Non-contact raman spectroscopy system and methods for using the same

Abstract

A system for non-contact Raman spectroscopy includes a substrate, such as a tray, and an objective. The substrate includes a base and a cover, such as a cover plate. The base includes a first and second well that define a first cavity and a second cavity, respectively. Each of the first cavity and the second cavity configured to receive a sample therein. The cover disposed over the first well and the second well such that the first cavity and the second cavity are each substantially sealed. The objective has an objective lens that is configured to separately analyze the samples in the first cavity and the second cavity through the cover with Raman spectroscopy.

Description

New PCT Application Sparta Biodiscovery Limited Vossius Ref.: AK4296 PCT S5NON-CONTACT RAMAN SPECTROSCOPY SYSTEM AND METHODS FOR USING THE SAMEBackground1. Technical Field

[0001] The present disclosure relates to spectroscopy and, more specifically, to systems and methods for performing non-contact Raman spectroscopy.2. Discussion of Related Art

[0002] Raman spectroscopy is a technique that can be used to identify molecules in a sample. Specifically, Raman spectroscopy uses vibrational modes of molecules to identify molecules within the sample and concentrations thereof. Raman spectroscopy provides averaged information across the sample, but lacks the sensitivity to resolve information at the single particle level.

[0003] In traditional or contact Raman spectroscopy or in Single Particle Automated Raman Trapping Analysis (SPARTA), a sample is trapped in a well and an objective lens of an objective is immersed into the sample to analyze the sample. After the sample is analyzed, the objective lens must be cleaned before the objective lens is used to analyze another sample. Cleaning the objective lens can prevent cross-contamination of the next sample. Once cleaned, the objective lens is immersed in the next sample to analyze that sample. Typically, the objective lens is manually cleaned by an operator. This manual cleaning may reduce the throughput of analyzing multiple samples.

[0004] The wells to receive or trap samples may be contained on a substrate having multiple wells. When a substrate includes multiple wells, the wells may need to be spaced apart to prevent contamination between analyzing of a first sample in a first well and analyzing of a second sample a second well. In some instances, the second sample is only placed into or trapped in the second well after the first sample is analyzed which can cause a further delay in analyzing multiple samples as a result of cleaning and trapping between each analysis.

[0005] Typically, the wells for samples are open wells. Such open wells may allow for evaporation of the sample such that a long analysis is not possible as a result of the evaporation. Further, the open wells may require large sample volumes to allow for immersion by the objective lens. In addition, an open sample well may not allow for analyzing of samples which require biosafety containment.

[0006] In view of the above, there is a continuing need for increasing the speed and efficiency of analyzing multiple samples with Raman spectroscopy. There may also be a need for reducing the sample volume required for an accurate analysis. There may also be a need to prevent or reduce evaporation from a sample during analysis. There may also be a need to allow for analysis of samples which require biosafety containment.Summary

[0007] In an aspect of the present disclosure, a substrate for Raman spectroscopy includes a base, a first well, and a cover. The first well defines a cavity that is configured to receive a sample therein. The cover is disposed over the first well such that the cavity of the first well is substantially sealed. The cover is configured to separate the sample in the cavity from an objective lens during analysis of the sample by Raman spectroscopy with the objective lens.

[0008] In aspects, the cavity of the first well may have a total volume in a range of 20 microliters to 150 microliters. The first well may be movably secured to the base. The cavity of the first well may be urged towards the cover. The first well may be pivotally secured to the base. The cover may be a cover plate that is secured to the base by walls that engaged the base. The wall may engage the first well to pivot the cavity upward into a bottom surface of the cover. The first well may be mounted on a biasing mechanism. The biasing mechanism may urge the first well towards the cover when the cover is engaged with the first well.

[0009] In some aspects, the first well includes walls that define a receiver, a channel, and the cavity. The receiver may be positioned outside of the cover such that the receiver is configured to receive a sample when the cover is secured to the base. The channel may be fluidly connecting the receiver to the cavity. The cover may be surrounded by walls. The wall may extend over a top surface of the cover to define a reservoir on the top surface of the cover. The reservoir may be configured to be flooded with an immersion medium.

[0010] In certain aspects, the substrate includes a ring that is disposed on a top surface of the cover. The ring may define a reservoir on the top surface of the cover. The reservoir may be configured to be flooded with an immersion medium.

[0011] In particular aspects, the substrate includes a plurality of wells that include the first well. Each of the plurality of wells may define a cavity that is configured to receive a sample. The cover that is disposed over each well of the plurality of wells. The first well may define a duct that is in fluid communication with the cavity. The duct may be configured to allow a sample to be added to the cavity when the cover is disposed over the first well. The base may be in the form of a tray that includes the first well.

[0012] In another aspect of the present disclosure, a system for Raman spectroscopy includes an objective that has an objective lens and a substrate as detailed herein. The objective lens is configured to analyze a sample disposed in a cavity of a first well of the substrate through a cover of the substrate. The objective lens may analyze the sample by Raman spectroscopy.

[0013] In aspects, the system includes an immersion medium that contacts a top surface of the cover and the objective lens. The immersion medium may be configured to eliminate an air gap between the objective lens and the cover.

[0014] In another aspect of the present disclosure, a system for Raman spectroscopy includes a substrate and an objective lens. The substrate includes a base and a cover. The base includes a first well and a second well. The first well defines a first cavity that is configured to receive a first sample therein. The second well defines a second cavity that is configured to receive a second sample therein. The cover is disposed over the first well and the second well such that the first cavity and the second cavity are each substantially sealed. The objective has an objective lens that is configured to separately analyze the samples in the first cavity and the second cavity through the cover with Raman spectroscopy.

[0015] In aspects, the first well and the second well are movably secured to the base. The cavities of the first well and the second well may be separately urged towards the cover. The first well and the second well may each be pivotally secured to the base. The cover may be a cover plate that is secured to the base by walls that engage the base. The walls may engage the first welland the second well to pivot the cavities upward into a bottom surface of the cover. The first well may be mounted on a first biasing mechanism and the second well may be mounted on a second biasing mechanism. The first biasing mechanism may urge the first well towards the cover when the cover is engaged with the first well and the second biasing mechanism may urge the second well towards the cover when the cover is engaged with the second well.

[0016] In some aspects, the first well may include walls that define a receiver, a channel, and the first cavity. The receiver may be positioned outside of the cover such that the receiver is configured to receive a sample when the cover is secured to the base. The channel may fluidly connect the receiver to the first cavity. The cover may be surrounded by walls. The walls may extend above a top surface of the cover to define a reservoir on the top surface of the cover that extends over the first well and the second well. The reservoir may be configured to be flooded with an immersion medium.

[0017] In certain aspects, the system includes a ring that is disposed on a top surface of the cover. The ring may define a reservoir on the top surface of the cover. The ring may be movable about the top surface of the cover such that the ring has a first position over the first well and a second position over the second well. The reservoir may be configured to be flooded with an immersion medium.

[0018] In another aspect of the present disclosure, a method of analyzing a sample with Raman spectroscopy includes positioning an objective lens over a first well and analyzing a sample in a cavity of the first well through a cover that is disposed between the objective lens and the cavity. The objective lens measures the sample with Raman spectroscopy without contacting or being immersed in the sample.

[0019] In aspects, the method includes adding the sample to the cavity of the first well with the cover positioned over the first well. The method may include immersing the objective lens in an immersion medium before analyzing the sample. Analyzing the sample in the cavity may include the immersion medium preventing air from being disposed between the objective lens and the cover.

[0020] In some aspects, the method may include flooding a reservoir defined on a top surface of the cover with an immersion medium before analyzing the sample in the cavity of the first well. The method may include immersing the objective lens in the immersion medium before analyzing the sample in the cavity of the first well.

[0021] In certain aspects, the method includes positioning the objective lens over a second well after analyzing the sample in the first well. The method may include analyzing a sample in a cavity of the second well through the cover is disposed between the objective lens and the cavity of the second well. The objective lens may measure the sample with Raman spectroscopy. The method may include placing a drop of an immersion medium on the objective lens of the cover between analyzing the sample in the cavity of the first well and analyzing the sample in the cavity of the second well. Positioning the objective lens over the second well may include dragging a droplet of an immersion medium from over the first well to over the second well with the objective lens. Analyzing the sample in the cavity of the second well may occur without wasting the objective lens after analyzing the sample in the cavity of the first well.

[0022] Further, to the extent consistent, any of the embodiments or aspects described herein may be used in conjunction with any or all of the other embodiments or aspects described herein.Brief Description of the Drawings

[0023] Various aspects of the present disclosure are described hereinbelow with reference to the drawings, which are not necessarily drawn to scale, which are incorporated in and constitute a part of this specification, wherein:

[0024] FIG. 1 is a schematic representation of a non-contact Raman spectroscopy system provided in accordance with the present disclosure including an objective having an objective lens, an immersion medium, and a well containing a sample to be analyzed;

[0025] FIG. 2 is a flow chart of a method of performing non-contact Raman spectroscopy provided in accordance with the present disclosure;

[0026] FIG. 3 is a schematic representation of a method of applying an immersion medium to the objective lens 22 of FIG. 1 provided in accordance with embodiments of the present disclosure;

[0027] FIG. 4 is a perspective view of a substrate provided in accordance with the present disclosure including three wells for holding samples to be analyzed;

[0028] FIG. 5 is a perspective view of the substrate of FIG. 4 including a cover disposed over the wells;

[0029] FIG. 6 is a perspective view of another substrate provided in accordance with the present disclosure including six wells for holding samples to be analyzed;

[0030] FIG. 7 is a perspective view of another substrate provided in accordance with the present disclosure including six wells for holding samples to be analyzed;

[0031] FIG. 8 is a top view of another substrate provided in accordance with the present disclosure including 5 wells for holding samples to be analyzed and a movable ring on a cover;

[0032] FIG. 9 is a perspective view of a base of the substrate of FIG. 8 with the cover removed;

[0033] FIG. 10 is a perspective view of a base of a substrate provided in accordance with the present disclosure;

[0034] FIG. 11 is a bottom, perspective view of a cover assembly provided in accordance with the present disclosure;

[0035] FIG. 12 is a perspective view of a substrate formed of the base of FIG. 10 and the cover assembly of FIG. 11 ;

[0036] FIG. 13 is a perspective view of a base provided in accordance with the present disclosure;

[0037] FIG. 14 is a bottom, perspective view of a substrate provided in accordance with the present disclosure including the base of FIG. 13;

[0038] FIG. 15 is a top, perspective cutaway view of the substrate of FIG. 14;

[0039] FIG. 16 is a perspective view of a substrate provided in accordance with the present disclosure;

[0040] FIG. 17 is a perspective view of another substrate provided in accordance with the present disclosure;

[0041] FIG. 18 is a graph of empirical data comparing contact and non-contact Raman spectroscopy with the non-contact Raman spectroscopy using the substrate of FIGS. 4 and 5;

[0042] FIG. 19 is a graph of empirical data comparing contact and non-contact Raman spectroscopy with the non-contact Raman spectroscopy using the substrates of FIGS. 6 and 7;

[0043] FIG. 20 is a perspective view of another substrate provided in accordance with the present disclosure; and

[0044] FIG. 21 is a perspective view of another substrate provided in accordance with the present disclosure.Detailed Description

[0045] The present disclosure will now be described more fully hereinafter with reference to example embodiments thereof with reference to the drawings in which like reference numerals designate identical or corresponding elements in each of the several views. These example embodiments are described so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. Features from one embodiment or aspect can be combined with features from any other embodiment or aspect in any appropriate combination. For example, any individual or collective features of method aspects or embodiments can be applied to apparatus, product, or component aspects or embodiments and vice versa. The disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. As used in the specification and the appended claims, the singular forms “a,” “an,” “the,” and the like include plural referents unless the context clearly dictates otherwise. In addition, while reference may be made herein to quantitative analyses, values, geometric relationships, or the like, unless otherwise stated, any one or more if not all of these may be absolute or approximate to account for acceptable variations that may occur, such as those due to manufacturing or engineering tolerances or the like.

[0046] Referring now to FIG. 1, a system for non-contact Raman spectroscopy is provided in accordance with embodiments of the present disclosure and is referred to generally as system 10. The system 10 includes an objective 20, a well 40, and a sample 90 disposed within the well 40. The objective 20 terminates in an objective lens 22 that is configured to analyze samples with Raman spectroscopy without contacting or being immersed in the sample, e.g., sample 90. As detailed below, the objective lens 22 may be immersed in an immersion medium 80 during analysis of a sample, e.g., sample 90.

[0047] The well 40 may be a single well or may be disposed on a substrate 30 having a plurality of wells 40. The term “substrate” may refer to a tray, a slide, or other structure that defines or supports a well. The well 40 includes one or more sidewalls 42 that form sides and possibly a bottom of a cavity 44 defined by the well 40. The well 40 may include a cover 50 that forms a top for the cavity 44. The cover 50 may be a cover plate, a cover slip, or other structure that forms a top or a cover for the cavity 44. The cover 50 may be a common cover for multiple wells 40 or may be a cover 50 for a single well 40 as described in greater detail below. The cover 50 may have a thickness that is configured to prevent distortion of an analysis of the sample 90. The thickness of the cover 50 may be in a range of 100 micrometers to 200 micrometers, e.g., 170 micrometers. The cover 50 may substantially seal the cavity 44 of the well 40. Substantially sealing the cavity 44 of the well 40 may allow for analyzing of samples which require biocontainment for safety purposes. Substantially sealing the cavity 44 of the well 40 may prevent or reduce evaporation of a sample within the well 40. Preventing or reducing evaporation of the sample may allow for longer analyzing times of the sample. The cover 50 may define a plurality of depressions 52 with each depression 52 configured to be positioned over the cavity 44 of a respective well 40. Each depression 52 may be configured to receive an immersion medium 80 as described below.

[0048] With reference to FIG. 2, a method of analyzing a sample with Raman spectroscopy 200 is disclosed in accordance with the present disclosure with reference to the objective 20 of FIG. 1. The method 200 includes adding a sample 90 to a cavity 44 of a well 40 (Step 210). Adding a sample 90 to the cavity 44 of a well 40 may include adding a plurality of samples, either the same or different to a plurality of wells 40 disposed on a substrate 30 having a plurality of wells 40. A single substrate 30 may have between 1 and 20 wells 40. In some embodiments, asingle substrate 30 may have 3, 6, or 9 wells 40. The method 200 may include placing a cover 50 over the well or wells 40 of the substrate 30 (Step 220). The cover 50 may substantially seal the cavity 44 of the wells 40. In some embodiments, the cover 50 is secured to the substrate 30 over the wells 40 such that the cavity 44 may be filled with the cover 50 secured to the substrate 30 over the wells 40. When each of the wells 40 are filled with a respective sample 90, the sample fills the cavity 44 of the respective well 40 such that the sample contacts an underside of the cover 50. The wells 40 may be filled with a range of between 10 microliters and 200 microliters of the sample, e.g., 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 150, 175 microliters of the sample. Contacting the underside of the cover 50 may prevent or remove air from within the well 40. Removing or reducing air within the well 40 may increase precision of the analysis of the system 10.

[0049] With the sample 90 in the well 40 and the cover 50, the objective lens 22 is positioned over a first well 40 of the substrate 30 to analyze the first sample disposed in the first well 40 (Step 240). Before analyzing the sample in the first well 40, an immersion medium 80 is positioned between the objective lens 22 and the cover 50 (Step 230). The immersion medium 80 may be water, oil, or other immersion medium 80 that interfaces with the objective lens 22 and the cover 50 to increase precision of the analysis of the sample in the well 40. The immersion medium 80 may be provided in a variety of means. For example, and with additional reference to FIG. 3, the objective lens 22 may be immersed into an immersion medium 80 that is in a reservoir 82 before being positioned over the first well in Step 240. In some embodiments, a droplet of the immersion medium 80 is manually or automatically applied to the cover 50 over each well 40. In certain embodiments, the entire top surface of the cover 50 is flooded with the immersion medium 80. In particular embodiments, one or more nozzles on the objective lens 22 may disperse an immersion medium on the objective lens 22 before each analysis such that a droplet of the immersion medium is disposed on the objective lens 22. The immersion medium may be replaced on the objective lens 22 before each analysis.

[0050] With the objective lens 22 positioned over the well 40 and the immersion medium 80 disposed between the objective lens 22 and the cover 50, the objective lens 22 is immersed into the immersion medium 80 (Step 250). The immersion medium 80 may fill the gap between the cover 50 and the objective lens 22. The immersion medium 80 may replace air in the gap to reducerefraction of light. Filling the gap between the objective lens 22 and the cover 50 may improve the precision of the analysis of the sample, e.g., improve the signal -to-noise ratio (SNR). The height of the objective lens 22 from the cover 50 may be determined by a signal received from the material of the cover 50, e.g., a glass signal when the cover 50 is formed of glass. The glass on the cover 50 may have a refractive index that essentially the same as the refractive index of the immersion medium 80.

[0051] The objective may be automatically moved to a sample well near the glass surface in order to measure a sample (Step 260). With the objective lens 22 immersed in the immersion medium 80, the objective lens 22 is used to analyze the sample in the first well 40. When the measurement is initiated, a physical shutter opens, which unblocks a high-power laser. In some embodiments, the laser may have a wavelength of 785 nm. The laser may be tightly focused into a sample through a high numerical aperture (NA) objective with a known working distance. In some embodiments, the NA may be 1.2. The objective lens 22 may use a flattop beam profile or may use a Gaussian beam profile to analyze the sample. As a result, a single particle in the sample is dragged into the confocal volume and trapped, and its Raman spectra is subsequently captured through the cover 50.

[0052] Once the Raman spectrum reaches a predetermined signal -to-noise ratio or meets a predetermined threshold, the physical shutter automatically closes, blocking the laser and releasing the trapped particle back into the sample (allowing particle diffusion) . After a brief delay (typically 1 second), the laser is unblocked again, and the next particle is trapped and analyzed. The predetermined threshold can either be automatically set or manually based on Raman spectral features. The automated process continues until a predetermined number of particles within the sample are analyzed depending on the sample and information to be extracted from the sample. For example, the particles are analyzed to quantify parameters such as sample heterogeneity, loading ratio, contamination, functionalization, etc. The high-throughput nature of capturing Raman spectra of several particles is a significant improvement over single-particle analysis.

[0053] In some embodiments, a beam expander may be used to match a beam size with the aperture of the objective lens 22. The beam expander may be a physical object inserted in the beam path before the objective that functions to expand the laser beam size to match the backaperture size of the objective lens. The beam expander may include two lenses but different configurations are possible. In some embodiments, the beam expander may use a Galilean Design having a biconcave lens and a plano-convex lens with specific focal distances that are placed a specific distance apart in a lens tube to achieve the correct beam expansion factor. Using a beam expander may allow for quick conversion between contact and non-contact Raman spectroscopy by swapping out objectives and / or beam expanders.

[0054] In certain embodiments, the objective is provided without a beam expander. In such a configuration, the objective may include collimators to control a beam diameter of the laser to match the back aperture of the objective. In particular embodiments, the objective of the laser may have a back aperture that is smaller than what is needed for a ‘non-contact’ objective. For this reason, there may be a need to expand the beam to achieve fully fill the back aperture. Fully filling the back aperture of the objective may achieve the right throughput and beam shape out of the objective to capture samples.

[0055] After analysis of the sample in the first well 40, the objective lens 22 is repositioned over a second well 40 on the substrate 30 (Step 270). Repositioning the objective lens 22 over the second well 40, may include immersing the objective lens 22 in another reservoir of immersion medium and is then positioned over the second well 40. Repositioning the objective lens 22 may include dispensing additional immersion medium onto the objective lens 22 before, during, or after the objective lens 22 is repositioned from over the first well 40 to over the second well 40. In some embodiments, the objective lens 22 drags the droplet of immersion medium 80 across the cover 50 from the first position to the second position without replacing or adding new immersion medium 80 between analysis. In embodiments where the cover 50 is flooded with immersion medium, the objective lens 22 may be repositioned between the first well 40 and the second well 40 without removing the objective lens 22 from within the immersion medium 80. With the objective lens 22 repositioned over the second well 40, the objective lens 22 is used to analyze the sample in the second well 40 (Step 260). The Steps 250-270 are repeated until samples in all the wells 40 are analyzed.

[0056] The method 200 may allow for automated analyzing multiple samples of individual particles with Raman spectroscopy. The method 200 provides detailed information at the single-particle level compared to traditional Raman measurements, in which the captured spectra are averaged over all contributing components or particles within the laser spot. The method 200 may be considered non-contact Raman spectroscopy in which the objective lens 22 is not immersed in the material being analyzed. Non-contact Raman spectroscopy as described in method 200 may improve the speed of analyzing multiple samples. Non-contact Raman spectroscopy may allow for analyzing of multiple samples (e.g., hundreds of individual particles) with less manual labor. Reducing the manual labor may reduce costs and / or time required for analyzing multiple samples. \

[0057] A capturing algorithm may be programmed to trap all particles within the sample and the laser's confocal volume, capturing their corresponding Raman spectra. Alternatively, the algorithm can be trained to trap only particles that meet specific criteria (e.g., a distinct Raman feature), which is particularly useful in cases where contaminants are present, and specific group / class of particles need to be analyzed.

[0058] According to another aspect, the present disclosure provides a computer program, distributable by electronic data transmission or by computer readable medium, comprising computer program code means adapted, when said program is loaded onto a particle trapping and data acquisition apparatus, to make the apparatus execute the procedure of any one of the methods defined above.

[0059] Although the method steps are described in a specific order, it should be understood that other steps may be performed in between described steps, described steps may be adjusted so that they occur at slightly different times, or the described steps may occur in any order unless otherwise specified.

[0060] Referring now to FIGS. 4 and 5, another substrate 330 is described in accordance with the present disclosure. The substrate 330 has a base 332 and three wells 340 that extend upward from the base 332. The substrate 330 is sized and dimensioned to be received in a traditional spectroscopy system or microscopy system. The wells 340 may be positioned within walls 334. Each of the wells 340 defines a cavity 344 that is configured to receive a volume of a sample. Each well 340 may define a duct 345 that is in fluid communication with the cavity 344. The duct 345 may allow for introduction of a sample into a well 340. In some embodiments, the duct 345 may be accessible when the cover 350 is positioned over the well 340. In certain embodiments,the duct 345 may allow an excess amount of a sample to flow out of the cavity 344 into the area defined between the wells 340 and the walls 334. The walls 334 define a slot 353 in one end and inner slits 354 that are configured to receive a cover 350. A bottom surface of the cover 350 may contact a top surface of the wells 340 to substantially seal the cavity 344 of the wells 340. The slot 353 and the inner slits 354 may be positioned such that the bottom surface of the cover 350 is in contact with or just above a top surface of the wells 340. When the cover 350 is positioned in the slot 353 and the inner slits 354, a sample within the cavity 344 of the well 340 may contact the bottom surface of the cover 350. In use, the cavity 344 of each well 340 may be filled with an automated or manual system. For example, a user or a robot may use a pipette to load a sample into each well 340. Once loaded, the cover 350 may be inserted into the slot 353 either by an automated system or manually. The substrate 330 is then positioned such that an objective lens 22 is positionable over each well 340 to analyze a sample disposed within the well 340. In use, a drop of an immersion medium 80 may be positioned on the cover 350 over one or each of the wells 340 or on the objective lens 22 to eliminate an air gap between the objective lens 22 and the cover 350. The objective lens 22 may drag the immersion medium 80 across the cover 350 from being positioned over one well 340 to be positioned over another well 340. In some embodiments, the immersion medium is positioned before each analysis.

[0061] With reference now to FIG. 6, another substrate 430 is described in accordance with the present disclosure. Elements of the substrate 430 may be similar to the elements of substrate 330 and are represented with similar labels with a leading “4” replacing the leading “3” of the substrate 330. Only the differences with the previous substrates will be detailed herein for brevity.

[0062] The substrate 430 includes a base 432 and six wells 440 that extend upward from the base 432. Each well 440 defines a cavity 444 that is configured to receive a sample. The substrate 430 includes walls 434 that extend upward from the base 432 to define a space above the substrate 430 and between the wells 440. The cover 450 is supported on the walls 434. The wells 440 may be ovular in shape with a portion extending from underneath the cover 450 such that a sample may be added to the cavity 444 when the cover 450 is secured to the substrate 430. The portion of the well 440 that extends beyond the cover 450 may be considered a receiver 448 of the well 440. The cover 450 may include walls 451 that extend above a top surface of the cover 450 to define a reservoir 452 on the top surface of the cover 450. The reservoir 452 may be configured to retainan immersion medium 80 on the top surface of the cover 450 such that the cover 450 is configured to be flooded with an immersion medium 80 to allow for an objective lens 22 to be immersed in the immersion medium 80 and to move between the wells 440 of the substrate 430 without needing to be reloaded with an immersion medium 80. In some embodiments, the objective lens 22 may be lifted or withdrawn from the immersion medium 80 before being repositioned over another well 440 and then be reimmersed or reinserted into the immersion medium 80 to analyze the sample in the other well 440. Withdrawing and reinserting the objective lens 22 from the immersion medium 80 may prevent the immersion medium 80 from flowing over the walls 451.

[0063] Referring to FIG. 7, another substrate 530 is described in accordance with the present disclosure. Elements of the substrate 530 may be similar to the elements of substrate 430 and are represented with similar labels with a leading “5” replacing the leading “4” of the substrate 430. Only the differences with the previous substrates will be detailed herein for brevity.

[0064] The substrate 530 includes a base 532 and six wells 540 that extend upward from the base 532. Each well 540 defines a cavity 544 that is configured to receive a sample. The substrate 530 includes supports 534 that extend from the base 532 and are configured to support the cover 550. The space between the supports 534 and the wells 540 may be substantially open. The cover550 is supported on the supports 534. The wells 540 may be ovular in shape with a portion extending from underneath the cover 550 such that a sample may be added to the cavity 544 when the cover 550 is secured to the substrate 530. The portion of the well 540 that extends beyond the cover 550 may be considered a receiver 548 of the well 540. The cover 550 may include walls551 that extend along the sides of the cover 550 that pass over the receivers 548 of the wells 540. The walls 551 may prevent immersion medium 80 from flowing into the cavity 544 of the wells 540 from on top of the cover 550. As shown, the walls 551 do not extend between sides of the cover 550 that do not extend over the wells 540; however, in some embodiments, the walls 551 may extend on all sides of the cover 550.

[0065] With reference to FIGS. 8 and 9, another substrate 630 is described in accordance with the present disclosure. Elements of the substrate 630 may be similar to the elements of substrate 530 and are represented with similar labels with a leading “6” replacing the leading “5” of the substrate 530. Only the differences with the previous substrates will be detailed herein for brevity.

[0066] The substrate 630 includes a base 632 and five wells 640 that extend upward from the base 632. Each well 640 includes a pair of walls 647 that extend upward from the base 630. The walls 647 define a receiver 648, a channel 649, and a cavity 644. The receiver 648 is positioned outside or beyond the cover 650 such that the receiver 648 is configured to receive a sample when the cover 650 is secured to the base 632. The channel 649 extends from the receiver 648 to the cavity 644. The channel 649 may extend from the bottom or lower portion of the receiver 648 and may be sloped downward to the cavity 644 such that a sample flows from the receiver 648 into the cavity 644 with a minimum of the sample remaining in the receiver 648 and the channel 649. The channel 649 may have a small width to minimize a volume of a sample within the channel 649. The substrate 630 includes supports 634 that extend from the base 632 and are configured to support the cover 650. The cover 650 is supported on the walls 647 to substantially seal the wells 640. For example, the cover 650 may be supported on the top surfaces of the walls 647. With the cover 650 in place, the receiver 648 of the wells 640 are positioned to receive a sample and flow the sample into the cavity 644 via the channel 649. The configuration of the wells 640 may allow for analysis of a small volume of a sample. The cover 650 may include walls 651 that extend along the sides of the cover 650 that pass over the walls 647 or along all sides of the cover 650. In certain embodiments, the cover 650 includes a ring 658 that defines a reservoir 652 on the top surface of the cover 650. The ring 658 may be sized and dimensioned to receive and space the objective 20 over the cover 650 such that the objective lens 22 is positioned above the cover 650. The ring 658 may be repositionable on the cover 650 such that the objective lens 22, and thus the ring 658, is movable along the cover 650 such that the objective lens 22 analyses a sample in each of the cavities 644 of the wells 640. The ring 658 may contain or hold an immersion medium 80 in the reservoir 652 as the ring 658 is moved about the top surface of the cover 650.

[0067] With reference to FIGS. 10-12, another substrate 730 is described in accordance with the present disclosure. The substrate 730 includes a frame or base 732 and a cover 750. Elements of the substrate 730 may be similar to the elements of substrate 630 and are represented with similar labels with a leading “7” replacing the leading “6” of the substrate 630. Only the differences with the previous substrates will be detailed herein for brevity.

[0068] With particular reference to FIG. 10, the base 732 includes four wells 740 that are received or secured to the base 732. The wells 740 and the base 732 may be formed as a singlemonolithic construction by additive manufacturing or by molding. In some embodiments, the wells 740 and the base 732 may be formed of separate elements that are joined together by adhesive, bonding, welding (ultrasonic welding), or other means. In certain embodiments, the base 732 includes pivots 736 that extend along the sidewalls of the base 732 and are configured to receive the wells 740 thereon. The wells 740 may be configured to pivotally mount to the pivots 736 such that the wells 740 may pivot or rotate about a central longitudinal axis defined by the respective pivot 736. For example, each well 740 may include a mount (not explicitly shown) that receive the pivot 736 to pivotally secure the well 740 to the pivot 736. In certain embodiments, the pivot 736 has a section of reduced or increased diameter that is configured to receive the respective well 740 such that when the well 740 is pivotally secured to the pivot 736, the well 740 is constrained to a single degree of freedom, e.g., rotation about the central longitudinal axis of the pivot 736. The substrate 730 includes supports 734 that extend from the base 732 and are configured to support the cover 750. The base 732 also includes a first half 738 of a retainer system that are configured to interface with the cover 750 to retain the cover 750 over the substrate 730 and the wells 740 as detailed below.

[0069] Each well 740 includes a pair of walls 747 that define a receiver 748, a channel 749, and a cavity 744. The receiver 748 is positioned outside or beyond the cover 750 such that the receiver 748 is configured to receive a sample when the cover 750 is secured to the base 732 as detailed below. The channel 749 extends from the receiver 748 to the cavity 744. The channel 749 may extend from the bottom or lower portion of the receiver 748 and may be sloped downward to the cavity 744 such that a sample flows from the receiver 748 into the cavity 744 with a minimum of the sample remaining in the receiver 748 and the channel 749. The channel 749 may have a small width to minimize a volume of a sample within the channel 749.

[0070] With particular reference to FIG. 11, the cover 750 is surrounded by walls 751 that receive the cover 750 therein. The walls 751 extend over the top surface of the cover 750 to draw the cover 750 towards the base 732. The walls 751 include the second half 757 of the retainer system that are configured to interface with the first halves 738 of the retainer system to secure the walls 751, and thus the cover 750, to the base 732. The cover 750 may define cutouts 758 that are configured to receive a portion of the wells 740 as detailed below. The walls 751 may extend above the cover 750 such that the walls 751 may form a reservoir 752 on the top surface of thecover 750. The reservoir 752 may prevent immersion medium 80 from flowing into the receivers 748 or allow the top surface of the cover 750 to be flooded with an immersion medium 80.

[0071] Referring now to FIG. 12, when the substrate 730 is assembled, the walls 751 engage the base 732 to secure the cover 750 to the base 732 such that the cavity 744 of the wells 740 are substantially sealed and positioned to be analyzed by an objective lens 22. The first halves 738 and the second halves 757 of the retainer system interact with one another to secure the cover 750 to the base 732. The first halves 738 and / or the second halves 757 may be clips or recesses that interact with an opposing clip or recess to secure the cover 750 to the base 732. The supports 734 may engage a bottom surface of the walls 751 and / or the bottom surface of the cover 750 to position the cover 750 relative to the base 732. The wells 740 are positioned such that the wells 740 are received in the cutouts 758 of the walls 751. The walls 751 extend beyond the pivots 736 such that when the walls 751 engage the top surface of the walls 747 of the wells 740, within the cutouts 758, the cavity 744 of the wells 740 is urged upward into the bottom surface of the cover 750. The urging of the wells 740 into the bottom surface of the cover 750 may form a seal between the walls 747 of the wells 740 and the bottom surface of the cover 750. As shown, the receivers 748 of the wells 740 are positioned outside of the walls 751 to allow the receivers 748 to receive a sample when the cover 750 is secured to the base 732. The substrate 730 may be provided as a single assembled unit which is assembled before use by a user or may be provided as a separate base 732 and a separate cover assembly including the cover 750 and the walls 751.

[0072] With reference to FIGS. 13-15, another substrate 830 is described in accordance with the present disclosure. The substrate 730 includes a frame or base 832 and a cover 850. Elements of the substrate 830 may be similar to the elements of substrate 730 and are represented with similar labels with a leading “8” replacing the leading “7” of the substrate 730. Only the differences with the previous substrates will be detailed herein for brevity.

[0073] With particular reference to FIG. 13, the base 832 includes four wells 840 that are received or secured to the base 832. The wells 840 and the base 832 may be formed as a single monolithic construction by additive manufacturing or by molding. In some embodiments, the wells 840 and the base 832 may be formed of separate elements that are joined together by adhesives, bonding, welding (ultrasonic welding), or other means. The base 832 may includebiasing mechanisms 860 with each biasing mechanism 860 positioned beneath a respective one of the wells 840. Each biasing mechanism 860 may be configured to urge the respective well 840 upward towards the cover 850 when the cover 850 is secured to the base 832. Each biasing mechanism 860 may include one or more biasing members 862 and a mount 868. The mount 868 may be supported by the biasing members 862 extending from the mount 868 to the base 832. The biasing members 862 may each have a first end 864, a second end 866, and a leg 865 that extends between the first and second ends 864, 866. The first end 864 is engaged with the top surface of the base 832 and the second end 866 is secured to a sidewall or bottom of the mount 868 to support the mount 868 over an opening 833 defined in the base 832. The opening 833 allows for downward movement of the mount 868 towards the bottom surface of the base 832. The leg 865 is formed of a material having a rigidity sufficient to resist movement of the mount 868 and an elasticity to urge the mount out of the opening 833 such that the well 840 mount to the base 868 is urged towards the cover 850. Each leg 865 may have an elongate construction that form a substantially S-shaped member to allow for elastic deformation as detailed above. The first ends 864 may rest on the top surface of the base 832 or may be secured to the top surface of the base 832 by adhesives, bonding, welding (ultrasonic welding), or other means. The second ends 866 may be secured to the mount 868 by adhesives, bonding, welding (ultrasonic welding), or other means. In certain embodiments, the entire biasing mechanism 860 may be monolithically formed with the base 832.

[0074] Continuing to refer to FIGS. 13-15, the cover assembly of the substrate 830 may be similar to the cover assembly of the substrate 730. When assembled, each well 840 includes walls 847 that define a receiver 848, a channel 849, and a cavity 844. The receiver 848 is positioned outside or beyond the cover 850 such that the receiver 848 is configured to receive a sample when the cover 850 is secured to the base 832. The walls 847 may extend more above the receiver 848 than other sections of the well 840 such that the walls 847 may be congruent with the top of the walls 851 when the cover 850 is secured to the base 832. When the substrate 830 is assembled, the walls 851 engage the base 832 to secure the cover 850 to the base 832 such that the cavity 844 of the wells 840 are substantially sealed and positioned to be analyzed by an objective lens 22. The supports 834 may engage a bottom surface of the walls 851 and / or the bottom surface of the cover 850 to position the cover 850 relative to the base 832. The walls 851 of the cover assembly engage the top surface of the walls 847 of the wells 840 to urge the wells 840 downward into the openings 833 of the base 832. The biasing mechanisms 860 may resist the movement of the wells840 downward such that a seal is maintained between the walls 847 and the bottom surface of the cover 850. The substrate 830 may be provided as a single assembled unit which is assembled before use by a user or may be provided as a separate base 832 and a separate cover assembly including the cover 850 and the walls 851.

[0075] With reference now to FIG. 16, another substrate 930 is described in accordance with the present disclosure. Elements of the substrate 930 may be similar to the elements of substrate 330 and are represented with similar labels with a leading “9” replacing the leading “3” of the substrate 330. Only the differences with the previous substrates will be detailed herein for brevity.

[0076] The substrate 930 includes a base 932 that defines six wells 940 that are concavities extending into the base 932 from a top surface thereof. Each well 940 includes a cavity 944 that is in the form of a conical or frustoconical depression that extends into the base 932. Each well 940 may include a first circular recess 941 defined in the base 932 that encircles the cavity 944 but is out of fluid communication with the cavity 944. Each well 940 may also include a second circular recess 943 defined in the base 932 that encircles the first circular recess 941. The second circular recess 943 is out of fluid communication with the cavity 944 and the first circular recess 941. The first and second circular recess 941, 943 may be configured to capture an overflow of a sample from the cavity 944. Capturing an overflow from the cavity 944 may prevent contamination of other wells 940 and / or an objective measuring the samples. The base 932 may include walls 934 that extend upward from the base 932 that are configured to support a cover (not explicitly shown) over the wells 940.

[0077] Referring now to FIG. 17, another substrate 1030 is described in accordance with the present disclosure. Elements of the substrate 1030 may be similar to the elements of substrate 930 and are represented with similar labels with a leading “10” replacing the leading “9” of the substrate 930. Only the differences with the previous substrates will be detailed herein for brevity.

[0078] The substrate 1030 includes a base 1032 that defines six wells 1040 that are concavities extending into the base 1032 from a top surface thereof. Each well 1040 includes a cavity 1044 that is in the form of a conical or frustoconical depression that extends into the base 1032. Each well 1040 may include a first circular recess 1041 defined in the base 1032 that encircles the cavity 1044 but is out of fluid communication with the cavity 1044. The base 1032 may define anoverflow recess 1043 that substantially surrounds the first circular recess 1041 of multiple wells 1040. The first circular recess 1041 and the overflow recess 1043 may be configured to capture an overflow of a sample from the cavities 1044. Capturing an overflow from the cavities 1044 may prevent contamination of other wells 1040 and / or an objective measuring the samples. The base 1032 may include walls 1034 that extend upward from the base 1032 that are configured to support a cover (not explicitly shown) over the wells 1040.

[0079] Referring now to FIGS. 18 and 19, empirical results of non-contact Raman spectroscopy are compared to that of traditional Raman spectroscopy. Starting with FIG. 18, the contact Raman spectroscopy is shown with lines 1 and 2 which provide major peaks around 1450 nanometers and 1300 nanometers and minor peaks at 700, 1050, and 1150 nanometers with a signal-to-noise ratio of 23.21 for line 1 and a signal -to-noise ratio of 28.17 for line 2. The noncontact Raman spectroscopy is represented with lines 3 and 4 and have similar peaks that are discernable at the same wavelengths with a signal-to-noise ratio of 25.49 for line 3 and a signal- to-noise ratio of 25.35 for line 4. The non-contact Raman spectroscopy was performed with the substrate 330. With reference to FIG. 19, the substrates 430 and 530 shown as Line B are compared to the contact Raman spectroscopy of line 2 of FIG. 18 shown as Line A. Substrates 430 and 530 provided similar peaks to that of contact Raman spectroscopy but had a drop in the signal-to-noise ratio of 25 percent to 30 percent with a signal-to-noise ratio of 17.87 compared to a signal-to-noise ratio of 25.35 for the contact Raman spectroscopy. It is noted that this drop of signal-to-noise ratio may be reduced or eliminated with additional revisions to the shape of the wells, a thickness of the cover, or different immersion mediums.

[0080] With reference now to FIG. 20, another substrate 1130 is described in accordance with the present disclosure. Elements of the substrate 1130 may be similar to the elements of substrate 930 and are represented with similar labels with a leading “ 11” replacing the leading “9” of the substrate 930. Only the differences with the previous substrates will be detailed herein for brevity.

[0081] The substrate 1130 includes a base 1132 that defines fifteen wells 1140 that are concavities extending into the base 1132 from a top surface thereof. Each well 1140 includes a cavity 1144 that is in the form of a conical or frustoconical depression that extends into the base 1132. Each well 1140 may include a first circular recess 1141 defined in the base 1132 thatencircles the cavity 1144 but is out of fluid communication with the cavity 1144. Each well 1140 may also include a second circular recess 1143 defined in the base 1132 that encircles the first circular recess 1141. The second circular recess 1143 is out of fluid communication with the cavity 1144 and the first circular recess 1141. The first and second circular recess 1141, 1143 may be configured to capture an overflow of a sample from the cavity 1144. Capturing an overflow from the cavity 1144 may prevent contamination of other wells 1140 and / or an objective measuring the samples. The base 1132 may include walls 1134 that extend upward from the base 1132 that are configured to support a cover (not explicitly shown) over the wells 1140.

[0082] With reference now to FIG. 21, another substrate 1230 is described in accordance with the present disclosure. Elements of the substrate 1230 may be similar to the elements of substrate 930 and are represented with similar labels with a leading “ 12” replacing the leading “9” of the substrate 930. Only the differences with the previous substrates will be detailed herein for brevity.

[0083] The substrate 1230 includes a base 1232 that defines fifteen wells 1240 that are concavities extending into the base 1232 from a top surface thereof. Each well 1240 includes a cavity 1244 that is in the form of a conical or frustoconical depression that extends into the base 1232. Each well 1240 may include a first circular recess 1241 defined in the base 1232 that encircles the cavity 1244 but is out of fluid communication with the cavity 1244. Each well 1240 may also include a second circular recess 1243 defined in the base 1232 that encircles the first circular recess 1241. The second circular recess 1243 is out of fluid communication with the cavity 1244 and the first circular recess 1241. The first and second circular recess 1241, 1243 may be configured to capture an overflow of a sample from the cavity 1244. Capturing an overflow from the cavity 1244 may prevent contamination of other wells 1240 and / or an objective measuring the samples. The base 1232 may include walls 1234 that extend upward from the base 1232 that are configured to support a cover (not explicitly shown) over the wells 1240.

[0084] The base 1232 further defines a receiver 1248 and a channel 1249 associated with each cavity 1244 that allow for filling of the associated cavity 1244 when a cover is supported over the base 1232. The receiver 1248 is positioned outside or beyond a portion of the base 1232 that is disposed under a cover such that the receiver 1248 is configured to receive a sample when a cover is secured to the base 1232. The channel 1249 extends from the receiver 1248 to the cavity 1244.The channel 1249 may extend from the bottom or lower portion of the receiver 1248 and may be sloped downward to the cavity 1244 such that a sample flows from the receiver 1248 into the cavity 1244 with a minimum of the sample remaining in the receiver 1248 and the channel 1249. The channel 1249 may have a small width to minimize a volume of a sample within the channel 1249. The receiver 1248 may be sized the same or larger than the channel 1249.

[0085] The substrates disclosed herein are shown with between three and twenty-four wells per substrate. It is contemplated that the substrates disclosed herein could be provided with less than three wells per substrate or more than twenty-four wells per substrate. For example, a substrate or plate could be provided with ninety-six wells to allow for automated analysis of ninety- six samples with one or more objective lenses. The increased automation may be a result of the non-contact Raman spectroscopy enabled by the well and cover design herein that allow for analysis without requiring cleaning of the objective lens between each analysis.

[0086] From the empirical data, the non-contact Raman spectroscopy, detailed herein, may be used as an alternative to contact Raman spectroscopy. The advantages of non-contact Raman spectroscopy may offset any possible decrease in the signal -to-noise ratio of non-contact versus contact Raman spectroscopy. For example, the non-contact Raman spectroscopy detailed herein may allow for increased throughput for analyzing samples. Increasing the throughput for analyzing samples may allow for more particles in each sample to be analyzed. The non-contact Raman spectroscopy may be automated without requiring cleaning of the objective lens between each analysis. Eliminating the need for cleaning between samples may increase throughput of analyzing samples. The non-contact Raman spectroscopy may be performed on samples in sealed wells such that samples that require safety procedures may be analyzed, e.g., biohazardous materials, biosafety level 2 (BSL-2), or containment level 2 (CL2). The non-contact Raman spectroscopy may also be used to analyze corrosive samples that could potentially damage the objective lens 22, e.g., samples having a high or low pH. For example, the non-contact Raman spectroscopy disclosed herein may be used to measure acidic samples without exposing the objective to the acidic conditions. The non-contact Raman spectroscopy detailed herein may be used to measure samples in solvent. The non-contact Raman spectroscopy detailed herein may be used to measure samples in a contained environment before transferring the sample to another environment. The sealed or substantially sealed configurations of the samples in the substratesdetailed herein may prevent or reduce evaporation of a sample within the wells. Reducing or preventing evaporation of a sample may allow for extended analyzing. As noted above, the workflow of trapping, capturing, and analyzing samples may be completely automated. Automating the process may improve throughput of analyzing samples. The wells of the substrates for non-contact Raman spectroscopy disclosed herein may allow for a smaller volume for each sample as compared to wells for substrates for contact Raman spectroscopy, e.g., 20 microliters to 150 microliters.

[0087] Identify some potential applications of the present disclosure in drug development, quality control, quality assurance, contamination detection, drug loading, surface localization, etc.

[0088] While several embodiments of the disclosure have been shown in the drawings, it is not intended that the disclosure be limited thereto, as it is intended that the disclosure be as broad in scope as the art will allow and that the specification be read likewise. Any combination of the above embodiments is also envisioned and is within the scope of the appended claims. Therefore, the above description should not be construed as limiting, but merely as exemplifications of particular embodiments. Those skilled in the art will envision other modifications within the scope of the claims appended hereto.

Claims

Claims1. A substrate for Raman spectroscopy, the substrate comprising: a base; a first well defining a cavity configured to receive a sample therein; and a cover disposed over the first well such that the cavity of the first well is substantially sealed, the cover configured to separate the sample in the cavity from an objective lens during analysis of the sample by Raman spectroscopy with the objective lens.

2. The substrate according to claim 1, wherein the cavity of the first well has a total volume in a range of 20 microliters to 150 microliters.

3. The substrate according to claim 1 or 2, wherein the first well is movably secured to the base, the cavity of the first well urged towards the cover.

4. The substrate according to claim 3, wherein the first well is pivotally secured to the base, the cover is a cover plate that is secured to the base by walls that engage the base, the walls engaging the first well to pivot the cavity upward into a bottom surface of the cover.

5. The substrate according to claim 3 or 4, wherein the first well is mounted on a biasing mechanism, the biasing mechanism urging the first well towards the cover when the cover is engaged with first well.

6. The substrate according to any one of claims 1 to 5, wherein the substrate defines a receiver and a channel that are in fluid communication with the first well, the receiver positioned outside of the cover such that the receiver is configured to receive a sample when the cover is secured to the base.

7. The substrate according to claim 6, wherein the first well includes walls that define the receiver, the channel, and the cavity.

8. The substrate according to any one of claims 1 to 7, wherein the cover is surrounded by walls, the walls extending above a top surface of the cover to define a reservoir on the top surface of the cover, the reservoir configured to be flooded with an immersion medium.

9. The substrate according to any one of claims 1 to 8, further comprising a ring disposed on a top surface of the cover, the ring defining a reservoir on the top surface of the cover, the reservoir configured to be flooded with an immersion medium.

10. The substrate according to any one of claims 1 to9, further comprising a plurality of wells including the first well, each of the plurality of wells defining a cavity configured to receive a sample, the cover disposed over each well of the plurality of wells.

11. The substrate according to any one of claims 1 to 10, wherein the first well defines a duct that is in fluid communication with the cavity, the duct configured to allow a sample to be added to the cavity when the cover is disposed over the first well.

12. The substrate according to any one of claims 1 to 11, wherein the base is in the form of a tray including the first well.

13. A system for Raman spectroscopy, the system comprising: an objective having an objective lens; and a substrate according to any one of claims 1 to 12, the objective lens configured to analyze a sample disposed in a cavity of a first well of the substrate through a cover of the substrate.

14. The system according to claim 13, further comprising an immersion medium contacting a top surface of the cover and the objective lens, the immersion medium configured to eliminate an air gap between the objective lens and the cover.

15. A system for Raman spectroscopy, the system comprising:a substrate including a base and a cover, the base including a first well and a second well, the first well defining a first cavity that is configured to receive a first sample therein, the second well defining a second cavity that is configured to receive a second sample therein, the cover disposed over the first well and the second well such that the first cavity and the second cavity are each substantially sealed; and an objective having an objective lens, the objective lens configured to separately analyze the samples in the first cavity and the second cavity through the cover with Raman spectroscopy.

16. The system according to claim 15, wherein the first well and the second well are movably secured to the base, the cavities of the first well and the second well separately urged towards the cover.

17. The system according to claim 15 or 16, wherein the first well and the second well are each pivotally secured to the base, the cover is a cover plate that is secured to the base by walls that engage the base, the walls engaging the first well and the second well to pivot the cavities upward into a bottom surface of the cover.

18. The system according to claim 15, 16 or 17, wherein the first well is mounted on a first biasing mechanism and the second well is mounted on a second biasing mechanism, the first biasing mechanism urging the first well towards the cover when the cover is engaged with first well and the second biasing mechanism urging the second well towards the cover when the cover is engaged with the second well.

19. The system according to any one of claims 15 to 18, wherein the first well includes walls that define a receiver, a channel, and the first cavity, the receiver positioned outside of the cover such that the receiver is configured to receive a sample when the cover is secured to the base, the channel fluidly connecting the receiver to the first cavity.

20. The system according to any one of claims 15 to 19, wherein the cover is surrounded by walls, the walls extending above a top surface of the cover to define a reservoir on the top surfaceof the cover that extends over the first well and the second well, the reservoir configured to be flooded with an immersion medium.

21. The system according to any one of claims 15 to 20, further comprising a ring disposed on a top surface of the cover, the ring defining a reservoir on the top surface of the cover, the ring being movable about the top surface of the cover such that the ring has a first position over the first well and a second position over the second well, the reservoir configured to be flooded with an immersion medium.

22. A method of analyzing a sample with Raman spectroscopy, the method comprising: positioning an objective lens over a first well; and analyzing a sample in a cavity of the first well through a cover disposed between the objective lens and the cavity, the objective lens measuring the sample with Raman spectroscopy without contacting or being immersed in the sample.

23. The method according to claim 22, further comprising adding the sample to the cavity of the first well with the cover positioned over the first well.

24. The method according to the claim 22 or 23, further comprising immersing the objective lens in an immersion medium before analyzing the sample, wherein analyzing the sample in the cavity includes the immersion medium preventing air from being disposed between the objective lens and the cover.

25. The method according to any one of claims 22 to 24, further comprising: flooding a reservoir defined on a top surface of the cover with an immersion medium before analyzing the sample in the cavity of the first well; and immersing the objective lens in the immersion medium before analyzing the sample in the cavity of the first well.

26. The method according to any one of claims 22 to 25, further comprising: positioning the objective lens over a second well after analyzing the sample in the first well; and analyzing a sample in a cavity of the second well through the cover disposed between the objective lens and the cavity of the second well, the objective lens measuring the sample with Raman spectroscopy.

27. The method according to claim 26, further comprising placing a drop of an immersion medium on the objective lens or the cover between analyzing the sample in the cavity of the first well and analyzing the sample in the cavity of the second well.

28. The method according to claim 26 or 27, wherein positioning the objective lens over the second well includes dragging a droplet of an immersion medium from over the first well to over the second well with the objective lens.

29. The method according to claim 26, 27, or 28, wherein analyzing the sample in the cavity of the second well occurs without washing the objective lens after analyzing the sample in the cavity of the first well.28

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