be determined based on commercially available sizes for a particular material.

[0116] The inventors have recognized that the intensity of the sharp Raman bands of a particular material can be used for intensity drift monitoring to trigger recalibration. Many materials degrade under laser exposure, making them unsuitable candidates for calibration standards. Therefore, diamond was chosen as a material that is highly stable under heavy laser exposure. Furthermore, hard materials such as diamond and silicon will provide peaks whose intensity varies with the probe's Z-axis position, allowing calibration using such materials to be used to adjust the Z-axis focusing accuracy of one or more probes.

[0117] The liquid orifice 1730 can be filled with a typical liquid / matrix used in a specific application, or with any of a variety of reference liquids. Compared to hard material calibration standards, reference liquids provide more reliable strength calibration because any Z-axis focus inaccuracy does not significantly affect strength. Raman peak intensity remains relatively stable over the Z-axis focusing range, as long as the light is focused below the liquid surface. However, liquid calibration standards can evaporate and may require user intervention to refill the associated calibration orifice.

[0118] Using different calibration standard sample materials can help identify one or more components that need adjustment or replacement. For example, the Raman peaks of a single material (such as diamond in this example) may not be sufficient to diagnose degradation or changes in various other system components in the optical path of a laser, fiber optic, probe, spectrometer, etc. Calibration standard sample materials (silicon wafers, liquids, etc.) with other peaks and / or multiple peaks can enable the diagnosis and subsequent compensation of specific components associated with corresponding portions of the spectrum.

[0119] Many components along the optical path, besides the laser or other light sources, can cause inaccurate measurements associated with component failure, misalignment, wear, aging, etc. Laser aging compensation is preferably performed automatically without user intervention, as the laser is the most common and frequent source of inaccuracy. Other conditions, such as damaged filters in the probe, spectrometer / camera misalignment, damaged or broken optical fibers, positioning errors in the positioning mechanism, etc., can also affect the resulting measurements. Therefore, using multiple calibration standard samples provided by the calibration plate 1700 offers additional diagnostic and calibration flexibility.

[0120] Figure 18This is a block diagram illustrating the operation of a system or method for simultaneously acquiring spectral measurement results. Control logic or functions executed by one or more controllers or computers are represented in the block diagram, providing representative control strategies, logic, or algorithms that can be implemented using one or more processing strategies, such as event-driven, interrupt-driven, multitasking, multithreading, etc. Therefore, the various steps or functions shown may be executed in the order shown, in parallel, or omitted in some cases. Although not always explicitly shown, those skilled in the art will recognize that one or more of the shown steps or functions may be repeatedly executed depending on the specific processing strategy used. Similarly, the order of processing is not necessary to achieve the features and advantages described herein, but is provided for ease of illustration and description. The control logic may be implemented primarily in software executed by a microprocessor-based controller, control module, microcontroller, or computer. Of course, depending on the specific application, the control logic may be implemented in one or more controllers in software, hardware, or a combination of software and hardware. When implemented in software, the control logic may be housed in one or more non-volatile computer-readable storage devices or media storing data representing code or instructions executed by a computer to control a vehicle or its subsystems. Computer-readable storage devices or media may include one or more of a plurality of known physical devices that utilize electrical, magnetic and / or optical storage devices to store executable instructions and associated data, operation variables, etc.

[0121] The system or method may include simultaneously focusing a laser into corresponding holes in a well plate containing at least two samples, the at least two samples being contained in holes spaced apart by at least one hole illuminated by an unfocused laser, as shown in 1802. Light emitted from the at least two samples is simultaneously directed to a spectrometer having a diffraction grating that redirects the light to a shared sensor at a wavelength-dependent angle, as shown in 1804. Box 1806 illustrates the acquisition of Raman spectral data associated with the at least two samples from the shared sensor. Box 1808 illustrates controlling the positioning of the well plate relative to the focused laser to simultaneously acquire Raman spectral data from at least two different samples contained in previously unchecked holes in the well plate.

[0122] Simultaneous focusing laser, as indicated in box 1802, may include simultaneously focusing light from different lasers associated with each of at least two samples. Box 1802 may also include simultaneously directing light from the source to at least two samples contained in corresponding wells of a well plate, each well being spaced apart by at least one well illuminated by an unfocused laser or light from the source. Box 1804 may further include simultaneously directing light emitted from samples illuminated by light from the source to a shared sensor of the spectrometer, while box 1806 may include acquiring fluorescence data associated with the at least two samples illuminated by light from the source from the shared sensor of the spectrometer.

[0123] In at least one embodiment, block 1804 includes simultaneously guiding light emitted by at least two samples by coupling light emitted by at least two samples to at least one optical fiber for each of the at least two samples, and arranging the optical fibers in a linear array at the input of the spectrometer, wherein the optical fibers associated with each sample are grouped together and the optical fibers associated with the other sample are separated by a distance corresponding to at least one optical fiber diameter.

[0124] Various implementations may also include positioning a calibration standard integrated within an orifice plate holder for irradiation by a focused laser via at least one controller, acquiring Raman spectral data from the calibration standard, and adjusting the wavelength of the focused laser based on a comparison of the Raman spectral data of the calibration standard with a previously stored peak wavelength of the calibration standard.

[0125] Figure 19 This is a block diagram illustrating the integration of a multiprobe spectroscopy system with an automated liquid handling system according to the present disclosure. The automated liquid handling system 1900 (also referred to as an automated laboratory robot) may include a liquid filling device 1910 to provide automated transfer of liquid into each well of a well plate 1912 within a reaction chamber 1930. The reaction chamber 1930 may provide for heating / cooling, mixing, stirring, quenching (dilution), etc., of liquid samples and may include a positioning or transport mechanism 1914 to move one or more well plates 1912 within the reaction chamber 1930. The positioning mechanism 1914 may be controlled to position the well plates 1912 relative to a probe 1916 for spectral analysis as described above, without requiring sample dilution or quenching for analysis. Alternatively, some applications may include a second positioning mechanism 1920 associated with the spectral analysis portion of the reaction chamber 1930, wherein the well plates 1912 are transferred from the positioning mechanism 1914 to the positioning mechanism 1920 for spectral analysis (without sample dilution or quenching). Also as Figure 19 As described, one or more lasers 1940, spectrometers / cameras 1942, light sources 1944, power supplies 1946, etc. are located outside the reaction chamber 1930 and connected by corresponding optical fibers 1950, wires, etc.

[0126] Figure 20This is a block diagram illustrating an embodiment of a Raman analyzer according to the present disclosure, wherein opposing Raman probes are focused on the same point of a moving or flowing liquid sample. System 2000 includes at least two Raman probes 2010, 2012, positioned along a common axis on opposite sides of a sample positioning device 2030 that guides a moving or flowing liquid sample 2040. In this representative embodiment, Raman probes 2010, 2012 have respective output ends positioned such that corresponding output lasers are focused on the same point of the moving liquid sample 2040. The sample positioning device 2030 can be implemented using tubing, a flow cell, or similar equipment. The opposing arrangement of Raman probes 2010, 2012 facilitates cross-collection of light from the sample 2040 to increase signal gain or time gain (reduce spectral acquisition time). For example, output light from Raman probe 2010 is acted upon by the sample 2040 and is collected by Raman probe 2010 and cross-collected by Raman probe 2012. Similarly, the output light from Raman probe 2012 is affected by sample 2040 and collected by both Raman probe 2012 and Raman probe 2010. Compared to a conventional arrangement with a single probe, a 4X signal or time gain can be achieved by providing a separate laser source for each Raman probe 2010, 2012 and having both probes focused on the same point on the sample. In various embodiments, the laser can be integrated with the Raman probe and contained within the housing of the Raman probe. Additional signal or time gain can be achieved using various combinations of probes and lasers. For example, a laser can be coupled to the input of multiple probes focused on the same point to provide a gain corresponding to the number of probes, even if one laser supplies five probes to obtain a 5X gain. Additional lasers can then be used to increase the gain, i.e., two lasers supplying five probes focused on the same point to obtain a 10X gain, and so on.

[0127] Alternatively, the Raman probe can be aligned along a common axis but focused on different points within the moving liquid, such as... Figure 29 As shown. This arrangement can be used when the sample may be altered or damaged by two or more lasers that might be focused on the same point. Because the probes are focused at different points, there is little or no cross-collection, and the corresponding signal or time gain is reduced accordingly. For example, for two opposing probes each coupled to a corresponding laser, if both probes were focused at the same point on the sample, the signal or time gain would be 2X instead of 4X.

[0128] Figure 29Another representative embodiment of a Raman analyzer with multiple probes focused at different points within a liquid sample is shown. In this embodiment, Raman probes 2910, 2912, 2914, and 2916 are positioned at the same location in the plane of the figure, perpendicular to the flow cell 2920. The flow cell 2920 contains a flowing liquid sample 2930, which flows (enters or exits the plane) perpendicular to the plane of the figure. Raman probes 2910, 2912, 2914, and 2916 focus at different points within the sample 2930 (generally indicated by 2940), such that there is no significant cross-collection of signals from relative probes 2910, 2914 or probes 2912, 2916. In the example shown, Raman probes 2910 and 2914 are positioned such that their respective outputs are opposite each other along a common axis. Similarly, Raman probes 2910 and 2914 are positioned such that their respective output ends are opposite each other along a common axis. Each probe has an associated laser source to provide 4X signal or time gain. As with various other embodiments shown, different numbers or locations of probes may be used depending on the specific application and implementation.

[0129] As described with respect to the previous embodiment, the outputs of at least two Raman probes are input to an imaging spectrometer and imaged on a shared two-dimensional sensor. Each Raman probe output can be coupled to the spectrometer input via one or more optical fibers, wherein the fibers are linearly arranged at the spectrometer input. Where it is necessary to reduce or eliminate crosstalk between images on the sensor, the fiber of one Raman probe can be spaced apart from the fibers of other Raman probes by a distance corresponding to one or more fiber diameters. Figure 20In one implementation, signals from Raman probes 2010 and 2012 can be combined or summed into a single sensor reading because both are focused on the same point on the sample. The spectrometer may include a shared shutterless CCD sensor, where the probe input is imaged over the entire vertical merging range of the sensor, or the sensor may operate in a two-dimensional mode, where sensor data is vertically summed by at least one controller. A shared CCD sensor can provide a lower-cost solution for applications requiring the combination or summation of signals from two or more probes. While these types of applications are not required, the spectrometer may alternatively include a shared two-dimensional scientific-grade CMOS sensor with similar summation of sensor signals from two or more probes. Similarly, where probe signals are summed or otherwise combined, optical fibers 2050 and 2052 from different probes can be positioned adjacent to each other in a linear array because crosstalk is not an issue. In one implementation, each probe has seven (7) optical fibers 2050 and 2052 to provide input light to the spectrometer. However, spectrometer input configurations with different probes separated from each other by one or more fiber diameters will also be acceptable, although not necessary.

[0130] Typically, all embodiments with at least two Raman probes may include a single high-power laser with a laser output divided or separated between the two or more probes, a low-power laser associated with each Raman probe (which may be integrated within the probe housing), a low-power laser with an output that combines to amplify the input to a single Raman probe, or any combination thereof. Laser configuration, power, etc., can vary based on specific application requirements and implementation details. Similarly, lasers, Raman probes, and spectrometers may be coupled via one or more optical fibers as described in more detail herein, but free-space coupling using associated optics of one or more system components may be suitable or acceptable in a particular application or embodiment. Similarly, embodiments may include two or more probes focused at the same point on the sample or positioned at the same location but focused at different points within the sample to provide the desired signal or time gain.

[0131] Figure 21This is a block diagram illustrating an embodiment of a Raman analyzer according to the present disclosure, wherein Raman probes are focused at different points or regions of a moving or flowing liquid sample. In a first representative embodiment, system 2100 includes at least two Raman probes 2110, 2112, the outputs of which are focused at different points on the same flowing liquid sample 2140 within a corresponding sample positioning device 2130. Alternatively, system 2100 may include Raman probes having outputs focused on different flowing liquid samples, such as Raman probe 2210 (and / or 2112) focused on liquid sample 2140 and Raman probe 2114 focused on different liquid samples 2142 flowing through another sample positioning device 2132. As previously discussed... Figure 20 As described in the embodiments, the moving or flowing liquid may be contained within pipes, flow cells, vials, or similar material handling equipment to move samples for analysis by Raman probes 2110, 2112, and 2114. However, Figure 21 Implementations typically require a spectrometer with a shared two-dimensional scientific-grade CMOS sensor to provide acceptable resolution for the individual spectra. A shutterless CCD sensor is unavailable in this configuration because CCDs exhibit vertical crosstalk readings even in two-dimensional mode, where the space between the fibers of the two probes prevents independent channel readouts. Therefore, in addition to using a shared scientific-grade CMOS sensor, the light from the sample delivered to the spectrometer by each of two or more Raman probes via one or more fiber inputs (as shown in 2150 and 2152) should be separated from each other by a distance of at least one fiber diameter (as shown in 2160) to substantially eliminate crosstalk between channels. Computer simulations of one implementation of a dual-probe 532nm system with a spectrometer featuring a scientific-grade CMOS sensor and an F / 2.3 optics system indicate no crosstalk, where each probe delivers light to the spectrometer via seven (7)-70 / 77 micrometer fibers, the seven (7)-70 / 77 micrometer fibers being linearly arranged and separated by five (5) ineffective or dead-zone fibers, substantially eliminating crosstalk between probe channels, as discussed in [reference to...]. Figure 27 To be shown and described in more detail.

[0132] Figure 22 This is a block diagram illustrating an embodiment of a Raman analyzer according to the present disclosure, wherein opposing Raman probes are focused on different points of a moving or flowing sample. System 2200 includes Raman probes 2210, 2212, having output ends aligned along a common axis 2220, wherein samples 2240, 2242 are moved therein via a material handling system generally indicated by 2230. Samples 2240, 2242 can be contained in vials or other containers. Figure 20The implementation scheme differs; the separator 2270 prevents Raman probes 2210 and 2212 from cross-collecting light and simultaneously collects Raman spectral data from different samples 2240 and 2242. Therefore, Figure 22 The spectrometer in the illustrated embodiment includes a single shared scientific-grade CMOS sensor, wherein input probe fibers 2250, 2252 are spaced apart by one or more fiber diameters 2260 to eliminate crosstalk, thereby independently and simultaneously reading signals from two Raman probes 2210, 2212.

[0133] Figures 23 to 2 6. Figure 30 A representative embodiment of a Raman analyzer according to this disclosure is shown, which includes a sample positioning device and a probe positioning mechanism for analyzing solid samples. System 2300 includes a probe positioning mechanism 2310 and a sample positioning device 2312 (e.g., Figure 24 As shown), they cooperate to change the relative positions between at least two Raman probes 2320, 2322 and the solid sample 2330, such as Figures 25 to 2 As shown in Figure 6, the probe positioning mechanism 2310 can be used to translate Raman probes 2310, 2312 along at least one axis. In the illustrated embodiment, the probe positioning mechanism 2310 can provide translational movement of Raman probes 2310, 2312 along orthogonal X, Y, and Z axes. Raman probes 2310, 2312 can be fixed relative to each other in the X and Y directions, having an adjustable Z position to provide optional autofocus capability. The autofocus function can be based on a distance sensor 2336 that detects the distance to the surface of sample 2330. Alternatively, the distance or Z-axis position can be adjusted to optimize or maximize the Raman spectral signal. Probes 2310, 2312 can be adjusted collaboratively or individually depending on the specific implementation. For example, autofocus or autoadjustment can be provided using corresponding signals from at least one programmable controller based on distance feedback from the distance sensor or signal strength from a scientific-grade CMOS sensor of the spectrometer. The sample positioning device 2312 can be a rotary stage 2400 with an associated computer control interface 2410. Figure 24 This is achieved by controlling the rotational position of sample 2330. At least one controller can be programmed to coordinate the control of probe positioning mechanism 2310 and sample positioning device 2312 to change the relative position of sample 2330 with two or more Raman probes 2320, 2322, thereby simultaneously generating independent Raman spectra for corresponding points on the sample. Alternatively, the sample positioning device can be implemented by a second translation stage having translational positioning along one or more axes.

[0134] Figure 25This diagram illustrates the operation of a system or method for simultaneously acquiring Raman spectral data from at least two Raman probes using a rotating sample positioning device and a translational probe positioning mechanism. (See diagram for example.) Figure 25 As shown, when the wafer is rotated by controlling the rotary stage, Raman spectral data are simultaneously acquired at two points 2510 and 2512 on wafer 2520 using corresponding Raman probes. Figure 24 The Raman probe is aligned with subsequent sample points 2514 and 2516 by controlling the rotation of wafer 2520, and with subsequent sample points 2522 and 2524 by controlling the translation of the probe along the X-axis. Various other sampling patterns can be provided by controlling the rotational position of sample wafer 2520 and the translational position of the Raman probe, such as... Figure 26A , Figure 26B and Figure 26C The overall representation is shown in the figure. Alternatively, one or more sampling patterns can be generated by maintaining a constant sample position and controlling the XY axes of the probe positioning mechanism, or by maintaining a constant probe position and controlling the rotation of the sample. The corresponding graphical representation of the scientific-grade CMOS sensor 2540 that records two Raman spectra also shows rows 2542 and 2544 of merged pixels corresponding to the two Raman channels.

[0135] Figure 27 The results of software simulations of a representative Raman analyzer embodiment according to this disclosure are shown, demonstrating that there is no crosstalk between Raman probe channels sharing a single two-dimensional scientific-grade CMOS sensor. Figure 27 The results shown represent a 532 nm dual-channel F / 2.3 system with no crosstalk between the two sets of fibers 2710 and 2712. Each set of fibers 2710 and 2712 comprises seven (7)-70 / 77 μm fibers arranged in a linear array adjacent to each other and spaced apart at the spectrometer inlet slit by five (5) invalid fibers 2720 to provide the spectral resolution shown. Depending on the desired spectral resolution for a particular application, the inlet slit may not be necessary. As illustrated in figure 2730, there is no crosstalk between the channel 1 (CH1) image 2732 and the channel 2 (CH2) image 2734 on the spectrometer's scientific-grade CMOS sensor.

[0136] Figure 30Another representative embodiment of a Raman analyzer is shown, which has two or more Raman probes focused at the same point on a solid sample. These Raman probes have cross-collection capabilities to increase signal or time gain. System 3000 includes two or more Raman probes whose outputs are focused at each sample point. Raman probes 3010 and 3012 are positioned to simultaneously illuminate and collect light from a first point 3020 on a solid wafer sample 3030, and Raman probes 3014 and 3016 are positioned to simultaneously illuminate and collect light from a second point 3022. As previously described, Raman probes 3010, 3012, 3014, and 3016 may have corresponding inputs coupled to different lasers (one for each probe), and in some applications, these inputs may be integrated into the probes. If the probes are configured for cross-collection (because Raman light emits in all directions), this will result in a signal or time gain equal to the number of probes multiplied by the number of lasers, i.e., 4X for each sample point 3020, 3030, where two probes are cross-collected per point and one laser is used for each probe. Alternatively, a single / common laser can be coupled to the inputs of two or more probes 3010, 3012, where different lasers are coupled to the inputs of two or more probes 3014, 3016 for applications sensitive to surface damage associated with increased lower power focused on the sample point. As described herein, configurations without cross-collection, with fewer lasers, or with a different number of probes per sample point will have a correspondingly lower signal or time gain.

[0137] As previously described, the probe positioning mechanism, generally indicated by arrow 3050, can be controlled to control the positioning of the probes relative to the solid wafer sample 3030. The positioning of probes 3010, 3012, 3014, and 3016 can be controlled independently and / or as a unit along one or more orthogonal axes. For example, the probes can have independent z-axis control, thereby adjusting the distance between the probes and the sample 3030 while moving together as a unit in x-axis or xy-axis positioning. Similarly, the sample positioning device can be used to provide rotational positioning and / or positioning along orthogonal x, y, and / or z axes. Probes 3010, 3012, 3014, and 3016 can be positioned to have an output perpendicular to the surface of the solid wafer sample 3030, for example... Figure 23 As described in the implementation plan, or can be positioned as such Figure 30 The angle that is roughly represented by the middle.

[0138] Figure 28This diagram illustrates the operation of a Raman analyzer or method for simultaneously acquiring Raman spectra from two or more points of a sample according to this disclosure. Control logic or functions executed by one or more controllers or computers are represented in the block diagram, providing representative control strategies, logic, or algorithms that can be implemented using one or more processing strategies, such as event-driven, interrupt-driven, multitasking, multithreading, etc. Therefore, the various steps or functions shown may be executed in the order shown, in parallel, or omitted in some cases. Although not always explicitly shown, those skilled in the art will recognize that one or more of the shown steps or functions may be repeatedly executed depending on the specific processing strategy used. Similarly, the order of processing is not necessary to achieve the features and advantages described herein, but is provided for ease of illustration and description. The control logic may be implemented primarily in software executed by a microprocessor-based controller, control module, microcontroller, or computer. Of course, depending on the specific application, the control logic may be implemented in software, hardware, or a combination of software and hardware in one or more controllers. When implemented in software, the control logic may be housed in one or more non-transient computer-readable storage devices or media that store data representing code or instructions executed by a computer to control a vehicle or its subsystems. The computer-readable storage device or media may include one or more of a plurality of known physical devices that utilize electrical, magnetic, and / or optical storage to hold executable instructions and associated data, operational variables, etc.

[0139] like Figure 28As shown, system or method 2800 includes simultaneously focusing a laser onto at least two points on a solid sample, as shown in 2810. This may include simultaneously focusing laser light from different lasers associated with each of the at least two points, as shown in 2812. The system or method also includes simultaneously directing light from the at least two points to an imaging spectrometer having a diffraction grating that redirects the light to a shared two-dimensional scientific-grade CMOS sensor at a wavelength-dependent angle, as shown in 2830. This may include coupling light from each point to at least one corresponding optical fiber, as shown in 2832, and arranging the optical fibers in a linear array at the input of the spectrometer, wherein at least one optical fiber associated with each point and an optical fiber associated with another point are separated by a distance corresponding to at least one optical fiber diameter, as shown in 2834. Raman spectral data associated with the at least two points from the shared two-dimensional scientific-grade CMOS sensor are simultaneously acquired, as indicated at 2850. The system or method also includes controlling the relative positioning between the sample and the laser via at least one controller to simultaneously acquire Raman spectral data from at least two different points on the solid sample, as shown in 2870. Controlling the relative positioning between the sample and the laser can include rotating the solid sample and / or translating the focused laser to simultaneously acquire Raman spectral data from at least two different points on the solid sample, as shown in 2872. The rotation of the solid sample can be controlled independently of the translation of the focused laser, as shown in 2874.

[0140] While representative embodiments have been described above and illustrated in the accompanying drawings, these embodiments are not intended to describe all possible forms covered by the teachings of this disclosure and the subject matter currently claimed. The language used in this specification is descriptive rather than restrictive, and it should be understood that various changes can be made without departing from the claimed subject matter. As previously stated, features of various representative embodiments can be combined to form other embodiments that may not be explicitly described or shown. While various embodiments may have been described as providing advantages or superiority over other embodiments or prior art embodiments in one or more desired characteristics, those skilled in the art will recognize that one or more features or characteristics can be compromised to achieve desired overall system properties, depending on the specific application and implementation. These properties may include, but are not limited to, strength, durability, lifespan, marketability, appearance, packaging, size, maintainability, weight, manufacturability, ease of assembly, etc. Therefore, an embodiment described as being less desirable than other embodiments or prior art embodiments in one or more characteristics is not necessarily outside the scope of this disclosure or claimed subject matter and may be desirable for a particular application.

Claims

1. A system for obtaining Raman spectra from a sample, the system comprising: At least one laser; At least two Raman probes, each Raman probe being positioned to irradiate and receive light from a corresponding point on the sample to simultaneously acquire the Raman spectrum of the sample, each Raman probe including an input end optically coupled to the at least one laser and a sample lens, the sample lens being configured to receive light from the input end, focus the light from the input end onto the corresponding point on the sample, collect light from the corresponding point on the sample, and guide the collected light to an output end; An imaging spectrometer optically coupled to the output of the at least two Raman probes, the imaging spectrometer including a diffraction grating configured to guide light collected by the at least two Raman probes to a single two-dimensional scientific-grade CMOS sensor; A sample positioning device configured to move the sample relative to the at least two Raman probes; A probe positioning mechanism configured to translate the at least two Raman probes along at least one axis; as well as At least one controller communicates with the at least one laser, the imaging spectrometer, the sample positioning device, and the probe positioning mechanism. The at least one controller is programmed to simultaneously acquire the Raman spectra of the corresponding points of the sample based on signals from the two-dimensional scientific-grade CMOS sensor of the spectrometer, and to control at least one of the sample positioning device and the probe positioning mechanism to change the positions of the at least two Raman probes relative to the sample.

2. The system according to claim 1, wherein, At least two of the at least two Raman probes are positioned to irradiate and receive light from each of the corresponding points of the sample.

3. The system according to claim 1, wherein, Each of the at least two Raman probes comprises: An achromatic collimating lens is positioned to receive light from the input end; A reflector is positioned to receive light from the achromatic collimating lens; A first dichroic long-pass filter is configured to reflect light from the mirror with a wavelength lower than a first cutoff wavelength to the sample lens, and transmit light from the sample lens with a wavelength higher than the first cutoff wavelength. A second dichroic long-pass filter is configured to transmit light with a wavelength higher than the second cutoff wavelength received from the first dichroic long-pass filter, and to reflect light with a wavelength lower than the second cutoff wavelength; and An achromatic collecting lens is configured to focus light transmitted through the second dichroic long-pass filter onto the output.

4. The system according to claim 3, wherein, The at least two Raman probes are fixed in position relative to each other and move together relative to the sample in response to a signal from the at least one controller.

5. The system according to claim 1, wherein, The sample positioning device includes a rotary table configured to rotate the sample in response to a corresponding signal from the at least one controller.

6. The system according to claim 5, wherein, The probe positioning mechanism is configured to translate the at least two Raman probes relative to the sample positioning device along orthogonal X, Y, and Z axes in response to a corresponding signal from the at least one controller.

7. The system according to claim 1, wherein, The probe positioning mechanism is configured to translate the at least two Raman probes in response to a corresponding signal from the at least one controller to control the distance between the probes and the sample.

8. The system according to claim 1, wherein, The output end of each of the Raman probes is optically coupled to the input end of the spectrometer via at least one fiber optic cable, wherein the fiber optic cable from the Raman probe is linearly arranged at the input end of the spectrometer, and wherein the fiber optic cable from each Raman probe is spaced apart from the fiber optic cable of the other Raman probe by a distance of at least one fiber diameter.

9. The system according to claim 1, wherein, The output end of each of the Raman probes is optically coupled to the input end of the spectrometer via multiple fiber optics, wherein the fiber from each Raman probe is spaced apart from the fiber of another Raman probe by multiple invalid fibers.

10. The system according to claim 1, wherein, The at least one laser includes a plurality of lasers, each of which is optically coupled to the input end of a single Raman probe in the Raman probe.

11. The system according to claim 1, wherein, The at least one laser includes a plurality of lasers, wherein each of the plurality of lasers is contained within the housing of a corresponding Raman probe in the Raman probe.

12. The system according to claim 1, wherein, The at least one laser includes a plurality of lasers, at least two of which are coupled to the input of a single Raman probe among the at least two Raman probes.

13. The system of claim 1, wherein the at least one laser comprises a plurality of lasers, and wherein at least one of the plurality of lasers is coupled to the input end of at least two Raman probes of the Raman probes.

14. The system according to claim 1, wherein, The at least two Raman probes are positioned such that their corresponding probe output ends are aligned along a common axis, and the system is configured to enable movement of the sample between the corresponding probe output ends.

15. The system according to claim 14, wherein, The sample positioning device includes a tube positioned between the corresponding probe outputs and configured to allow the sample to flow through the tube.

16. The system according to claim 15, wherein, The at least one controller is programmed to combine scientific-grade CMOS signals from the at least two Raman probes to generate combined Raman spectra.

17. The system according to claim 14, wherein, The lasers output from the at least two Raman probes are focused on the same point on the sample.

18. The system according to claim 14, wherein, The sample positioning device includes a conveyor configured to move a vial containing the sample between the corresponding probe outputs.

19. A system for obtaining Raman spectra of a moving liquid sample, the system comprising: At least one laser; At least two Raman probes, each positioned on opposite sides of the moving liquid sample along a common axis, are used to illuminate and receive light from corresponding points of the moving liquid sample, thereby simultaneously acquiring the Raman spectrum of the sample. Each Raman probe includes an input end optically coupled to the at least one laser and a sample lens, the sample lens being configured to receive light from the input end, focus the light from the input end onto the corresponding point of the sample, collect light from the corresponding point of the sample, and guide the collected light to an output end. An imaging spectrometer is optically coupled to the output of at least two Raman probes via multiple fiber optics, the fibers of the at least two Raman probes being linearly arranged at the input of the imaging spectrometer. The imaging spectrometer includes a diffraction grating configured to guide light collected by the at least two Raman probes to a single two-dimensional sensor. as well as At least one controller, which communicates with the at least one laser and the imaging spectrometer, is programmed to simultaneously acquire the Raman spectra of the corresponding points of the sample based on signals from the two-dimensional sensor of the spectrometer.

20. The system according to claim 19, wherein: The moving liquid sample includes moving liquid through a tube located between the output ends of the at least two Raman probes; Each of the at least two Raman probes focuses its corresponding output laser beam onto the same point on the moving liquid sample moving through the tube; and The at least one controller is programmed to sum the signals from the two-dimensional sensor corresponding to the outputs of the at least two Raman probes to generate a combined Raman spectrum.

21. The system according to claim 20, wherein, The two-dimensional sensor includes a shutterless CCD sensor.

22. The system according to claim 20, wherein, The two-dimensional sensor includes a scientific-grade CMOS sensor.

23. The system according to claim 19, wherein, The at least one laser includes a plurality of lasers, each of which is optically coupled to the input end of a single Raman probe in the Raman probe.

24. The system according to claim 19, wherein, The at least one laser includes a plurality of lasers, wherein each of the plurality of lasers is contained within the housing of a corresponding Raman probe in the Raman probe.

25. The system according to claim 19, wherein, The at least one laser includes a plurality of lasers, at least two of which are coupled to the input of a single Raman probe among the at least two Raman probes.

26. The system according to claim 19, wherein, The at least one laser includes a plurality of lasers, and at least one of the plurality of lasers is coupled to the input end of at least two Raman probes of the Raman probes.

27. The system according to claim 19, wherein: The moving liquid sample includes a vial containing the liquid sample, which is moved via a material handling system past the outputs of the at least two Raman probes. The at least two Raman probes focus the corresponding output laser at points within different vials; The two-dimensional sensor includes a scientific-grade CMOS sensor; The optical fibers associated with each Raman probe are positioned adjacent to each other and spaced apart from the optical fibers associated with other Raman probes by a distance corresponding to at least one optical fiber diameter; and The at least one controller is programmed to use signals from the scientific-grade CMOS sensor to simultaneously and independently generate individual Raman spectra for each Raman probe readout.

28. The system of claim 27, comprising a separate laser coupled to the input end of each of the at least two Raman probes.

29. The system according to claim 19, wherein, Each of the at least two Raman probes comprises: Laser; An achromatic collimating lens is positioned to receive light from the laser. A reflector is positioned to receive light from the achromatic collimating lens; A first dichroic long-pass filter is configured to reflect light from the mirror with a wavelength lower than a first cutoff wavelength to the sample lens, and transmit light from the sample lens with a wavelength higher than the first cutoff wavelength. A second dichroic long-pass filter is configured to transmit light with a wavelength higher than the second cutoff wavelength received from the first dichroic long-pass filter, and to reflect light with a wavelength lower than the second cutoff wavelength; and An achromatic collecting lens is configured to focus light transmitted through the second dichroic long-pass filter onto the output.

30. A system for obtaining Raman spectra of flowing liquid samples, the system comprising: At least two lasers; At least two Raman probes are positioned along the path of the flowing liquid sample to illuminate and receive light from a corresponding point on the flowing liquid sample, thereby simultaneously acquiring the Raman spectrum of the sample. Each Raman probe includes an input end optically coupled to a corresponding laser of the at least two lasers and a sample lens. The sample lens is configured to receive light from the input end, focus the light from the input end onto the corresponding point on the sample, collect the light from the corresponding point on the sample, and guide the collected light to an output end. An imaging spectrometer is optically coupled to the output of at least two Raman probes via a plurality of fiber optics, the fibers of the at least two Raman probes being linearly arranged at the input of the imaging spectrometer, wherein the fibers associated with each probe are positioned adjacent to each other, and the imaging spectrometer includes a diffraction grating configured to guide light collected by the at least two Raman probes to a single two-dimensional scientific-grade CMOS sensor. as well as At least one controller, which communicates with the at least one laser and the imaging spectrometer, is programmed to simultaneously acquire the Raman spectra of the corresponding points of the sample based on signals from the two-dimensional CMOS sensor of the spectrometer.

31. The system according to claim 30, wherein, The Raman probe has output ends aligned in opposite directions along a common axis, and the flowing liquid passes between the output ends.

32. The system according to claim 31, wherein, The at least one controller is programmed to sum the signals from the two-dimensional CMOS sensor to generate a combined Raman spectrum corresponding to the at least two Raman probes.

33. The system according to claim 30, wherein, The optical fiber associated with each probe and the optical fiber associated with other Raman probes coupled to the imaging spectrometer are spaced apart by a distance corresponding to at least two optical fiber diameters.

34. A method comprising: The laser is simultaneously focused on at least two points on a solid sample; Light from the at least two points is simultaneously directed to an imaging spectrometer with a diffraction grating that redirects the light to a shared two-dimensional scientific-grade CMOS sensor at a wavelength-dependent angle. Acquire Raman spectral data associated with the at least two points from the shared two-dimensional scientific-grade CMOS sensor; and The rotation of the solid sample and the translation of the focused laser are controlled by at least one controller to simultaneously acquire Raman spectral data from at least two different points on the solid sample.

35. The method according to claim 34, wherein, Simultaneous focusing lasers include simultaneously focusing light from different lasers associated with each of the at least two points.

36. The method of claim 35, wherein simultaneously guiding light from the at least two points comprises: The light from each of the points is coupled to at least one corresponding optical fiber; as well as The optical fibers are arranged in a linear array at the input of the spectrometer, with at least one optical fiber associated with each point and an optical fiber associated with another point separated by a distance corresponding to at least one optical fiber diameter.

37. The method of claim 36, wherein, The rotation of the solid sample is controlled independently of the translation of the focused laser.

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