Multitrack Raman Well Plate Reader

Abstract

A microplate reader acquires Raman measurements from liquid samples contained in non-adjacent wells in a well plate. At least two Raman probes are positioned perpendicular to the microplate to acquire Raman spectral data of the non-adjacent liquid samples. Each probe includes a lens coupled to a laser and a spectrometer to focus the laser light into the sample and collect light from the sample for the spectrometer. The spectrometer may include a 2D imaging sensor and simultaneously image light from multiple probes that are spaced apart from each other to reduce crosstalk. A positioner moves the microplate or the probes to acquire data from different subsets of the non-adjacent samples and may also change the laser focus within the wells during data acquisition. Multiple fluorescent probes simultaneously acquire fluorescent data from the same sample or non-adjacent samples. The Raman and fluorescent probes are fiber coupled and positioned within the reaction chamber of the liquid handler system.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. Section 119(e) to U.S. patent application Ser. No. 63 / 293,733, filed December 24, 2021, which is incorporated by reference in its entirety.

[0002] The present disclosure relates generally to a multi-track well plate reader that provides Raman measurements alone or in combination with fluorescence, absorbance, and / or other spectroscopy-based measurements of samples contained in well plates. [Background technology]

[0003] Well plate or microplate readers are commonly used in life science and pharmaceutical applications for quantification of biological and chemical assays in microplates that can have hundreds or even thousands of wells (cavities, tubes, channels, etc.). The optical properties of the sample may change as biological, chemical, biochemical, or physical reactions occur (usually at controlled temperatures). Absorbance, fluorescence intensity, and luminescence are typical detection methods. Automated readers of well plates (or similarly arrays of vials) can be used to provide high throughput (high speed) testing per minute on typical well plates containing, for example, 8, 48, 96, or 384 wells. White light fluorescence, luminescence, and absorbance methods are used in many commercially available plate readers with well plates of various formats, including transparent, opaque, translucent, and black, but Raman plate readers are rare and slow due to single-well readout modes using low-power lasers and low-sensitivity CCDs. Well plates may exhibit different background contributions from the selected material based on the type of read (fluorescence, Raman, absorbance, etc.) Therefore, well plate properties (material, color, etc.) should be evaluated and selected specifically for Raman applications at the selected laser wavelength.

[0004] Raman microscopes have been used as microprobes for well plate reading. Another strategy involves scanning 96 to 384 wells with one Raman probe that moves relative to the well plate (XY stage). This solution involves 96 translational movements for a 96 well plate, which compromises the overall throughput, i.e., the time to acquire data across the well plate. This strategy often sacrifices sensor exposure time per well to increase throughput (wells processed / min), typically resulting in a low sensitivity solution using CCD sensors and low power lasers. In such cases, a linear (or two-dimensional (2D), vertical binning) CCD is sufficient to acquire a single spectrum when a single probe is used to record signals from the 96 wells one by one.

[0005] Another strategy for fast Raman well-plate reading proposes a lens array optical setup (8x24 matrix, 4.5mm separation) combined with a non-polarizing beam splitter cube that splits the output of a single laser to each of the multiple wells resulting in a laser power per channel of only about 7.5mW. This approach provides for static simultaneous acquisition of 192 wells. However, stray light / crosstalk between adjacent wells is increased compared to other strategies using individual standard Raman probes that are carefully designed to eliminate crosstalk between the laser and Raman paths. Splitting the laser in free space can also have stability issues, especially for low concentration sample solutions, resulting in a lower sensitivity per channel and a lower signal-to-noise ratio (SNR) associated with low laser power. Another drawback of this approach is the inability to perform simultaneous acquisition of fluorescence or other spectroscopy on the well-plate array. The laser power cannot be adjusted up or down either to a single probe or to a subsection of the array of 192 wells.

[0006] Various other approaches are described, for example, in published patent applications U.S. Patent Application Publication No. 2017 / 0010153A1, as well as U.S. Pat. Nos. 8,520,204 and 6,266,140. Summary of the Invention [Means for solving the problem]

[0007] A system and method for simultaneously acquiring spectroscopic measurements from at least two liquid samples contained in an array of sample holders, such as a microplate or well plate, may include an imaging spectrometer having a two-dimensional sensor that receives light emitted by the at least two samples and diffracted by a diffraction grating of the spectrometer during illumination by a focused laser light directed at the input of the spectrometer. The spectrometer measurements may include Raman and fluorescence measurements as well as a variety of other measurements acquired using the same or different spectrometers. To reduce sample-to-sample crosstalk and improve signal-to-noise ratio (SNR), the at least two samples may be separated or spaced apart by at least one sample holder or well of the array that is not illuminated by the focused laser light during the measurements. To reduce channel-to-channel crosstalk and further improve SNR, the light imaged onto the spectrometer sensor from each sample is spatially separated from any other samples corresponding to the Raman and / or fluorescence measurements. The spectroscopic probe may be fiber coupled to a spectrometer and an illumination laser or other light source to facilitate positioning of the probe within the filling and reaction chamber of the automated liquid handler system, where the laser, other light source, one or more controllers, and spectrometer are located outside the reaction chamber. Calibration standards may be integrated into the well plate holder for periodic automated calibration and adjustment of laser wavelength, intensity, and / or focusing.

[0008] In one embodiment, a system for acquiring Raman spectra from liquid samples in an array of sample holders includes at least one laser and at least two Raman probes, each positioned perpendicular to the array of sample holders and aligned to simultaneously acquire Raman spectra of liquid samples in non-adjacent sample holders, each Raman probe including an input optically coupled to the at least one laser and a sample lens configured to receive light from the input, focus the light from the input into an associated liquid sample, collect light from the associated liquid sample, and direct the collected light from the associated liquid sample to an output, and a Raman probe optically coupled to the output of the at least two Raman probes. the spectrometer includes a diffraction grating configured to direct light collected by the lobes to a sensor; a mechanical positioning mechanism configured to adjust a relative position of the array of sample holders and the at least two Raman probes; and at least one controller in communication with the at least one laser, the spectrometer, and the mechanical positioning mechanism, the at least one controller being programmed to generate Raman spectra based on signals from the spectrometer sensor of liquid samples in the at least two non-adjacent sample holders, and to control the positioning mechanism to change the position of the at least two Raman probes relative to the array of sample holders to simultaneously acquire Raman spectra of liquid samples in different non-adjacent sample holders of the array.

[0009] In various embodiments, each Raman probe includes an achromatic collimating lens positioned to receive light from the input, a mirror positioned to receive light from the achromatic collimating lens, a first dichroic longpass filter configured to reflect light from the mirror having a wavelength less than a first cutoff wavelength to the sample lens and transmit light from the sample lens having a wavelength greater than the first cutoff wavelength, a second dichroic longpass filter configured to transmit light received from the first dichroic longpass filter having a wavelength greater than a second cutoff wavelength and reflect light having a wavelength less than the second cutoff wavelength, and an achromatic focusing lens configured to focus light transmitted through the second dichroic longpass filter to the output. The Raman probe may also include at least one bandpass filter positioned in the optical path between the achromatic collimating lens and the mirror. In various embodiments, the output of each Raman probe is optically coupled to the input of the spectrometer by at least one optical fiber, and the optical fibers from the Raman probes are arranged in a linear arrangement at the input of the spectrometer, with the optical fibers from each Raman probe spaced apart from the optical fibers of another of the Raman probes by at least one optical fiber diameter. The spacing may be provided by one or more inactive or dead optical fibers that do not carry light arranged in the linear array of fibers between the fibers associated with the Raman probes or the fluorescent probes. The optical fibers from the Raman probes may be arranged closer to the center of the linear array than the output optical fibers from the fluorescent probes, such that the light from the Raman probes is directed closer to the optical axis of the spectrometer sensor than the light from the fluorescent probes.

[0010] In one or more embodiments, the spectrometer includes a diffraction grating including a reflective aberration-corrected holographic concave grating, and the sensor includes a two-dimensional imaging sensor such as a sCMOS sensor or a CCD sensor with a shutter. In one embodiment, the Raman and fluorescent probes are all optically coupled to a single spectrometer with a single two-dimensional sensor. Other embodiments include spectrometers with separate diffraction gratings for each connected probe, and a linear CCD sensor is shared by two or more probes. Embodiments can also include separate spectrometers optimized for either fluorescence or Raman data and optically coupled to the associated probe. The spectrometer can include slits located in the optical path between the optical fiber from each Raman probe and the diffraction grating of the spectrometer. The width of the slits may be selectable from a number of fixed width slits by at least one controller, or may have a variable width controlled by at least one controller.

[0011] The mechanical positioning mechanism may be configured to move the array of sample holders tied to at least two Raman probes (and fluorescent probes, if present) to orthogonal X and Y positions corresponding to the sample holders in the array. The Z axis position may be controlled to change the position of the focused laser light within the sample. The positioning mechanism may include a well plate holder having one or more integrated calibration reference samples, such as diamond samples. Additionally, the well plate holder may be configured to hold a calibration plate having one or more calibration reference samples, such as diamond, silicon, etc., and / or wells containing liquid calibration samples customized for a particular application. The at least one controller may be programmed to change the relative position of the Raman probe and the array of sample holders to vary the position of the focused laser light within liquid samples contained in non-adjacent sample holders while acquiring data from the spectrometer sensor to generate a Raman spectrum, thereby providing, reducing, or eliminating localized heating and / or photobleaching of the sample, improving sample homogeneity and / or averaging of measurements from different locations within the sample.

[0012] Various embodiments may include at least one laser associated with each Raman probe or a small number of Raman probes, e.g., 2-4 probes. The laser may be a single mode laser or may be a multimode laser. Separate light sources may be provided for the fluorescent probes, and multiple fluorescent probes are connected to a single light source. The Raman and fluorescent probes may be implemented as non-contact probes or as immersion probes. The Raman and fluorescent probes may be located either above or below the sample, and may be located perpendicular to the array of samples. In one embodiment, the laser source light focused on or within the sample is spatially offset from the Raman probe, at an angle or perpendicular to the array of samples. An illumination light source may be located opposite the collection light source of the probe, and may provide transmission Raman or fluorescence measurements.

[0013] In one embodiment, an additional laser is optically coupled to the input of an additional Raman probe, the additional Raman probe having an output coupled to a spectrometer and positioned to acquire a Raman spectrum of a reference sample not included in the array of sample holders.

[0014] In one or more embodiments, a system for obtaining Raman spectra from liquid samples contained within a well plate comprises at least one laser optically coupled to at least two fibers to selectively focus laser light into samples contained within at least two wells of the well plate, the at least two wells being separated by at least one well that is not illuminated by the focused laser light, the system comprises at least two Raman probes, each Raman probe aligned with an associated one of the at least two wells of the well plate that are illuminated by the focused laser light, each Raman probe including a lens configured to focus light from an associated sample onto an output of the Raman probe, the system comprises at least two Raman probes aligned with an associated one of the at least two wells of the well plate that are illuminated by the focused laser light, and each Raman probe includes a lens configured to focus light from an associated sample onto an output of the Raman probe, The system includes a spectrometer including a diffraction grating optically coupled to an output of the spectrometer and configured to direct light collected by the at least two probes to a shared sensor, the system includes a mechanical positioning mechanism configured to adjust a relative position of the well plate and the at least two Raman probes, and at least one controller in communication with the spectrometer and the mechanical positioning mechanism, the at least one controller being programmed to generate Raman spectra for liquid samples 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 of the at least two probes and the well plate to simultaneously acquire Raman spectra for liquid samples in at least two different wells of the well plate. The relative position may include a three-dimensional position of the well plate relative to the probes. Each of the at least two fibers may be tilted relative to an associated one of the at least two Raman probes that collects light emitted from the associated sample to focus laser light into an associated one of the at least two wells. The fibers may be positioned to focus laser light into the sample from a reverse direction relative to the Raman probes. The system may include a separate laser associated with the fibers.The spectrometer may include a two-dimensional sensor, in which the output of the Raman probes is connected to the input of the spectrometer by corresponding optical fibers arranged in a linear array at the input of the spectrometer, the optical fibers associated with each probe being positioned adjacent to one another and spaced from the fibers associated with the other probes by a distance corresponding to at least one fiber diameter.

[0015] In various embodiments, the system includes a number of fluorescent probes corresponding to the number of the Raman probes, each fluorescent probe being fixed for movement with an associated Raman probe, the fluorescent probe aligned with an associated well of a well plate spaced apart by at least one well that is not aligned with any other fluorescent or Raman probe. The fluorescent probes may be optically coupled to an input of a spectrometer, and the at least one controller may be further programmed to simultaneously acquire fluorescent data from the shared sensor. In some embodiments, the fluorescent probes may be aligned with the same wells as the Raman probes, and a simultaneous Raman exposure is sequentially followed by a simultaneous fluorescent exposure for each subset or group of wells associated with the probe. The shared sensor may include a two-dimensional sCMOS sensor or a CCD sensor with a shutter.

[0016] In one or more embodiments, the outputs of the at least two Raman probes are connected to the input of the spectrometer by corresponding optical fibers, and each of the plurality of fluorescent probes is coupled to the input of the spectrometer by at least one optical fiber, and the optical fibers of each Raman probe are arranged linearly and grouped together, and the optical fibers of each fluorescent probe are arranged linearly and grouped together, and the grouped optical fibers of each Raman probe are spaced apart from the grouped optical fibers of any other Raman probe or any fluorescent probe by a distance corresponding to at least one fiber diameter, and the grouped optical fibers of each fluorescent probe are spaced apart from the grouped optical fibers of any other fluorescent probe or any Raman probe by a distance corresponding to at least one fiber diameter. The grouped optical fibers of each Raman probe may be located closer to the center of the linear arrangement than the grouped optical fibers of each fluorescent probe. The Raman probe, the fluorescent probe, the at least two fibers coupled to the at least one laser, and the mechanical positioning mechanism may be located inside a reaction chamber of the automated liquid handler system, and the spectrometer and the at least one laser are located outside the reaction chamber. The mechanical positioning mechanism may include a well plate holder with integrated Raman calibration reference samples positionable by at least one controller to align with one or more of the Raman probes. The wavelength and / or intensity of a laser associated with a corresponding probe may be adjusted in response to measurements obtained from the calibration reference sample. In some embodiments, the system may include a second spectrometer optimized for fluorescence measurements having an input optically coupled to the output of the multiple fluorescent probes.

[0017] Embodiments may also include a method including simultaneously focusing laser light 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 into which the laser light is not focused, simultaneously directing light emitted by the at least two samples to a spectrometer having a diffraction grating that redirects the light at a wavelength-dependent angle to a shared sensor, acquiring Raman spectroscopy data associated with the at least two samples from the shared sensor, and controlling, by at least one controller, a position of the well plate relative to the focused laser light to simultaneously acquire Raman spectroscopy data from at least two different samples contained in as yet untested wells of the well plate. Simultaneously focusing the laser light may include simultaneously focusing light from different lasers associated with each one of the at least two samples. The method may also include simultaneously directing light from a light source to at least two samples contained in corresponding wells of a well plate, each well separated by at least one well that is not illuminated by the focused laser light or light from the light source, simultaneously directing light emitted by the samples illuminated by the light from the light source to a shared sensor of a spectrometer, and acquiring fluorescence data from the shared sensor of the spectrometer associated with the at least two samples illuminated by the light from the light source.

[0018] In at least one embodiment, simultaneously directing light emitted by at least two samples includes coupling the emitted light into at least one optical fiber at each of the at least two samples and arranging the optical fibers in a linear array at the input of the spectrometer, the optical fibers associated with each sample being grouped together and separated from optical fibers associated with another sample by a distance corresponding to at least one optical fiber diameter.

[0019] Various embodiments may also include positioning, by at least one controller, a calibration standard integrated within the well plate holder to be illuminated by the focused laser light, acquiring Raman spectroscopic data from the calibration standard, and adjusting the wavelength of the focused laser light based on the Raman spectroscopic data of the calibration standard compared to a peak wavelength previously stored for the calibration standard.

[0020] One or more embodiments according to the present disclosure may provide related advantages. For example, various embodiments provide high throughput Raman measurements obtained using Raman spectroscopy and spatially offset Raman spectroscopy (SORS) and various combinations of Raman spectroscopy with absorbance measurements, fluorescence intensity, luminescence, time resolved fluorescence, AlphaScreen, and many other types of spectroscopic and non-spectroscopic measurements of samples contained in well plates. Embodiments may provide whole well imaging using various types of two-dimensional (2D) sensors such as one or more CCD, CMOS, or scientific CMOS (sCMOS) sensors. By using a laser at the highest power / well or with a laser power split between a few wells (any typical UV, VIS, and NIR laser, typically 532 nm and 785 nm), the laser power / channel is kept reasonably high, facilitating tuning to the laser wavelength and / or intensity for the associated probe. Allocating a Raman probe to each channel ensures the best sensitivity, low stray light, and maximum SNR. The system may provide automated calibration using one or more calibration standards incorporated into the plate holder or a calibration well plate containing wells configured to contain one or more calibration standards and / or liquid calibration samples. The fiber-coupled Raman probe may incorporate an automated plate handler system located inside the filling and reaction chamber and coupled to a remotely located laser and spectrometer to facilitate real-time or near real-time spectral acquisition for live monitoring of reactions in the well plate without pre-analysis quenching or dilution. Various embodiments may include integration with an automated plate handler system with automated transfer of the well plate from the filling and reaction chamber for Raman data acquisition by the multi-probe system. [Brief description of the drawings]

[0021] [Figure 1] A representative scan pattern of a well plate with four Raman probes is shown. [Diagram 2] A prior art strategy for full frame readout of a CMOS sensor is presented. [Figure 3A] The readout of a 2D CCD sensor is shown. [Figure 3B] 1 shows the readout of a shutterless 2D CMOS or sCMOS sensor. [Figure 3C] A plan view of a typical 2D sCMOS sensor is shown. [Figure 4] 1 shows components of a multi-probe Raman well-plate reader. [Diagram 5] 1 illustrates components of another embodiment of a multi-probe Raman well plate reader. [Figure 6] 1 illustrates a system or method for simultaneously acquiring Raman and fluorescence data from a well plate. [Figure 7A] A linear fiber arrangement with one active fiber per channel and an optional slit input to the spectrometer is shown. [Figure 7B] 1 shows another configuration of linearly arranged fibers with three active fibers per channel and an optional slit at the spectrometer input, where both ends of the fibers are grouped into triangular clusters. [Figure 7C] An alternative configuration of linearly arranged fibers is shown, with seven active fibers per channel and an optional slit at the spectrometer input, where both ends of the fibers are grouped into hexagonal clusters. [Figure 7D] 13 shows another configuration of a dual column linear fiber arrangement with three active fibers per channel and an optional slit at the spectrometer input, where the resulting spectrum is laterally shifted onto the spectrometer sensor. [Figure 8]FIG. 1 is a simplified diagram showing a well plate reader with combined ram and whole well imaging. [Figure 9A] FIG. 1 is a perspective view showing components within a typical Raman probe, including input and output fiber connectors. [Figure 9B] FIG. 9B is an optical layout of the representative probe of FIG. [Figure 10] Representative fluorescent probes are shown. [Figure 11] A typical setup for spatially offset Raman spectroscopy (SORS) is shown. [Figure 12] 1 shows a SORS system that may be used to simultaneously obtain Raman measurements from multiple wells of a well plate in accordance with the present disclosure. [Figure 13] 1 shows an exemplary imaging spectrometer for use in a well plate reader according to the present disclosure. [Figure 14A] 1 shows an exemplary non-imaging spectrometer with a shared CCD sensor for use in a well plate reader according to the present disclosure. [Figure 14B] FIG. 1 is a simplified functional block diagram showing sensor control for a non-imaging spectrometer having a shared CCD sensor. [Figure 14C] FIG. 13 is a cutaway view showing the lower left / right optical chambers of a non-imaging spectrometer with a shared CCD sensor. [Figure 15] FIG. 1 is a partial perspective view showing various components of a representative well plate reader. [Figure 16] 1 illustrates an exemplary positioning mechanism having a well plate holder with integrated calibration reference samples according to the present disclosure. [Figure 17] 1 shows an exemplary calibration plate having calibration reference samples for one or more probes of a well plate reader according to the present disclosure. [Figure 18] FIG. 1 is a block diagram illustrating the operation of a system or method for simultaneously acquiring spectroscopy measurements. [Figure 19] FIG. 1 is a block diagram illustrating the integration of a multi-probe spectroscopy system with an automated liquid handler system according to the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The embodiments of the present disclosure are described herein. However, it should be understood that the disclosed embodiments are merely examples, and that other embodiments may take various alternative forms. The figures are not necessarily to scale, and some features may be exaggerated or minimized to show the details of certain components. Therefore, specific structural and functional details disclosed herein should not be interpreted as limiting, but merely as a representative basis for teaching those skilled in the art to variously adopt the subject matter described in the claims. As will be appreciated by those skilled in the art, various features shown and described with reference to any one of the figures can be combined with features shown in one or more other figures to produce embodiments not expressly shown or described. The combinations of features shown provide representative embodiments for typical applications. However, various combinations and modifications of features are consistent with the teachings of the present disclosure and may be desirable for specific applications or implementations.

[0023] As described in this disclosure, a well plate refers to a flat plate or tray with multiple wells spaced apart from one another and configured to contain liquid samples, typically ranging in volume from nanoliters to milliliters of liquid. Well plates often contain 96,384 or 1,536 wells, but may be customized for specific applications. Well plates may also be referred to as multi-well plates, microplates, microtiter plates, microwell plates, or similar variations. Well plates may be made of molded plastics, such as polystyrene, polypropylene, or polycarbonate, or may contain more complex structures to provide desired properties for certain types of measurements. Well plates may be transparent or colored, for example with white or black pigments, to provide desired optical properties suited to certain desired measurements. Similarly, well plate measurements may be taken with the cells open or a lid or cover may be placed over the cells after the samples have been deposited.

[0024] One or more configurations of well plates according to the present disclosure provide fast readout of the well plate using multiple Raman probes arranged in an array, with a laser associated with each well being read to facilitate highest power laser operation. Commercially available UV, VIS, or NIR lasers may be used. Many applications use lasers operating at 532 nm or 785 nm. This configuration allows for individual laser power adjustments at each mapped / associated portion of the well scanned by the laser. For example, in a well plate having 96 wells with 4 probes, each with an associated laser, it is possible to individually adjust the laser power at each of the 4 areas containing 24 wells. This facilitates the processing of wells containing different sample concentrations while reducing or eliminating the risk of localized heating and / or photobleaching of sample material during reading. In some configurations, one or more lasers may include power splits (e.g., using associated fibers) into a small number of wells to maintain the desired laser power / channel with a fully light-shielded Raman probe associated with each channel to ensure highest sensitivity, low stray light, and desired SNR. The probe array can consist of Raman probes, a combination of Raman and fluorescent probes, or a variety of other types of probes.

[0025] Two-dimensional (2D) CCD sensors can be used in known configurations using commercially available spectrographs and CCD camera devices to simultaneously acquire spectra from multiple wells. For example, an Isoplane spectrometer can be configured with a Pixis CCD camera, both of which are available from Teledyne Princeton Instruments, Inc. Conventional CMOS sensors have exhibited too much dark current and insufficient thermoelectric (TE) cooling for use in high-speed Raman setups. One or more embodiments according to the present disclosure can use compact scientific CMOS (sCMOS) sensors / cameras such as Sylent-Blue from HORIBA Instruments Incorporated, which achieve low read noise in the range of <1e- to 2e- and low dark current (<1e- / pixel sec), integrated into the camera with air or water TE vacuum cooling to reach -25°C to -40°C. This allows noise-free Raman spectra to be acquired simultaneously in the described embodiments. Such sCMOS sensors are typically back-illuminated (BSI) (also known as back-illuminated (BI) or back-illuminated (BT)) sensors to achieve high quantum efficiency (QE), close to 95% in the visible range.

[0026] Scientific CMOS sensors do not require a shutter when using 2D CCDs (in the Isoplane / Pixis configuration the object is carefully avoided), because such problems exist with full-frame CCDs and can only be solved by using interline CCDs (shutterless), which unfortunately feature a very low quantum efficiency (QE). Frame transfer CCDs are typically not used due to their prohibitive cost and size, but may be used for special applications. Many imaging spectrometers of the compact imaging section rectangle available from HORIBA Instruments Incorporated combine high spectral resolution (necessary for Raman) with a multi-fiber input capability (placed along the slit). The CiCi-Raman-532 HORIBA spectrometer, in which the CCD is replaced by a Sylent TE cooled CMOS camera, features 4 to 12 channels and has high sensitivity per channel when custom fiber bundles are appropriately configured (see possible arrays with 1-7 steps of active fiber per channel, separated by 1, 2, 3 or more dead fibers used for spacing and minimized crosstalk well below 1%) and slit widths are optimized (optional for high resolution requirements). The active fiber channels can be linearly arranged in two or more rows that are shifted or offset from each other.

[0027] As recognized by the inventors of the present disclosure, the need to provide a high throughput well plate reader can be addressed by combining Raman probes / lasers and fluorescence (FLUO) probes / light sources in a hybrid array to allow simultaneous acquisition of Raman and fluorescence measurements from different wells. This avoids crosstalk and shortens the long processing times associated with sequential acquisition if simultaneous acquisition is not used. Additionally, all spectra can be collected on the same imaging spectrometer with an sCMOS sensor, which may be optionally cooled. Fluorescence typically covers a wide spectral range, while Raman covers a narrow range (3,000-4,000 cm).-1 ) in comparison to the Raman-only configuration, the hybrid system may require translation between the probes and the well plate so that one combined array of Raman and fluorescent probes can be positioned to cover the entire well. Because the Raman and fluorescent probes are not positioned to focus simultaneously in the same well, there is no significant stray light or crosstalk (such as crosstalk from laser to fluorescent measurement or from fluorescent measurement to Raman) and there is no need to switch the Raman and fluorescent measurement light sources on and off consecutively. Thus, the total acquisition time for the entire well plate is greatly reduced. Depending on the particular configuration, all the probes may be positioned above the well plate or some may be positioned below the well plate. Two arrays of probes (Raman and fluorescent) may be provided, which are not adjacent and may move on the same stage or may move on two separate stages, where one array is positioned above the well plate and the other below the well plate. The latter configuration may require wells with flat, transparent well bottoms for best performance.

[0028] In some configurations, a hybrid system may include both a Raman probe and a fluorescent probe positioned to read the same well. For example, the Raman and fluorescent probes may be aimed at the same well at a slight angle (away from normal), and a Raman and fluorescent reading may be performed sequentially for that well before the stage (or probe, depending on the application) is adjusted to read the next well.

[0029] An exemplary array of sample holders embodied as a well plate 100 and associated scanning patterns for a multi-probe plate reader are shown in FIG. 1. The well plate 100 is shown in a top / bottom or plan view, with 12 columns and 8 rows of wells 102 that may be filled with liquid samples. The wells 102 are arranged in a rectangular array that extends into or out of the plane of the drawing, providing a total of 96 wells. As mentioned above, well plates or microplates are commercially available in different sizes, with common configurations including arrays of 48, 96, 192, 384, or 1,536 wells. Although a rectangular array is shown, other configurations are possible, such as, for example, an array with offset alternating rows or an array with two different arrays of the same or different shapes / configurations separated by a space. Those skilled in the art will recognize that the claimed subject matter is generally independent of the particular configuration or number of wells or other sample holders in the array. Similarly, well plates may be made of various types of materials, as mentioned above, that have properties suitable for particular types of liquid samples. The material of the well plate or other sample holder may be selected to provide desirable optical properties for spectroscopic analysis. For example, black or other optically opaque or absorbing wells may be used to reduce or eliminate stray light and well-to-well crosstalk during exposure for reading while simultaneously acquiring spectroscopic measurements from multiple wells. Various system optical components may be selected or adjusted to optimize one or more desired system parameters, such as sensitivity, exposure time and associated throughput, SNR, etc., based on the optical properties of the selected well plate material.

[0030] The well plate 100 may include a cover (not shown) that is placed over the wells to contain the liquid samples. The cover may remain in place or may be removed during spectroscopic analysis depending on the particular application and implementation. In this example, the reader includes four Raman probes configured to simultaneously read four wells having initial positions as indicated by filled dots 110, 112, 114, and 116. In this example, the well plate is translated in the xy plane by an associated mechanical positioning mechanism that includes a well plate holder in a pattern as represented by arrow 120 such that each probe reads one of the associated 24 wells and the four probes simultaneously read corresponding four wells of the well plate. Alternatively, the probes may be translated relative to the well plate. The use of multiple probes simultaneously captures spectra from multiple wells, reducing the number of xy movements compared to applications using fewer probes or a single probe, and increasing system throughput.

[0031] As shown in FIG. 1, the four probes are aligned in non-adjacent wells to reduce stray light from other probes and associated inter-well crosstalk. In the exemplary configuration shown, probe location 110 is separated by five wells from probe location 112 and by four wells from probe location 114. The probe spacing may vary depending on the specific requirements of each application, which may include optical properties of the liquid sample, well plate material, laser wavelength, sample concentration, etc. In the example of FIG. 1, adjacent wells of probe location 110 include wells at locations A2, B1, and B2, and all other wells are not adjacent to probe location 110 (A1). Similarly, adjacent wells of probe location 116 include wells at locations D6, D7, D8, E6, E8, F6, F7, and F8, and all other wells are not adjacent to location 116.

[0032] Figure 2 shows the smear that can occur when reading a prior art full frame CCD (vertical transfer) that does not use a shutter. As mentioned above, frame transfer CCDs are typically not used due to prohibitive cost and size, but may be used in special applications.

[0033] FIG. 3A shows a readout of a scientific CCD sensor with a shutter (not shown) that may be used in various applications according to the present disclosure. The shutter may be closed during sensor readout to reduce or eliminate smear. FIG. 3B and FIG. 3C show a shutterless scientific CMOS (sCMOS) sensor used in various embodiments according to the present disclosure to provide a high-speed direct readout without smear. As generally represented in FIG. 3B, a 2D CMOS or sCMOS sensor includes a 2D array 200 of pixels 210, each with associated readout circuitry 212, providing a high-speed direct readout without shutters and without smear. The 2D sCMOS sensor allows multiple spectral readouts along a height in the astigmatism-free focal plane of an imaging spectrometer, as described in further detail herein.

[0034] FIG. 4 illustrates components of a representative multi-probe well plate reader 400 that includes a Raman probe 410 and provides simultaneous readings of samples contained in four non-adjacent wells having initial locations at A1, A7, E1, and E7 of a well plate 420. The quad configuration of FIG. 4 includes four fiber-coupled 532 nm, 150 mW single mode lasers 430 and probes 410 that are in fixed positions relative to one another and positioned vertically above the well plate 420 as indicated by the filled circles. Each probe 410 is optically coupled by at least one optical fiber 432 to provide excitation light from a corresponding laser 430 perpendicular to the plane of the well plate 420. The laser light is focused into the liquid sample contained in the corresponding well that extends into the plane of the drawing. In various applications, the laser light is focused just below the surface of the liquid sample to minimize the effect of the laser light on the sample and to minimize absorption and improve the SNR of the Raman measurements. Other applications may focus the laser light deeper within the liquid sample and / or take measurements from two or more depths within the sample.

[0035] The Raman signal is collected through a common (same) lens 434 as the excitation light from the laser 430. The output from the probes is provided by at least one corresponding optical fiber 480 associated with each probe to a single four-channel imaging spectrometer 440 including a 2D imaging cooled camera 450 with a scientific CCD (combined with a shutter) or a 2D CMOS sensor 460 (without a shutter). As shown, the output light from the probes can be fed to a single spectrometer input by a linear array 470 of fibers 480, 482, 484, 486, with fiber spacing of at least one fiber diameter to reduce inter-channel crosstalk. The spacing of the active fibers can be provided by one or more dead or inactive fibers 492 that do not carry light and may or may not extend partially or completely within the fiber bundle to the corresponding probe 410. Other mechanical spacers may be used instead of dead fibers. In the representative example shown, each active fiber 480, 482, 484, and 486 is spaced apart by three fiber diameters corresponding to the inactive or dead fiber represented at 492. The active fibers are arranged linearly to form a line to the spectrometer and may optionally be coupled to an entrance slit (best shown in Figures 7A-7D). The entrance slit may be a fixed slit. In other embodiments, the slit is selected from one of a number of fixed slits and moved into position by an associated controller, such as by translation or rotation of a multi-slit wheel or device. Alternatively, a variable width slit may be controlled by a corresponding controller to provide the desired slit width.

[0036] Each active fiber 480, 482, 484, and 486 forms a corresponding line 480', 482', 484', and 486' on the sensor 460, with the unread rows of the sensor 492' coinciding with the inactive spacing fibers 492. The Raman signals 496 correspond to selected binned rows of sensor pixels that may be binned by associated software or hardware (FPGA / FW). For example, each line 480', 482', 484', and 486' may correspond to multiple rows of sensor pixels. The readouts associated with a particular line or channel are binned or grouped together to form a corresponding Raman spectrum 496, and the Raman data is acquired simultaneously to generate the four corresponding Raman spectra. After acquiring Raman measurements of samples in wells A1, A7, E1, and E7, at least one controller controls an associated mechanical positioning mechanism, such as a 2D or 3D stage with a plate holder that holds well plate 420, to the next position to simultaneously acquire data from samples in wells A2, A8, E2, and E8. This process is repeated as shown by the representative scan pattern to acquire data for all samples contained in well plate 420. A reset or home signal returns well plate 420 to the initial position shown. As described above, the relative position of well plate 420 and Raman probe 410 may be controlled by one or more controllers by moving probe 410, well plate 420, or both.

[0037] The spectrometer 440 may be implemented with a near 1:1 imaging spectrometer, including a reflective aberration-corrected holographic convex grating 442 or various other concentric spectrometer configurations (Dyson, Offner, toroid-based, Schmidt-Czerny-Turner, grating imaging spectrometers, etc.) all featuring low to no astigmatism. The number of fiber channels in such a multi-track configuration is limited by the aberrations of the spectrometer (affecting crosstalk and resolution) and the height of the sensor 460 of the cooled camera 450. In addition, the number of fiber channels is also limited by the size of the physical array of probes that can be attached to the top or bottom of the well plate and the desired cost of the system.

[0038] 5 illustrates an exemplary embodiment 500 similar to the embodiment shown and described with respect to FIG. 4, but including 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., pigtails) coupled to the probes 510 by optical fibers 532. Alternatively, fewer lasers may be used, each with an associated splitter to carry an excitation beam to two or more probes 510. In this embodiment, twelve non-adjacent probes 510 are positioned perpendicular to the plane of the well plate 520 to read associated spaced wells. The wells are spaced from one another in each direction, with at least one well not receiving focused laser light from the probe 510 during the current measurement.

[0039] A representative scan pattern 502 is shown in which each of the twelve probes 510 acquires Raman data from one of six wells of the well plate during each measurement cycle. For example, in the initial position shown in FIG. 5, measurements of samples contained in wells A1, A5, A9, C1, C5, C9, E1, E5, E9, G1, G5, and G9 are taken simultaneously. After each measurement, the relative position of the probe and well plate is changed by moving the well plate, moving the probe, or redirecting the excitation light from the probe. In one example, after simultaneously acquiring measurements from a first subset of wells, at least one controller operates at least one mechanical positioning mechanism to change the relative position of the well plate 520 and the probe 534. In this example, measurements are acquired simultaneously from the next subset of twelve wells, corresponding to wells A2, A6, A10, C2, C6, C10, E2, E6, E10, G2, G6, and G10. The process is repeated until measurements have been taken from samples in all wells of the well plate 520. Of course, the positioning / location of the probe 510 and the reading or scanning pattern 502 may vary depending on the application and implementation.

[0040] The example of FIG. 5 is similar to the example of FIG. 4, in which the output of the probe 510 is optically coupled by corresponding optical fibers (which may include one or more fibers per probe) to a multi-channel spectrometer 540 having an imaging camera 550 along with a 2D CCD or CMOS sensor 560. One end of the output fibers 580, 581, ..., 590, 591 from the probe 510 are arranged linearly spaced apart from each other by at least one fiber diameter (three fiber diameters in this example) at the input of the spectrometer 540, as shown at 570. Spacing of the fibers 580-591 may be provided by one or more inactive or non-functional fibers 592. The light from each fiber forms a corresponding line 580', 581', ..., 590', 591' on the 2D sensor 560. Each line may correspond to multiple rows of sensor pixels, and each column of the multiple rows are binned or combined together to generate a corresponding Raman spectrum 596. Each line is separated by multiple rows of unread sensor pixels corresponding to non-functioning fibers 592' to reduce inter-channel crosstalk and increase SNR. The location of the fibers corresponding to a particular Raman probe relative to the fibers corresponding to another probe is chosen for convenience and can vary from application to application. For example, fiber 580 can be located anywhere in the linear arrangement rather than in a top or bottom position. In applications having more than one fiber associated with the output from one probe, the fibers from each probe are grouped together and located adjacent to each other and spaced apart from the other probe fibers by one or more fiber diameters, as shown and described in more detail with respect to FIG.

[0041] FIG. 6 illustrates the operation of a system or method 600 for simultaneously performing Raman and fluorescence measurements from samples contained in different wells of a well plate 620. The double-quad configuration of FIG. 6 includes four Raman probes 610 (A-D) and four fluorescence probes 612 (A-D). Each probe 610, 612 is positioned to obtain a corresponding measurement from a sample contained in a different non-adjacent well. In this example, four lasers 630 provide excitation light to a corresponding Raman probe 610, i.e., each laser 630 provides excitation light to one of the Raman probes 610. Four different illumination sources 636 provide illumination of the corresponding wells for the fluorescence measurements. The fluorescence illumination sources 636 may be LEDs, laser diodes, or filtered white light, and may provide, for example, the wavelengths (UV-VIS-NIR) desired for illumination.

[0042] 6 requires additional positioning of the well plate 620 in the xy plane where only fluorescence measurements are acquired, for the fluorescent probes 612 (A-D) to read wells between the initial positions of the fluorescent probes (A3, A9, E3, and E9) and the initial positions of the Raman probes (A1, A7, E1, and E7), but may provide an overall acceleration of 75-80% with simultaneous fluorescence and Raman measurements in other wells. In the exemplary configuration shown, additional relative movement of the well plate and probe is required for the fluorescent probe 612 to scan wells in columns 1, 2, 7, and 8. One skilled in the art would recognize similar requirements for different spacing or other orientations of the Raman probes 610 and fluorescent probes 612. As a non-limiting example, the configuration may include Raman probe 610, fluorescent probe 612 offset in the xy array by one or more positions in the x-axis (row) and / or y-axis (column) directions relative to the initial position of Raman probe 610(A-D) measuring wells A1, A7, E1, and E7, and fluorescent probe 612(A-D) measuring wells B2, F2, B8, and F8. Probe positioning may vary based on the requirements of a particular application and implementation.

[0043] The configuration of FIG. 6 utilizes a fiber bundle 680 connected from the output of the Raman probe 610 to the input of the imaging spectrometer 640 and a fiber bundle 682 connected from the output of the fluorescent probe 612 to the input of the imaging spectrometer 640. One end of the fibers 680, 682 are arranged in a linear arrangement at the input of the spectrometer 640. The fibers associated with each probe (whether Raman or fluorescent) are separated by at least one fiber diameter 692 by the fibers associated with another probe to reduce inter-channel crosstalk. The spacing of the fluorescent probes can be different from the spacing of the Raman probes. Additionally, because the spectral resolution along the height of the sCMOS sensor 660 decreases with distance from the optical center, the fibers associated with the Raman probes 610 (A-D) are located in the center of the linear arrangement and the fibers associated with the fluorescent probes 612 (A-D) are located on either side of the Raman probe fibers. In other words, the fiber associated with the Raman probe 610 is located closer to the center of the linear arrangement than the fiber associated with the fluorescent probe 612, and the corresponding imaged lines of light 612A', 612B', etc. will be further from the optical axis of the sensor 660 than the lines of light being imaged 610A', 610B'. Thus, the angle of incidence (AOI) for the fluorescent readout (low resolution requirement) is furthest from the optical center of the sensor. This configuration works well in hybrid configurations including both Raman and fluorescent probes, since the resolution required for fluorescence is typically much lower (on the order of 1 / 5 to 1 / 15) than that required for Raman. As with the example above, each line of light being imaged may correspond to multiple rows of sensor pixels that are grouped or binned by column to generate corresponding Raman and fluorescent spectra 696 and 698. The number of pixel rows associated with each of the fluorescent lines being imaged may be different than the number of pixel rows associated with each of the Raman lines being imaged. As shown in the example of FIG. 6, a single spectrometer with a single diffraction grating and 2D sensor can be used to simultaneously capture Raman and fluorescence measurements from samples in multiple wells.

[0044] Although the inventors have recognized that positioning or aligning probes on non-adjacent wells reduces well-to-well crosstalk while acquiring measurements, various applications may compromise crosstalk and SNR by acquiring fluorescence measurements from the same wells as Raman measurements to achieve a desired throughput or other overall system performance.

[0045] 7A-7D show various linear configurations of fibers for coupling light emitted in response to excitation light by samples contained in a well plate to the input of a spectrometer. As shown in FIG. 7A, the first configuration includes four probes, with active fibers 702, 704, 706, and 708 in each of the four channels corresponding to simultaneous acquisition from the wells. Each active fiber 702, 704, 706, and 708 is separated by a number of inactive fibers 710. As mentioned above, the inactive fibers are used for convenience and may be replaced by any suitable spacer. However, the use of inactive fibers provides a low-cost and precise diameter that facilitates the formation of a linear arrangement of fibers, and the positioning of such fiber arrays is a common task for fiber assembly providers. Other mechanical spacers may be used if the desired spacing distance between active fibers is not an integer multiple of the fiber diameter.

[0046] In the example of the first configuration of FIG. 7A, three inactive fibers 710 are located between adjacent active fibers, i.e., between fibers 702 and 704, between 704 and 706, and between 706 and 70, in a linear arrangement at the input of the spectrometer, although other configurations may include more or fewer inactive fibers or other spacers. An optional slit 720 may be used to provide the desired spectral resolution for any of the configurations shown. As mentioned above, the slit 720 may be of a fixed width, may be selected by a controller from multiple slits having different widths, or may be a variable width slit controlled by an associated controller. The number of inactive spacing fibers may be selected to reduce crosstalk below a threshold desired for the particular selected imaging spectrometer, and generally, greater spacing between fibers corresponds to lower inter-channel crosstalk.

[0047] 7B shows an example of two or more active fibers per well / channel separated by one or more inactive fibers. In the representative example shown, three active fibers 730, 734, 738, 742 are provided for corresponding channels 1-4, with two inactive fibers 732, 736, 740 separating each group or set of three active fibers. The active fibers 742 are 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 to the spectrometer. An optional slit 746 may be provided, as described above.

[0048] 7C includes seven active fibers 760 per channel with four inactive fibers 762 separating each active fiber group, the fibers arranged in a linear array at the input to the spectrometer. The active fibers 760 are arranged in a cluster 764 at one end to capture the Raman output signal from the probe, and at the opposite end of the linearly arranged fibers with additional inactive fibers 762 or similar spacers at the input to the spectrometer. An optional slit 766 may be provided as described above.

[0049] 7D includes three active fibers 770 in each channel separated by one or more inactive fibers 772. The fibers are arranged in two parallel rows 780, 782 at the input to the spectrometer and may be used without slits or, optionally, with a patterned slit or slit array as shown. This shifts the spectrum on the 2D sensor, as represented by spectrum 786 compared to spectrum 788, but has the advantage that strong peaks such as those represented by 790 and 792, which would otherwise be aligned and overlap on the same sensor pixel, do not interfere with each other.

[0050] As mentioned above, high throughput well plate readers may combine Raman with other types of measurements. The fiber arrangements shown in Figures 7A-7D may be used to couple various types of probes to each spectrometer. For example, an application having a fluorescent probe may use any of the fiber arrangements shown for the fluorescent probe in combination with a Raman probe, as described above. The number of fibers per fluorescent channel may differ from the number of fibers.

[0051] FIG. 8 is a simplified diagram of a well plate reader combining Raman and whole-well imaging. As shown, a Raman probe 800 is used to read one well 802 of a well plate 804, while an LED 810 or filtered broadband source is used to simultaneously read a second non-adjacent well 812. Light from the probe is directed by a first mirror 830 and a second dichroic mirror or beam splitter 832, respectively, to a camera 820 having a 2D sCMOS sensor, which may be cooled or uncooled, to provide whole-well imaging. As shown in the example of FIG. 8, the Raman probe 800 illuminates the liquid sample in the corresponding well 802 from one side of the well plate 804 (the top side in this example) with light emitted by or transmitted through the sample collected from the opposite side of the well plate 804 (the bottom side in this example). Similarly, an additional light source, such as an LED 810, illuminates a non-adjacent well 812 from above the well plate 804 with emission and transmission light collected from below the well plate 804. Optical fibers may be used to carry illumination light from one or both probes to the associated wells 802, 812 and / or to collect light and carry the collected light to a camera 820, depending on the particular application and implementation.

[0052] 9A and 9B show components of a representative Raman probe 900, including an input fiber connector 910 and an output fiber connector 912. The input 910 is connected to a laser source (not shown). The probe 900 may be located either above the well plate or below the well plate, depending on the particular application and implementation, as described above. Similarly, in some applications, the illumination / exposure components configured to direct laser light to the sample may be separate from the components used to collect light emitted by or transmitted through the sample, as described above.

[0053] In the example shown in Figures 9A and 9B, various optical components are contained within a housing 902 (shown with the cover removed in Figure 9A). Light from a laser source passes through an input 910, an achromatic collimating lens 914, and one or more optical bandpass filters 916, 918 to a mirror 920. The bandpass filters can be positioned at an angle with respect to the optical axis of the bandpass filter 916 to reduce or eliminate back reflections of the laser light back into the laser input that could otherwise have the effect of destabilizing the laser. The laser light is reflected by the mirror 920 to a dichroic longpass filter 930, which reflects the wavelengths of the laser light to a plano-convex sample lens 932, which focuses the light into the liquid sample contained within each well. Light emitted by the liquid sample is collected by the same plano-convex sample lens 932 and directed to the dichroic longpass filter 930. The dichroic long-pass filter 930 transmits the light (including the wavelength shifted from the reflected laser light) to a second dichroic long-pass filter 934 (or a notch filter), which transmits the filtered light to an achromatic focusing lens 940. The achromatic focusing lens 940 focuses the light into one or more optical fibers coupled to the 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 such as those shown and described with reference to Figures 7A-7D may be used to collect the signal light and couple the Raman probe to the input of the spectrometer. Other Raman probe configurations may be used as known to those skilled in the art. For example, a dip probe may be used that is immersed in the sample contained in the well being measured, but may require self-cleaning between measurements to avoid cross-contamination of the samples.

[0054] FIG. 10 shows a representative fluorescent probe 1000. A light source 1010 (LED, filtered broadband, etc.) is coupled to a conduit of the probe 1010 by a fiber bundle 1012 and illuminates a liquid sample 1004 contained in one of the wells of a well plate from either above or below depending on the particular application and implementation. Fluorescence emitted by the liquid sample is then coupled by one or more fibers 1014 to the input of an associated imaging spectrometer 1016 in a linear arrangement with one or more other fluorescent and / or Raman probes, as described above. The optical fibers at the tip 1020 of the probe 1000 may be arranged in a cluster with at least one central illumination fiber 1022 coupled to the fiber bundle 1012 and the light source 1010 surrounded by a group of collection fibers of the fiber bundle 1014 coupled to the spectrometer 1016, or vice versa. The fluorescent probe 1000 may be configured as a remote sensing (as shown) or as a dip / dip probe. One or more fibers in the probe may be angled with respect to the sample surface to reduce backscattering of the illumination light.

[0055] 11 and 12 show representative configurations of a spatially offset Raman spectrometer (SORS) embodiment of a well plate reader according to the present disclosure. The SORS configuration can be used to eliminate background from the well plate material (below, sides, etc.) and any contribution from a lid / cover that may be applied to the well plate. SORS allows probing of liquid samples at different liquid levels within the wells of a well plate. FIG. 11 shows the relative positions of an excitation laser beam 1110 and a collection probe 1120 located above the well and spatially offset by a distance Δ. The SORS embodiment can be used to obtain measurements within an upper layer 1130 and / or a lower layer 1140 of the liquid sample. Alternatively, the upper layer 1130 can correspond to a well plate cover, and measurements are obtained only below the well plate cover, within a selected depth of the liquid sample.

[0056] FIG. 12 shows a configuration that can be used for line scanning to determine the desired spatial offset. The use of an imaging spectrometer with a large number of pixels in the vertical (spatial) axis facilitates the line scanning configuration. The line scanning configuration may be particularly suitable for applications involving a well plate cover / lid placed on the top of the well or in configurations where, for example, the Raman signal is acquired through the bottom of the well. The SORS system 1200 includes a laser 1210 coupled by 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 well plate and offset from the collection optics. The laser focusing unit 1214 generates a scan line 1230 on or in the well 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 an imaging spectrometer 1250. One or more optical elements, such as a lens 1252, long pass filters 1252, 1254, and an input slit 1256, may be used to provide the Raman signals 1234 to the spectrometer input to generate a scattering image 1270 for subsurface evaluation. Related signals / data are provided to one or more controllers, such as a computer 1280.

[0057] FIG. 13 shows a representative spectrometer for use in a well plate reader according to the present disclosure. The spectrometer 1300 includes a reflective aberration corrected convex holographic grating (ACHG) 1310 and a camera 1320 having a cooled 2D sensor 1322. The camera may be implemented with a Sylent-Blue scientific CMOS camera available from HORIBA Instruments Incorporated, which includes a sCMOS sensor with 2048×2048 active pixels with an active light sensitive area of ​​22.528 mm×22.528 mm, and may include a Peltier cooled vacuum head or alternatively an argon purged nitrogen back-filled sCMOS sensor. The imaging spectrometer 1300 includes a fiber optic input 1312 that may accept multiple linearly arranged fibers from associated probes with any slits, as described above. Light from two or more coupled fiber probes 1316 is reflected by a mirror 1314 to the diffraction grating 1310. The diffraction grating 1310 images the diffracted light from the two or more probes onto a 2D sensor 1322 of a camera 1320 to simultaneously acquire multiple Raman spectra from samples in associated wells of a well plate. The imaging spectrometer 1300 and camera 1320 may be in communication with one or more controllers or computers (not shown) to acquire relevant data from the sensor 1322 and to control various features of the spectrometer 1300 (such as selectable or variable slits) and camera 1320.

[0058] 14A-14C show an exemplary non-imaging spectrometer 1400 for use in a well plate reader according to the present disclosure. The non-imaging spectrometer 1400 may be implemented, for example, by a PoliSpectra® Quad multi-channel grating spectrometer available from Horiba Instruments Incorporated. The PoliSpectra® Quad spectrometer integrates four VS70 spectrometers, each with a flat-field aberration-corrected holographic concave grating. This high-throughput spectrometer 1400 includes four fiber inputs 1402, 1404, 1406, 1408, four concave aberration-corrected gratings 1410, 1412, 1414, 1416, and two imaged linear CCD sensors 1420, 1422 to provide high-speed simultaneous acquisition of four channels with minimal crosstalk. The spectrometer 1400 includes four optical chambers, each with an associated fiber input and grating. Light from the upper fiber inputs 1402, 1404 is directed to corresponding photons 1410, 1412 and then to an associated portion of a shared linear CCD sensor 1420. Similarly, light from the lower fiber inputs 1406, 1408 is directed to respective gratings 1414, 1416, as best shown in Figure 14C. Light from each grating 1414, 1416 is directed to a corresponding portion 1422A, 1422B of a shared linear CCD sensor 1422.

[0059] As shown in the simplified functional block diagram of FIG. 14B, the main electronics board 1450 is designed to simultaneously read the linear CCD sensors 1420, 1422, providing 1-8X binning capability to acquire data from all four channels simultaneously. The signals from the CCD sensors 1420 and 1422 are provided to analog conditioning circuits 1460, 1462, respectively. The conditioned sensor signals are provided to each channel of a 16-bit analog-to-digital converter (ADC) 1470. The generated digital data is captured in temporary computer-readable storage 1480 for reading, analysis, and storage by one or more connected controllers or computers (not shown).

[0060] 15 is a partial perspective view showing various components of a representative well plate reader. In this example, a system subassembly 1500 includes Raman probes 1510, 1512, 1514, 1516 fixed relative to one another and aligned with corresponding non-adjacent wells of a well plate 1520. A mechanical positioning mechanism 1530 is positionable under control of a number of motors 1540 that control the position 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 described above, at least one controller can control the position of the well plate 1520 relative to the focused laser light from the Raman probes 1510, 1512, 1514, 1516 to simultaneously acquire Raman spectroscopy data from different samples contained in corresponding wells of the well plate 1520. In some configurations, the positioning mechanism 1530 is controlled to vary the relative positions of 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 light from each probe within an associated well during acquisition of Raman measurements to mix the liquid sample and / or acquire data from multiple points within the liquid sample. The positioning mechanism 1530 is then controlled to align untested wells of the well plate with the probes in a predetermined pattern, as described above.

[0061] FIG. 16 illustrates an exemplary positioning mechanism 1600 having a well plate holder 1606 with at least one integrated calibration reference sample 1610, 1612 according to the present disclosure. The positioning mechanism 1600 includes at least one motor 1620 that moves the well plate holder 1606 in at least two orthogonal directions and optionally a third orthogonal direction. One or more associated controllers may control the positioning mechanism 1600 to periodically align the integrated calibration reference sample 1610, 1612 with a corresponding probe located above or below the well plate holder 1606 during the calibration process. Measurements obtained by the system from the calibration reference may be compared to previously established and stored Raman data for the material of the reference. Detected deviations in peak wavelength or intensity may be used to adjust the wavelength / intensity of the corresponding laser or other light source associated with the probe. The number and material of the reference samples may vary based on the number and type of probes being calibrated, as well as the maximum available movement of the positioning mechanism to position the probe relative to the reference and the time designated to complete the calibration of all probes. Different reference samples may be provided for different types of probes and / or to calibrate the probes at desired specified wavelengths. In one representative example, diamond calibration samples 1610, 1612 are fitted to corresponding arms / legs of the well plate holder 1606, and each of the diamond calibration samples is used during the calibration of two probes.

[0062] One or more controllers of a typical well plate reader system may be programmed to automatically calibrate the probe periodically, such as once a day. The controller may be programmed to align an associated calibration standard with the probe, collect measurement data, compare the measurement data to previously stored data for the reference material, and control the wavelength and / or intensity of the corresponding light source based on the comparison. For example, if the calibration process detects a variation in wavelength (X-axis) for one or more peak wavelengths between the measured calibration sample material and the known sample material spectrum, the controller automatically corrects for this variation by shifting the wavelength of the laser to reduce the difference between the measured peak wavelength and the reference peak wavelength of the material. Similarly, the calibration process may detect variations in intensity and adjust the light source intensity accordingly. Alternatively, the system may suggest a different calibration process using a calibration plate with multiple reference samples held by the well plate holder 1606. The calibration plate may be used to calibrate the intensities of various light sources against each other to match throughput across different probes.

[0063] FIG. 17 illustrates a representative calibration well plate 1700 having at least one calibration reference sample for one or more probes of a well plate reader according to the present disclosure. The calibration well plate 1700 has a size based on a corresponding well plate that may be secured to a well plate holder of a well plate reader system as described herein. In the illustrated example, the calibration well plate 1700 is also sized similarly to a 96 well plate and configured to be positioned within a corresponding well plate holder. Additionally, the calibration well plate 1700 provides a representative example of a well plate reader system having four Raman probes. Thus, the number and location of the various calibration samples are selected based on application requirements and may vary based on the particular application and implementation.

[0064] In the example of Figure 17, a calibration well plate 1700 includes symmetrically positioned calibration standard samples and calibration wells, with 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 that may be filled with any liquid calibration sample desired for a particular application. The size of the calibration standard samples may be based on commercially available sizes for a particular material.

[0065] The inventors have realized that the intensity of the sharp Raman bands of certain materials can be used to monitor the intensity variation to trigger recalibration. Many materials will degrade under laser exposure and are therefore poor candidates for calibration standards. Diamond was therefore selected as a material that is very stable over a significant number of laser exposures. In addition, hard materials such as diamond and silicon will provide peaks whose intensity varies with the Z-axis position of the probe, so that calibration using such materials can be used to adjust the Z-axis focusing precision of one or more probes.

[0066] The liquid well 1730 can be filled with either a typical liquid / base used in a particular application or some reference liquid. Any Z-axis focusing inaccuracies have less effect on the intensity. Thus, a reference liquid provides a more reliable intensity calibration than a hard material calibration standard. The Raman peak intensity remains relatively stable over the Z-axis focusing range as long as the light is focused below the surface of the liquid. However, liquid calibration standards will evaporate and user intervention may be required to refill the associated calibration well.

[0067] The use of different calibration standard sample materials can be useful in identifying one or more components in need of adjustment or replacement. For example, the Raman peak of a single material (such as diamond in this example) may be insufficient to diagnose degradation or changes in various other system components in the optical path of a laser, fiber, probe, spectrometer, etc. Calibration standard sample materials (e.g., silicon wafers, liquids, etc.) with other peaks and / or multiple peaks can facilitate diagnosis and subsequent compensation of specific components associated with corresponding portions of the spectrum.

[0068] Many components along the optical path other than the laser or other light source can contribute to inaccurate measurements associated with component failure, misalignment, wear, aging, etc. Lasers are the most common and frequent source of inaccuracies. For this reason, laser aging compensation is preferably performed automatically without user action. Other situations such as broken filters in the probe, spectrometer / camera shifts, broken or damaged fibers, positioning errors due to the positioning mechanism, etc. can also all affect the measurements taken. Thus, the use of multiple calibration reference samples provided by the calibration plate 1700 provides additional flexibility in diagnostics and calibration.

[0069] FIG. 18 is a block diagram showing the operation of a system or method for simultaneously acquiring spectroscopic measurements. The control logic or functions executed by one or more controllers or computers are represented in the block diagram. The block diagram provides a representative control strategy, logic, or algorithm that may be implemented using one or more processing strategies, such as event-driven, interrupt-driven, multitasking, multithreading, etc. Thus, the various steps or functions shown may be performed in the order shown, in parallel, or may be omitted in some cases. Although not always explicitly shown, one skilled in the art will recognize that one or more of the steps or functions shown may be repeatedly performed depending on the particular processing strategy being used. Similarly, the order of processing is not necessarily required to achieve the features and advantages described herein, and 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, the control logic may be implemented in software, hardware, or a combination of software and hardware in one or more controllers depending on the particular application. If implemented in software, the control logic may be provided in one or more non-transitory computer-readable storage devices or media that store data representing code or instructions executed by the computer to control the means or its subsystems. The computer-readable storage devices or media may include one or more of several known physical devices that utilize electrical, magnetic, and / or optical storage to hold executable instructions and associated data, operating variables, and the like.

[0070] The system or method may include simultaneously focusing laser light into at least two samples contained in corresponding wells of a well plate, as represented at 1802. The at least two samples contained in the wells are separated by at least one well where the laser light is not focused. Light emitted by the at least two samples is simultaneously directed to a spectrometer having a diffraction grating, as represented at 1804. The diffraction grating redirects the light at a wavelength-dependent angle to a shared sensor. Block 1806 represents acquiring Raman spectroscopy data associated with the at least two samples from the shared sensor. Block 1808 represents controlling a position of the well plate relative to the focused laser light to simultaneously acquire Raman spectroscopy data from at least two different samples contained in untested wells of the well plate.

[0071] The simultaneously focusing laser light represented by block 1802 may include simultaneously focusing light from different lasers associated with each one of the at least two samples. Block 1802 may also include simultaneously directing light from a light source to at least two samples contained in corresponding wells of a well plate, each well being separated by at least one well that is not illuminated by the focused laser light or light from the light source. Block 1804 may also include simultaneously directing light emitted by the samples illuminated by the light from the light source to a shared sensor of a spectrometer. Meanwhile, block 1806 may include acquiring fluorescence data from a shared sensor of a spectrometer associated with the at least two samples illuminated by the light from the light source.

[0072] In at least one embodiment, block 1804 includes simultaneously directing light emitted by at least two samples by coupling the emitted light into at least one optical fiber at each of the at least two samples and arranging the optical fibers in a linear array at an input of the spectrometer, the optical fibers associated with each sample being grouped together and separated from optical fibers associated with another sample by a distance corresponding to at least one optical fiber diameter.

[0073] Various embodiments may also include positioning, by at least one controller, a calibration standard integrated within the well plate holder illuminated with the focused laser light, acquiring Raman spectroscopic data from the calibration standard, and adjusting the wavelength of the focused laser light based on the Raman spectroscopic data of the calibration standard compared to a previously stored peak wavelength of the calibration standard.

[0074] FIG. 19 is a block diagram illustrating the integration of a multi-probe spectroscopy system with an automated liquid handler system according to the present disclosure. The automated liquid handler system 1900 (also referred to as an automated lab robot) may include a liquid filling instrument 1910 that provides robotic pipetting of liquid into each well of a well plate 1912 in a reaction chamber 1930. The reaction chamber 1930 may provide heating / cooling, mixing, agitation, quenching (dilution) of the liquid sample, etc., and may include a positioning or transport mechanism 1914 that moves one or more well plates 1912 within the reaction chamber 1930. The positioning mechanism 1914 may be controlled to position the well plate 1912 relative to the probe 1916 for spectroscopic analysis without requiring dilution or quenching for analysis, as described above. Alternatively, some applications may include a second positioning mechanism 1920 associated with the spectroscopic analysis section of the reaction chamber 1930. The well plate 1912 is transferred from the positioning mechanism 1914 to the positioning mechanism 1920 for spectroscopic analysis (without dilution or quenching of the sample). As also shown in FIG. 19, one or more lasers 1940, spectrometer / camera 1942, light source 1944, power supply 1946, etc. are located outside the reaction chamber 1930 and are connected by corresponding fibers 1950, wires, etc.

[0075] Although exemplary embodiments have been described and illustrated above, these embodiments are not intended to describe all possible forms encompassed by the teachings of the present disclosure. The terms used herein are terms of description rather than limitation, and it is understood that various modifications may be made without departing from the spirit of the claims. As noted above, features of various exemplary embodiments may be combined to form further embodiments not expressly described or shown. Although various embodiments may be described as offering advantages or being preferred over other embodiments or prior art implementations in terms of one or more desired characteristics, those skilled in the art will recognize that one or more features or characteristics may be compromised to achieve desired overall system attributes depending on the particular application and implementation. These attributes may include, but are not limited to, strength, durability, life cycle, marketability, appearance, packaging, size, usefulness, weight, manufacturability, ease of assembly, and the like. Thus, an embodiment described as less desirable than other embodiments or prior art implementations in terms of one or more characteristics is not necessarily outside the spirit of the present disclosure or claims, and may be desirable for a particular application.

Claims

1. 1. A system for acquiring Raman spectra from liquid samples in an array of sample holders, comprising: at least one laser; at least two Raman probes, each positioned perpendicular to the array of sample holders and aligned to simultaneously acquire Raman spectra of the liquid samples in non-adjacent sample holders, each Raman probe including an input optically coupled to the at least one laser and a sample lens configured to receive light from the input, focus the light from the input into an associated liquid sample, collect light from the associated liquid sample, and direct the collected light from the associated liquid sample to an output; a spectrometer including a diffraction grating optically coupled to the outputs of the at least two Raman probes and configured to direct light collected by the Raman probes to a sensor; a mechanical positioning mechanism configured to adjust a relative position of the array of sample holders and the at least two Raman probes; at least one controller in communication with the at least one laser, the spectrometer, and the mechanical positioning mechanism; The at least one controller generating a Raman spectrum based on signals from the sensor of the spectrometer of the liquid samples in the at least two non-adjacent sample holders; and programmed to control the mechanical positioning mechanism to change the position of the at least two Raman probes relative to the array of sample holders to simultaneously acquire Raman spectra of the liquid samples in different non-adjacent sample holders of the array. system.

2. Each of the at least two Raman probes comprises: an achromatic collimating lens positioned to receive light from the input; a mirror positioned to receive light from the achromatic collimating lens; a first dichroic longpass filter configured to reflect light from the mirror having wavelengths less than a first cutoff wavelength to the sample lens and to transmit light from the sample lens having wavelengths greater than the first cutoff wavelength; a second dichroic longpass filter configured to transmit light received from the first dichroic longpass filter having wavelengths greater than a second cutoff wavelength and to reflect light having wavelengths less than the second cutoff wavelength; an achromatic focusing lens configured to focus light transmitted through the second dichroic longpass filter to the output; The system of claim 1 , comprising:

3. The system of claim 2 , wherein each of the at least two Raman probes further comprises at least one bandpass filter located in an optical path between the achromatic collimating lens and the mirror.

4. the diffraction grating comprises a reflective aberration-corrected holographic concave grating; the mechanical positioning mechanism is configured to move the array of sample holders relative to the at least two Raman probes in mutually orthogonal X, Y, and Z directions; The system of claim 1 , wherein the sensor comprises a two-dimensional imaging sensor.

5. The system of claim 4 , wherein the two-dimensional imaging sensor comprises an sCMOS sensor.

6. an output of each Raman probe is optically coupled to an input of the spectrometer by at least one optical fiber; 2. The system of claim 1 , wherein the optical fibers from the Raman probes are arranged in a linear fashion at the input of the spectrometer, and the optical fiber from each Raman probe is spaced apart from the optical fiber of another one of the Raman probes by at least one optical fiber diameter.

7. 7. The system of claim 6, wherein the output of each Raman probe is coupled to the output of the spectrometer by a plurality of optical fibers, and the optical fibers from each Raman probe are separated from the optical fibers of other ones of the Raman probes by a plurality of inactive optical fibers.

8. 8. The system of claim 7, further comprising a slit located in an optical path between the optical fiber from each Raman probe and the diffraction grating of the spectrometer.

9. The system of claim 8 , wherein a width of the slit is controlled by the at least one controller.

10. The system of claim 7 , wherein the at least one controller is programmed to position one of a plurality of fixed-width slits in the optical path.

11. 2. The system of claim 1, wherein the at least one controller is programmed to change a relative position of the Raman probe and the array of sample holders to change a position of a focused laser light within the liquid sample contained in the non-adjacent sample holder while acquiring data from a spectrometer sensor to generate the Raman spectrum.

12. The system of claim 11 , wherein the at least one laser comprises a single mode laser.

13. The system of claim 1 , wherein the at least one laser comprises a plurality of lasers, each of the plurality of lasers optically coupled to the input of one of the Raman probes.

14. The system of claim 1 , wherein all of the Raman probes are optically coupled to a single spectrometer having a single two-dimensional sensor.

15. The system of claim 1 , wherein the sensor of the spectrometer comprises a CCD sensor.

16. The CCD sensor includes a two-dimensional sensor, 16. The system of claim 15, wherein the spectrometer further comprises a shutter located in an optical path between the CCD sensor and the diffraction grating.

17. The system of claim 1 , wherein the at least two Raman probes are located within a reaction chamber of an automated liquid handler.

18. the spectrometer and the at least one laser are located outside the reaction chamber of the automated liquid handler; the inputs of the at least two Raman probes are optically coupled to the at least one laser by a first plurality of optical fibers; 20. The system of claim 17, wherein the outputs of the at least two Raman probes are optically coupled to the spectrometer by a second plurality of optical fibers.

19. At least one light source; and a plurality of fluorescent probes, each of the plurality of fluorescent probes is optically coupled to the at least one light source by at least one input optical fiber and optically coupled to the spectrometer by at least one output optical fiber; 10. The system of claim 1, wherein the output optical fibers are arranged in a linear array at the input of the spectrometer and spaced apart from each other by at least one optical fiber diameter.

20. an output of each Raman probe is optically coupled to an input of the spectrometer by at least one optical fiber; the optical fiber from the Raman probe is aligned linearly with the output optical fiber from the fluorescent probe at the input of the spectrometer; 20. The system of claim 19, wherein the optical fiber from each Raman probe is spaced from the optical fiber of another one of the Raman probes by at least one optical fiber diameter.

21. 21. The system of claim 20, wherein the optical fiber from the Raman probe is positioned closer to a center of the linear array than the output optical fiber from the fluorescent probe.

22. 21. The system of claim 20, wherein light from the Raman probe is directed closer to an optical axis of the sensor of the spectrometer than light from the fluorescent probe.

23. 20. The system of claim 19, wherein each of the fluorescent probes is positioned perpendicular to the array of sample holders aligned with non-adjacent sample holders relative to the Raman probe and other fluorescent probes.

24. the Raman probe is positioned above the array of sample holders; 20. The system of claim 19, wherein the fluorescent probes are located below the array of sample holders.

25. the array of sample holders comprises a microplate; 2. The system of claim 1 , wherein the mechanical positioning mechanism comprises a microplate holder having at least one integrated calibration standard positionable by the at least one controller to align one or more of the Raman probes with a calibration standard and calibrate the at least one laser.

26. 26. The system of claim 25, wherein the at least one controller adjusts a wavelength or intensity of the at least one laser in response to measuring a Raman spectrum of the at least one integrated calibration standard.

27. The system of claim 1 , further comprising at least one additional spectrometer optically coupled to the at least one additional Raman probe.

28. The system of claim 1 , wherein the at least two Raman probes are positioned above the array of sample holders.

29. further comprising an additional laser optically coupled to the additional Raman probe; 10. The system of claim 1, wherein the additional Raman probe is coupled to the spectrometer and has an output positioned to obtain a Raman spectrum of a reference sample not included in the array of sample holders.

30. 2. The system of claim 1, wherein the spectrometer comprises a non-imaging spectrometer including a separate diffraction grating associated with each of the plurality of Raman probes and a single linear sensor associated with each pair of diffraction gratings.

31. 31. The system of claim 30, wherein each linear sensor comprises a CCD.

32. At least one light source; a plurality of probes, each probe having an input optically coupled to the at least one light source; a second spectrometer optically connected to the outputs of the plurality of probes; The system of claim 1 further comprising:

33. 33. The system of claim 32, wherein each of the plurality of probes comprises a fluorescent probe.

34. 1. A system for obtaining Raman spectra from a liquid sample contained within a well plate, comprising: at least one laser optically coupled to the at least two fibers to selectively focus laser light into samples contained in at least two wells of the well plate, the at least two wells being separated by at least one well that is not illuminated by the focused laser light; at least two Raman probes, each aligned with an associated one of the at least two wells of the well plate illuminated by the focused laser light, each Raman probe including a lens configured to focus light from an associated sample onto an output of the Raman probe; a spectrometer optically coupled to the outputs of the at least two Raman probes, the spectrometer including a diffraction grating configured to direct light collected by the at least two probes to a shared sensor; a mechanical positioning mechanism configured to adjust a relative position between the well plate and the at least two Raman probes; at least one controller in communication with the spectrometer and the mechanical positioning mechanism; The at least one controller generating a Raman spectrum for the liquid sample in the at least two wells based on a signal from the shared sensor of the spectrometer; The system is programmed to control the mechanical positioning mechanism to change the relative positions of the at least two probes and the well plate to simultaneously acquire Raman spectra of the liquid samples in at least two different wells of the well plate.

35. 35. The system of claim 34, wherein each of the at least two fibers that focuses laser light into an associated one of the at least two wells is oriented at an angle relative to an associated one of the at least two Raman probes.

36. 35. The system of claim 34, wherein the at least two fibers are positioned to focus the laser light into the sample from opposite directions relative to the at least two Raman probes.

37. 35. The system of claim 34, wherein the at least one laser comprises a separate laser associated with each of the at least two fibers.

38. the shared sensor includes a two-dimensional sensor; 35. The system of claim 34, wherein the outputs of the at least two Raman probes are connected to the input of the spectrometer by corresponding optical fibers arranged in a linear array with the optical fibers associated with each probe positioned adjacent to one another at the input of the spectrometer and spaced a distance corresponding to at least one fiber diameter from the fibers associated with the other probes.

39. Further comprising a number of fluorescent probes corresponding to the at least two Raman probes; 35. The system of claim 34, wherein each fluorescent probe is in a fixed position relative to an associated Raman probe, the fluorescent probe aligned with an associated well of the well plate separated by at least one well that is not aligned with any other fluorescent probe or with either the Raman probe.

40. 40. The system of claim 39, wherein the multiple fluorescent probes are optically coupled to an input of the spectrometer, and the at least one controller is further programmed to simultaneously acquire fluorescent data from the shared sensors.

41. the shared sensor includes a two-dimensional sensor; the outputs of the at least two Raman probes are connected by corresponding optical fibers to the inputs of the spectrometer; each of the plurality of fluorescent probes is coupled to an input of the spectrometer by at least one optical fiber; The optical fibers of each Raman probe are arranged in a linear array and grouped together. The optical fibers of each fluorescent probe are arranged in a linear array and grouped together. the grouped optical fibers of each Raman probe are spaced from the grouped optical fibers of any other Raman probe or any fluorescent probe by a distance corresponding to at least one fiber diameter; 41. The system of claim 40, wherein the grouped optical fibers of each fluorescent probe are spaced apart from the grouped optical fibers of any other fluorescent probe or any Raman probe by a distance corresponding to at least one fiber diameter.

42. the Raman probe, the fluorescent probe, the at least two fibers coupled to the at least one laser, and the mechanical positioning mechanism are located within a reaction chamber of an automated liquid handler system; 42. The system of claim 41, wherein the spectrometer and the at least one laser are located outside the reaction chamber.

43. 43. The system of claim 42, wherein the mechanical positioning mechanism comprises a well plate holder having an integrated Raman calibration reference sample positionable by the at least one controller into alignment with one or more of the Raman probes.

44. 42. The system of claim 41, wherein the grouped optical fibers of each Raman probe are located closer to the center of the linear arrangement than the grouped optical fibers of each fluorescent probe.

45. 40. The system of claim 39, further comprising a second spectrometer having an input optically coupled to the output of the plurality of fluorescent probes.

46. simultaneously focusing laser light 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 to which the laser light is not focused; simultaneously directing light emitted by the at least two samples to a spectrometer having a diffraction grating that redirects the light at a wavelength-dependent angle to a shared sensor; acquiring Raman spectroscopy data associated with the at least two samples from the shared sensor; controlling, with at least one controller, a position of the well plate relative to the focused laser light to simultaneously acquire Raman spectroscopy data from at least two different samples contained in untested wells of the well plate; The method includes:

47. 47. The method of claim 46, wherein simultaneously focusing laser light comprises simultaneously focusing light from different lasers associated with each one of the at least two samples.

48. simultaneously directing light from a light source to at least two samples contained in wells corresponding to said well plate, each sample being separated by at least one well that is not illuminated by said focused laser light or light from a light source; simultaneously directing light emitted by the sample illuminated by light from the light source to the shared sensor of the spectrometer; acquiring fluorescence data from the shared sensor of the spectrometer associated with the at least two samples illuminated by light from the light source; 47. The method of claim 46, further comprising:

49. Simultaneously directing the light emitted by the at least two samples includes: coupling the emitted light into at least one optical fiber in each of the at least two samples; arranging the optical fibers in a linear array at an input of the spectrometer; 47. The method of claim 46, wherein the optical fibers associated with each sample are grouped together and separated from optical fibers associated with other samples by a distance corresponding to at least one optical fiber diameter.

50. by the at least one controller Positioning a calibration standard integrated within the well plate holder to be illuminated by the focused laser light; obtaining Raman spectroscopic data from said calibration standards; 47. The method of claim 46, further comprising adjusting a wavelength of the focused laser light based on the Raman spectroscopic data of the calibration standard compared to a previously stored peak wavelength for the calibration standard.

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