Patent Application
20250189375
This disclosure generally relates to a multi-track analyzer that provides Raman measurements alone or in combination with fluorescence, absorbance, and/or other spectroscopic-based measurements of liquid samples that may be contained in a well plate, in vials, in flow cell(s) or of a solid sample, such as a wafer or disk.
Raman measurements may be used alone or in combination with fluorescence, absorbance, and/or other spectroscopic-based measurements for quality control and analysis of various substances during manufacturing. Liquid samples may be contained in a well plate, in separate vials, in flow cell(s), or moving through tubing, for example. Analysis of solid samples on a disk or wafer may also be performed.
Well plate or microplate readers are commonly used in life sciences and pharmaceutical drug applications for the quantification of biological and chemical assays in a microplate which can have hundreds or thousands of wells (cavities, tubes, channels, etc.). Optical properties of the samples may change as biological, chemical, biochemical, or physical reactions take place (usually at controlled temperatures). Absorbance, fluorescence intensity, and luminescence are typical detection methods. Automated readers for a well plate (or similarly, an array of vials) may be used to provide high throughput (high speed) testing per minute for typical well plates containing 8, 48, 96, or 384 wells, for example. While fluorescence, luminescence, and absorbance methods are used by many commercially available plate readers with well plates of various formats, including transparent, opaque, semi-transparent, and black colors, Raman plate readers are rare and slow due to a single well readout mode using a low-power laser and low sensitivity CCDs. Well plates may exhibit various background contributions from the selected material based on the type of reading (fluorescence, Raman, absorbance, etc.). As such, well plate characteristics (material, color, etc.) should be evaluated and selected specifically for Raman applications at the selected laser wavelengths.
A Raman microscope has been used as a microprobe for well plate reading. Another strategy involves scanning 96 to 384 wells with one Raman probe moving in relation to the well plate (with an X-Y stage). This solution involves 96 translation movements for a 96 well plate which reduces the overall throughput, i.e. time for data acquisition for the entire well plate. This strategy often sacrifices sensor exposure time per well to increase throughput (in wells processed/minute) and is typically a low sensitivity solution using a CCD sensor and low-power laser. In such case, a linear (or two-dimensional (2D), vertically binned) CCD is sufficient for single spectrum acquisition in the case of a single probe being used to record the signals from 96 wells one-by-one.
Another strategy for high-speed Raman well plate reading proposes a lens array optical setup (8×24 matrices with a separation of 4.5 mm), combined with non-polarized beam splitter cubes that split the output of a single laser to each of the multiple wells resulting in a laser power of only about 7.5 mW per channel. This approach provides static simultaneous acquisition of 192 wells. However, it is subject to increased stray light/crosstalk between adjacent wells relative to other strategies using individual standard Raman probes, which are carefully designed to eliminate crosstalk between the laser path and Raman path. The splitting of the laser in free space may also have stability issues with reduced sensitivity per channel and low signal-to-noise ratio (SNR) associated with low laser power, particularly for low concentration sample solutions. Another disadvantage of this approach is that it does not allow the simultaneous acquisition of fluorescence or other spectroscopic methods on a well plate array. The laser power may also not be adjusted up or down for a single probe or sub-section of the array of 192 wells.
Various other approaches are described in published patent application US2017/0010153A1 and in U.S. Pat. Nos. 8,520,204 and 6,266,140, for example.
Various life science applications, such as pharmaceutical drug development or manufacturing, may include vials of product moving on conveyor belts or liquid flowing through tubing, flow cells, square or round or hexagonal, etc.
Semiconductor, from development to manufacturing, may include wafer process monitoring and control, materials analysis, which may use a three-axis positioning mechanism to fully map a wafer using Raman, photoluminescence, or ellipsometry, but requires significant processing time when using a single probe due to crosstalk and signal to noise limitations. Slurries and liquid, plasmas, here again may be analyzed through flow cells, tubing or through a window or pluralities of windows.
Another application for the energy sector, particularly Hydrogen monitoring, can make use of Raman signal monitoring. Raman spectra may be acquired through windows on a chamber, flow cell, tubing, or using an immersion probe, although contamination becomes an issue in many applications. The challenge in all these applications is to achieve the best signal to noise, while keeping the system components' cost reasonable.
As described in greater detail herein, a multi-track Raman analyzer and method include at least two Raman probes providing light from corresponding spots of a solid, liquid, or gas sample to the input of an imaging spectrometer having a shared two-dimensional sensor to simultaneously acquire Raman data for the corresponding spots and generate associated Raman spectra. Various types of sample holders and sample/probe positioning devices or mechanisms may be used to analyze several spots or areas of a single or multiple samples. The system and method may be used to analyze liquid samples ranging from clear solutions to slurries, solids/powders, gas or gas and liquid samples, and plasmas used for wafer manufacturing. Solid samples may include semiconductor products or wafers, food, packaging, recycled items, energy production (H2), etc. Applications may include pharmaceutical manufacturing having a conveyor belt with two or more rows of vials, semiconductor process liquid flow cells containing wafer cleaning liquid or slurry, semiconductor wafers, etc. with analysis from side, top, or bottom, for example. Liquids flowing in tubing or flow cells may be analyzed with different samples flowing in parallel or a single flow cell with two or more probes aligned for cross-collection/excitation. Sample/probe positioning may be provided for rotational and/or translational relative movement between the probes and sample(s) with optional probe autofocus based on signals from a distance sensor detecting distance to a desired spot or surface of the sample, or based on Raman signal optimization or maximization.
In various embodiments, a system for obtaining Raman spectra from a sample includes at least one laser, at least two Raman probes each positioned to illuminate and receive light from a corresponding spot of the sample to simultaneously acquire Raman spectra of the sample, 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, to focus the light from the input on the corresponding spot of the sample, to collect light from the corresponding spot of the sample, and to direct the collected light to an output, an imaging spectrometer optically coupled to the outputs of the at least two Raman probes, the imaging spectrometer including a diffraction grating (reflective or transmission) configured to direct light collected by the at least two Raman probes to a single two-dimensional scientific CMOS sensor, a sample positioning device configured to move the sample relative to the at least two Raman probes, a probe positioning mechanism configured to translate the at least two Raman probes along at least one axis, and at least one controller in communication with the at least one laser, the imaging spectrometer, the sample positioning device, and the probe positioning mechanism, the at least one controller programmed to simultaneously acquire Raman spectra for the corresponding spots of the sample based on signals from the two-dimensional scientific CMOS sensor of the spectrometer, and to control at least one of the sample positioning device and the probe positioning mechanism to change position of the at least two Raman probes relative to the sample. Each of the at least two Raman probes may include: an achromatic collimating lens positioned to receive light from the input; a mirror positioned to receive light from the achromatic collimating lens; a first dichroic longpass filter configured to reflect light from the mirror having wavelengths below a first cutoff wavelength to the sample lens, and to transmit light from the sample lens having wavelengths above the first cutoff wavelength; a second dichroic longpass filter configured to transmit light received from the first dichroic longpass filter having wavelengths above a second cutoff wavelength and to reflect light having wavelengths below the second cutoff wavelength; and an achromatic collection lens configured to focus light transmitted through the second dichroic longpass filter to the output. The Raman probes may be positionally fixed relative to one another and move together relative to the sample in response to a signal from the at least one controller. The sample positioning device may include a rotational stage configured to rotate the sample in response to a corresponding signal from the at least one controller. The probe positioning mechanism may be configured to translate the Raman probes along orthogonal X, Y, and Z axes relative to the sample positioning device in response to corresponding signals from the at least one controller. In various embodiments, the probe positioning mechanism is configured to translate the at least two Raman probes to control distance between the probes and the sample in response to corresponding signals from the at least one controller.
In one or more embodiments, the output of each of the Raman probes is optically coupled to an input of the spectrometer by at least one optical fiber. The optical fibers from the Raman probes may be linearly arranged at the input of the spectrometer with optical fibers from each Raman probe spaced from optical fibers of another of the Raman probes by at least one optical fiber diameter. In one embodiment, the output of each of the Raman probes is optically coupled by a plurality of optical fibers to the input of the spectrometer with the optical fibers from each Raman probe spaced from optical fibers of another of the Raman probes by a plurality of inactive optical fibers. In one embodiment, seven (7) active optical fibers for a first Raman probe are arranged linearly adjacent one another with five (5) inactive fibers between the active fibers of the first Raman probe and active optical fibers of a second Raman probe.
In one or more embodiments, the system includes a plurality of lasers with each of the plurality of lasers optically coupled to the input of a single different one of the Raman probes. In another embodiment, the system includes a plurality of lasers with each laser integrated with a corresponding Raman probe. The integrated laser may be contained within a housing of a corresponding one of the Raman probes. In another embodiment, the system includes a plurality of lasers with at least two of the plurality of lasers coupled to the input of a single one of the Raman probes. In yet another embodiment, a single laser is coupled to the inputs of at least two of the Raman probes.
Embodiments may also include a system having at least two Raman probes positioned with corresponding probe outputs aligned along a common axis wherein the system is configured to facilitate movement of the sample between the corresponding probe outputs. The sample positioning device may be implemented by a tube, cuvette, or flow cell positioned between the corresponding probe outputs and configured to flow the sample through the tube, cuvette, or flow cell. The controller may be programmed to sum or otherwise combine the scientific CMOS signals from the Raman probes to generate combined Raman spectra with laser light output from the at least two probes focused on the same spot of the sample. The sample positioning device may comprise a conveyor or other material handling system configured to move vials containing samples between the opposing probe outputs. The scientific CMOS sensor's integrated electronics may also be designed and programmed to combine Raman spectra as needed, in each channel, and to ignore the inter-channel readout areas.
Various embodiments may include a system for obtaining Raman spectra of moving liquid samples that includes: at least one laser; at least two Raman probes each positioned along a common axis on opposite sides of the moving liquid samples to illuminate and receive light from a corresponding spot of the moving liquid samples to simultaneously acquire Raman spectra of the samples, 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, to focus the light from the input on the corresponding spot of the sample, to collect light from the corresponding spot of the sample, and to direct the collected light to an output; an imaging spectrometer optically coupled to the outputs of the at least two Raman probes by a plurality of optical fibers, the optical fibers of the at least two Raman probes linearly arranged at an input of the imaging spectrometer, the imaging spectrometer including a diffraction grating configured to direct light collected by the at least two Raman probes to a single two-dimensional sensor; and at least one controller in communication with the at least one laser and the imaging spectrometer, the at least one controller programmed to simultaneously acquire Raman spectra for the corresponding spots of the sample based on signals from the two-dimensional sensor of the spectrometer.
In various embodiments, the moving liquid samples comprise liquid moving through a tube (round, square, or other shape) positioned between the outputs of the Raman probes with the Raman probes each having corresponding output laser light focused on the same spot of the moving liquid samples moving through the tube. The controller may be programmed to sum signals from the two-dimensional sensor corresponding to outputs of the at least two Raman probes to generate combined Raman spectra. In one embodiment, the two-dimensional sensor is a shutterless CCD sensor. In another embodiment, the two-dimensional sensor is a scientific CMOS sensor.
Various embodiments of the system include a plurality of lasers with each of the plurality of lasers optically coupled to the input of a single one of the Raman probes. In other embodiments, each Raman probe includes an integrated laser that may be contained within a housing of the probe. In one embodiment, the system includes a plurality of lasers with at least two of the plurality of lasers coupled to the input of a single one of the Raman probes. In another embodiment, the system includes a laser coupled to the inputs of at least two of the Raman probes. In other embodiments, a laser is integrated within the Raman probe In various embodiments, the moving liquid samples comprise vials containing the liquid samples with the vials moving between the outputs of opposing Raman probes via a material handling system with the opposing Raman probes having corresponding output laser light focused at a spot within different vials. In these embodiments, the two-dimensional sensor comprises a scientific CMOS sensor, the optical fibers associated with each Raman probe are positioned adjacent to one another and spaced by a distance corresponding to at least one fiber diameter from fibers associated with other Raman probes, and the controller is programmed to generate separate Raman spectra using signals from the scientific CMOS sensor for each Raman probe read simultaneously and independently. The system may include a separate laser coupled to the input of each Raman probe. In various embodiments, each Raman probe includes an integrated laser, an achromatic collimating lens positioned to receive light from the laser, a mirror positioned to receive light from the achromatic collimating lens, a first dichroic longpass filter configured to reflect light from the mirror having wavelengths below a first cutoff wavelength to the sample lens, and to transmit light from the sample lens having wavelengths above the first cutoff wavelength, a second dichroic longpass filter configured to transmit light received from the first dichroic longpass filter having wavelengths above a second cutoff wavelength and to reflect light having wavelengths below the second cutoff wavelength, and an achromatic collection lens configured to focus light transmitted through the second dichroic longpass filter to the output.
Embodiments may also include a system for obtaining Raman spectra of flowing liquid samples including at least two lasers, at least two Raman probes each positioned along a path of the flowing liquid samples to illuminate and receive light from a corresponding spot of the flowing liquid samples to simultaneously acquire Raman spectra of the samples, each Raman probe including an input optically coupled to a respective one of the at least two lasers, and a sample lens configured to receive light from the input, to focus the light from the input on the corresponding spot of the sample, to collect light from the corresponding spot of the sample, and to direct the collected light to an output, an imaging spectrometer optically coupled to the outputs of the at least two Raman probes by a plurality of optical fibers, the optical fibers of the at least two Raman probes linearly arranged at an input of the imaging spectrometer with optical fibers associated with each probe positioned adjacent to one another, the imaging spectrometer including a diffraction grating configured to direct light collected by the at least two Raman probes to a single two-dimensional scientific CMOS sensor, and at least one controller in communication with the at least one laser and the imaging spectrometer, the at least one controller programmed to simultaneously acquire Raman spectra for the corresponding spots of the sample based on signals from the two-dimensional CMOS sensor of the spectrometer. In one embodiment, the Raman probes have outputs aligned along a common axis in opposite directions with the flowing liquid passing between the outputs and the controller is programmed to sum the signals from the two-dimensional cMOS sensor to generate combined Raman spectra corresponding to the at least two Raman probes. In various embodiments, the optical fibers associated with each probe are spaced by a distance corresponding to at least two fiber diameters from fibers associated with other Raman probes coupled to the imaging spectrometer and the controller is programmed to generate separate Raman spectra associated with each Raman probe.
Embodiments may also include a method including simultaneously focusing laser light on at least two spots of a solid sample, simultaneously directing light from the at least two spots to an imaging spectrometer having a diffraction grating (reflective or transmission) that redirects light at a wavelength-dependent angle to a shared two-dimensional scientific CMOS sensor, acquiring Raman spectroscopy data associated with the at least two spots from the shared two-dimensional scientific CMOS sensor, and controlling, by at least one controller, rotation of the solid sample and translation of the focused laser light to simultaneously acquire Raman spectroscopy data from at least two different spots of the solid sample. Simultaneously focusing laser light may include simultaneously focusing light from a different laser associated with each one of the at least two spots. Simultaneously directing light from the at least two spots may include coupling the light from each of the spots to at least one corresponding optical fiber and arranging the optical fibers in a linear array at an input of the spectrometer, the at least one optical fiber associated with each spot separated from optical fibers associated with another spot by a distance corresponding to at least one optical fiber diameter. In various embodiments, rotation of the solid sample is controlled independently from translation of the focused laser light. Translation of the focused laser light may include translation in orthogonal X, Y, and/or Z directions relative to the solid sample.
A system and method for simultaneously obtaining 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 with a two-dimensional sensor receiving light emitted by the at least two samples during illuminate by focused laser light that is directed to an input of the spectrometer and diffracted by a diffraction grating of the spectrometer. The spectroscopic measurements may include Raman and fluorescence measurements, in addition to various other measurements obtained with the same spectrometer or a different spectrometer. To reduce sample-to-sample crosstalk and improve signal-to-noise ratio (SNR), the at least two samples may be separated or spaced by at least one sample holder or well of the array that is not illuminated by focused laser light during the measurements. To reduce channel-to-channel crosstalk and further improve SNR, light imaged on the spectrometer sensor from each of the samples is spatially separated from light of any other samples corresponding to Raman and/or fluorescence measurements. Spectroscopic probes may be fiber coupled to the spectrometer and illuminating laser(s) or other light source(s) to facilitate positioning of the probes within the filling and reaction chamber of an automated liquid handling system, with the laser(s), other light source(s), one or more controllers, and spectrometer(s) positioned outside the reaction chamber. A calibration standard may be integrated within a well plate holder for automated periodic calibration and adjustment of the laser wavelength, intensity, and/or focus.
In one embodiment, a system for obtaining Raman spectra from liquid samples within an array of sample holders includes 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, to focus the light from the input within an associated liquid sample, to collect light from the associated liquid sample, and to direct the collected light from the associated liquid sample to an output, 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 Raman probes to a sensor, a mechanical positioning mechanism configured to adjust a relative position between 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 programmed to generate Raman spectra based on signals from the sensor of the spectrometer for the liquid samples of the at least two non-adjacent sample holders, and control the positioning mechanism to change 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.
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 wavelengths below a first cutoff wavelength to the sample lens, and to transmit light from the sample lens having wavelengths above the first cutoff wavelength, a second dichroic longpass filter configured to transmit light received from the first dichroic longpass filter having wavelengths above a second cutoff wavelength and to reflect light having wavelengths below the second cutoff wavelength, and an achromatic collection lens configured to focus light transmitted through the second dichroic longpass filter to the output. The Raman probes may also include at least one bandpass filter positioned in an optical path between the achromatic collimating lens and the mirror. In various embodiments, the output of each Raman probe is optically coupled to an input of the spectrometer by at least one optical fiber, with the optical fibers from the Raman probes linearly arranged at the input of the spectrometer, with optical fibers from each Raman probe spaced from 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 placed in the linear array of fibers between fibers associated with a Raman or fluorescence probe. The optical fibers from the Raman probes may be arranged nearer to a center of the linear array than the output optical fibers from the fluorescence probes so that light from the Raman probes is directed nearer to an optical axis of the sensor of the spectrometer than light from the fluorescence probes.
In one or more embodiments, the spectrometer(s) include a diffraction grating comprising a reflective aberration corrected holographic concave grating and the sensor comprises a two-dimensional imaging sensor, such as a scientific CMOS sensor, or a CCD sensor with a shutter. In one embodiment all of the Raman probes and fluorescence probes are optically coupled to a single spectrometer having a single two-dimensional sensor. Other embodiments include a spectrometer having a separate diffraction grating for each connected probe with a linear CCD sensor shared by two or more probes. Embodiments may also include a separate spectrometers optimized for either fluorescence/photoluminescence data or Raman data and optically coupled to associated probes. The spectrometer may include a slit positioned in an optical path between the optical fibers from each Raman probe and the diffraction grating of the spectrometer. The slit width may be selectable from a plurality of fixed-width slits by at least one controller, or may have a variable width controlled by at least one controller.
The mechanical positioning mechanism may be configured to move the array of sample holders relative to the at least two Raman probes (and fluorescence probes when present) to orthogonal X and Y positions corresponding to the sample holders in the array. A Z-axis position may also be controlled to vary position of focused laser light within the samples. The positioning mechanism may include a well plate holder having one or more integrated calibration standard samples, such as a diamond sample. In addition, the well plate holder may be configured to hold a calibration plate having one or more calibration standard samples, such as diamond, silicon, and/or wells containing a customized liquid calibration sample for a particular application. The at least one controller may be programmed to change relative position between the Raman probes and the array of sample holders to vary position of focused laser light within the liquid samples while acquiring data from the spectrometer sensor to generate the Raman spectra to provide reduce or eliminate localized heating and/or photo bleaching of the sample and to improve homogeneity of the sample and/or averaging of measurements from different positions within the samples.
Various embodiments may include at least one laser associated with each Raman probe, or with a small number of Raman probes, such as two to four probes, for example. The laser may be a single mode or multi-mode laser. A separate light source may be provided for the fluorescence/photoluminescence probes with multiple fluorescence/photoluminescence probes connected to a single light source. Raman and fluorescence/photoluminescence probes may be implemented as non-contact, or as immersion probes. The Raman probes and fluorescence/photoluminescence probes may be positioned either above or below the samples, and may be positioned perpendicular to the array of samples. In one embodiment, the laser source light focused on or within the samples is spatially offset from the Raman probe, either at an angle or perpendicular to the array of samples. Illuminating light sources may be positioned opposite to collecting light sources of the probes to provide transmission Raman or fluorescence measurements.
In one embodiment, an additional laser is optically coupled to an input of an additional Raman probe, the additional Raman probe having an output coupled to the spectrometer, and positioned to acquire Raman spectra of a reference sample not contained in the array of sample holders.
In one or more embodiments, a system for obtaining Raman spectra from liquid samples contained within a well plate includes at least one laser optically coupled to at least two fibers to selectively focus laser light within samples contained within at least two wells of the well plate, the at least two wells separated by at least one well that is not illuminated by focused laser light, at least two Raman probes, each Raman probe 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 on 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, and at least one controller in communication with the spectrometer and the mechanical positioning mechanism, the at least one controller programmed to generate Raman spectra based on signals from the shared sensor of the spectrometer for the liquid samples within the at least two wells, and control the positioning mechanism to change relative position between the at least two probes and the well plate to simultaneously acquire Raman spectra of the liquid samples within at least two different wells of the well plate. The relative position may include three-dimensional positioning of the well plate relative to the probes. Each of the at least two fibers may focus laser light within an associated one of the at least two wells at an angle relative to an associated one of the at least two Raman probes collecting light emitted from associated samples. The fibers may be positioned to focus the laser light within the samples from an opposite direction relative to the Raman probes. The system may include a separate laser associated with each of the fibers. The spectrometer may include a two-dimensional sensor wherein the outputs of the Raman probes are connected to an input of the spectrometer by corresponding optical fibers arranged in a linear array at the input of the spectrometer with optical fibers associated with each probe positioned adjacent to one another and spaced by a distance corresponding to at least one fiber diameter from fibers associated with other probes.
In various embodiments, the system includes a plurality of fluorescence probes corresponding in number to the plurality of Raman probes, each fluorescence probe being fixed for movement with an associated Raman probe, the fluorescence probes aligned with associated wells of the well plate that are spaced by at least one well not aligned with any other fluorescence probe or any of the Raman probes. The fluorescence probes may be optically coupled to the input of the spectrometer and the at least one controller may be further programmed to simultaneously obtain fluorescence data from the shared sensor. In some embodiments, the fluorescence probes may be aligned with the same wells as the Raman probes with simultaneous Raman exposure sequentially followed by simultaneous fluorescence exposure for each subset or group of wells associated with the probes. The shared sensor may include a two-dimensional scientific CMOS sensor or CCD sensor with a shutter.
In one or more embodiments, the outputs of the at least two Raman probes are connected to an input of the spectrometer by corresponding optical fibers, each of the plurality of fluorescence probes is coupled by at least one optical fiber to the input of the spectrometer, optical fibers of each Raman probe are linearly arranged and grouped together, optical fibers of each fluorescence probe are linearly arranged and grouped together, grouped optical fibers of each Raman probe are spaced by a distance corresponding to at least one fiber diameter from grouped optical fibers of any other Raman probe or any fluorescence probe, and grouped optical fibers of each fluorescence probe are spaced by a distance corresponding to at least one fiber diameter from grouped optical fibers of any other fluorescence probe or any Raman probe. The grouped optical fibers of each Raman probe may be positioned closer to a center of the linear arrangement than the grouped optical fibers of each fluorescence probe. The Raman probes, the fluorescence probes, the at least two fibers coupled to the at least one laser, and the mechanical positioning mechanism may be positioned inside a reaction chamber of an automated liquid handing system, with the spectrometer and the at least one laser positioned outside of the reaction chamber. The mechanical positioning mechanism may include a well plate holder having an integrated calibration standard sample positionable by the at least one controller to align with one or more of the probes. Wavelength and/or intensity of a laser associated with the corresponding probe(s) may be adjusted in response to measurements obtained from the calibration standard sample. In some embodiments, the system may include a second spectrometer optimized for fluorescence measurements having an input optically coupled to outputs of the plurality of fluorescence probes.
Embodiments may also include a method comprising simultaneously focusing laser light within at least two samples contained in corresponding wells of a well plate, the at least two samples contained in wells spaced by at least one well without focused laser light, simultaneously directing light emitted by the at least two samples to a spectrometer having a diffraction grating that redirects 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, positioning of the well plate relative to the focused laser light to simultaneously acquire Raman spectroscopy data from at least two different samples contained in previously unexamined wells of the well plate. Simultaneously focusing laser light may include simultaneously focusing light from a different laser associated with each one of the at least two samples. The method may also include simultaneously directing light from a source to at least two samples contained in corresponding wells of the well plate, each well spaced by at least one well not illuminated by the focused laser light or light from the source, simultaneously directing light emitted by the samples illuminated by the light from the source to the shared sensor of the 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 source.
In at least one embodiment, simultaneously directing light emitted by the at least two samples comprises coupling the emitted light to at least one optical fiber for each of the at least two samples, and arranging the optical fibers in a linear array at an input of the spectrometer, the optical fibers associated with each sample grouped together and separated from optical fibers associated with another sample by a distance corresponding to at least one optical fiber diameter.
Various embodiments may also include, by the at least one controller, positioning a calibration standard integrated within a well plate holder to be illuminated by the focused laser light, acquiring Raman spectroscopy data from the calibration standard, and adjusting wavelength of the focused laser light based on the Raman spectroscopy data of the calibration standard compared to a previously stored peak wavelength for the calibration standard.
One or more embodiments according to the disclosure may provide associated advantages. For example, various embodiments provide for high throughput Raman measurements obtained using Raman spectroscopy and spatially offset Raman spectroscopy (SORS) as well as various combinations of Raman spectroscopy with absorbance measurements, fluorescence intensity, luminescence, time-resolved fluorescence, alpha screen, and many other types of spectroscopic and non-spectroscopic measurements of samples contained in a well plate. Embodiments may provide whole well imaging using various types of two-dimensional (2D) sensors, such as one or more CCD, CMOS or scientific CMOS (scientific CMOS) sensors. Using a laser at its full power/well (any typical UV, VIS and NIR laser, commonly 532 nm and 785 m), or with laser output split between a few wells maintains the laser output/channel reasonably high and facilitates adjustments to laser wavelength and/or intensity for associated probes. Allocating a Raman probe for each channel ensures best sensitivity, low stray light, and maximized SNR. The system may provide auto-calibration using one or more calibration standards embedded in the plate holder, or a calibration well plate containing one or more calibration standards and/or wells configured to contain liquid calibration samples. Fiber-coupled Raman probes may be incorporated with an automated plate handler system positioned inside the filling and reaction chamber and coupled to remotely positioned lasers and spectrometer to facilitate real-time or near real-time spectra acquisition for live monitoring of reactions within the well plate without quenching or dilution prior to analysis. Various embodiments may include integration with an automated plate handler system having automated transfer of a well-plate from a filling and reaction chamber to an analysis chamber for Raman data acquisition by the multi-probe system.
Embodiments of the present disclosure are described herein. It is to be understood, however, that the disclosed embodiments are merely examples and other embodiments can take various and alternative forms. The figures are not necessarily to scale; some features could be exaggerated or minimized to show details of particular components. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the claimed subject matter. As those of ordinary skill in the art will understand, various features illustrated and described with reference to any one of the figures can be combined with features illustrated in one or more other figures to produce embodiments that are not explicitly illustrated or described. The combinations of features illustrated provide representative embodiments for typical applications. Various combinations and modifications of the features consistent with the teachings of this disclosure, however, could be desired for particular applications or implementations.
As described in this disclosure, a well plate refers to a flat plate or tray having multiple wells spaced from one another and configured to contain a liquid sample that typically ranges in volume of nanoliters to milliliters of liquid. Well plates often contain 96, 384, or 1,536 wells, although may be customized for particular applications. A well plate may also be referred to as a multi-well plate, a microplate, a microtiter plate, a microwell plate, or similar variations. Well plates may be made of molded plastic, such as polystyrene, polypropylene, or polycarbonate, or may include a more complex construction to provide desired properties for particular types of measurements. Well plates may be transparent or pigmented with white or black pigments, for example, to provide desired optical characteristics suitable for particular desired measurements. Similarly, well plate measurements may be obtained with open cells or may have a lid or cover placed over the cells after depositing the samples.
One or more configurations of a well plate reader according to the disclosure provide rapid readout of a well plate using multiple Raman probes arranged in an array with a laser associated with each well being read to facilitate full-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 individual laser power adjustment for each of the respective mapped/related portion of wells scanned by that laser. For example, a well plate having 96 wells including 4 probes each having an associated laser would allow laser power to be individually adjusted for each of the 4 areas including 24 wells. This facilitates processing of wells containing different sample concentrations while reducing or eliminating the risk of localized heating and/or photo bleaching of the sample materials during reading. In some configurations, one or more lasers may include an output split among a small number of wells (using associated fibers, for example) to maintain a desirable laser output/channel with a full light-tight Raman probe associated with each channel to ensure best sensitivity, low stray light, and desired SNR. The probe array may consist of Raman probes or a combination of Raman and fluorescence probes, or various other types of probes.
A two-dimensional (2D) CCD sensor may be used in a known configuration using a commercially available spectrograph and CCD camera arrangement to acquire spectra from a large number of wells simultaneously. For example, an Isoplane™ (Roper Princeton Instruments) spectrometer may be configured with a Pixis Isoplane™ (Roper Princeton Instruments) CCD camera, both available from Teledyne Princeton Instruments, Inc. Conventional CMOS sensors have exhibited too much dark current and insufficient thermoelectric (TE) cooling, to be used in high-speed Raman set-ups. One or more embodiments according to this disclosure may use a compact scientific CMOS (scientific CMOS) sensor/camera, such as the Sylent-Blue from HORIBA Instruments Incorporated that achieves low read noise in the range of sub 1-e to 2e- and low dark current (sub 1e-/pixelsec) integrated in cameras reaching −25 C to −40 C with air or water TE vacuum cooling. This allows almost noise-free Raman spectra to be acquired in the described embodiments simultaneously. Such scientific CMOS sensors would typically be backside-illuminated (BSI) (also known as back-illuminated (BI) or back-thinned (BT)) sensors to achieve high quantum efficiency (QE), near 95% in the visible range.
Scientific CMOS sensors eliminate the need for a shutter when using a 2D CCD (a subject carefully avoided in the Isoplane/Pixis configuration) since such problem is present with full frame CCDs, and is only eliminated with an Interline CCD (shutterless), which unfortunately features very low quantum efficiency (QE). Frame transfer CCDs are typically not used due to their unaffordable costs and size, but may be used for specialized applications. An imaging spectrometer, such as many of the compact imaging spectrometers available from HORIBA Instruments Incorporated, combines high spectral resolution (required for Raman) with multiple fiber input (arranged along the slit) capability. A CiCi-Raman-532 HORIBA spectrometer, in which the CCD is replaced with the Sylent TE cooled CMOS camera features 4 to 12 channels, with a high sensitivity per channel if properly configured with custom fiber bundles (see possible arrays with 1 to 7 active fibers per channel, separated by 1, 2, 3 or more dead fibers used for spacing and crosstalk minimized well below 1%) and optimized slit widths (optional for high resolution requirements) according to one or more embodiments described herein. Active fiber channels may be linearly arranged in two or more columns shifted or offset from one another.
As recognized by the inventors of this disclosure, the need to provide high throughput well plate readers may be addressed by combining Raman probes/lasers and fluorescence (FLUO) probes/sources in a hybrid array, allowing simultaneous acquisitions of Raman and fluorescence measurements from different wells, which avoids crosstalk as well as reduces the lengthy processing time otherwise associated with sequential acquisitions. In addition, all spectra may be collected on the same imaging spectrometer with a scientific CMOS sensor that may be optionally cooled. The fluorescence typically covers broad spectral regions while Raman is for narrow ranges (3,000-4,000 of 1/cm). In comparison with a Raman only configuration, a hybrid system may require translations between the probes and the well plate so that the two combined arrays of Raman and fluorescence probes can be positioned to cover all wells. Because the Raman and fluorescence probes are not positioned to focus into the same wells at the same time, there is no significant stray light or crosstalk (such as crosstalk from the laser to fluorescence measurements, or fluorescence measurements to Raman), and there is no need to turn the light sources for the Raman and fluorescence measurements ON/OFF consecutively. As such, there is a significant reduction of total acquisition time for the entire well plate. All probes may be positioned above the well plate, or some probes may be positioned below the well plate depending on the particular configuration. Two arrays of probes may be provided (Raman and fluorescence), which may be non-adjacent, moving on the same stage or on two separate stages if one array is located on top of the well plates and the other is located below the well plates. The latter configuration may require wells having flat transparent well bottoms for best performance.
In some configurations, a hybrid system may include both Raman and fluorescence probes that are positioned to read the same well. For example, a Raman probe and a fluorescence probe may be aimed at the same well at slight angles (away from perpendicular) with the Raman and fluorescence readings performed sequentially for that well prior to the stage (or probes, depending on the application) being adjusted to read the next well.
A representative array of sample holders implemented as a well plate 100 and associated scanning pattern for a multi-probe plate reader is illustrated in
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 (4) Raman probes configured to simultaneously read four wells with an initial position as indicated by solid dots 110, 112, 114, and 116. In this example, the well plate is translated in the x-y plane by an associated mechanical positioning mechanism including a well plate holder in a pattern as represented by the arrows 120 so that each probe will read one of its associated 24 wells, with the four probes reading a corresponding four wells of the well plate simultaneously. Alternatively, the probes may be translated relative to the well plate. Use of multiple probes simultaneously captures spectra from multiple wells, reducing the number of x-y movements relative to applications with fewer probes or a single probe, to increase system throughput.
As illustrated in
The Raman signal is collected through a common (same) lens 434 as the excitation light from laser 430 with output from the probes provided by at least one corresponding optical fiber 480 associated with each probe to a single 4-channel imaging spectrometer 440 that includes a 2D imaging cooled camera 450 having a scientific CCD (combined with shutter) or a 2D CMOS sensor 460 (no shutter). As illustrated, the output light from the probes may be supplied by a linear array 470 of fibers 480, 482, 484, 486 to a single spectrometer input with spacing of at least one fiber diameter therebetween to reduce channel-to-channel crosstalk. Spacing between active fibers may be provided by one or more dead or inactive fibers 492 that do not carry light and may or may not extend partially or fully within a fiber bundle to a corresponding probe 410. Other mechanical spacers may be used in place of dead fibers. In the representative example illustrated, each active fiber 480, 482, 484, and 486 is spaced by three fiber diameters corresponding to inactive or dead fibers represented at 492. The active fibers are linearly arranged to form a line into the spectrometer and may optionally be coupled to an entrance slit (best shown in
Each active fiber 480, 482, 484, and 486 results in a corresponding line 480′, 482′, 484′, and 486′ on the sensor 460 with unread rows of the sensor 492′ matching the inactive spacing fibers 492. The Raman signal 496 corresponds to selected binned rows of sensor pixels, which 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. Readings associated with a particular line or channel are binned or grouped together to generate the corresponding Raman spectrum 496 to simultaneously acquire the Raman data to generate the corresponding four Raman spectra. After acquiring the Raman measurements for 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 having a plate holder holding 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 indicated by the representative scan pattern to acquire data for all samples contained in well plate 420. A reset or home signal returns the well plate 420 to the initial position indicated. As previously described, the relative position between well plate 420 and Raman probes 410 may be controlled by one or more controllers by moving probes 410, well plate 420, or both.
The spectrometer 440 may be implemented by a near 1:1 imaging spectrometer that includes a reflective concave aberration-corrected holographic grating 442, or various other concentric spectrometer configurations (Dyson, Offner, toroid-based, Schmidt-Czerny-Turner, grating-imaging spectrometer, etc.) all featuring low to no astigmatism. The number of fiber channels for such a multi-track configuration is limited by 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 fit on top or bottom of the well plate and by the desired cost of the system.
A representative scanning pattern 502 is shown so that 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 illustrated in
The example of
The configuration illustrated in
The configuration of
While the present inventors have recognized that the positioning or alignment of probes over 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 well as Raman measurements to achieve a desired throughput or other overall system performance.
In the example of the first configuration of
As previously described, the high throughput well plate reader may combine Raman with other types of measurements. The fiber arrangements illustrated in
In the example illustrated in
As illustrated in the simplified functional block diagram of
One or more controllers of a representative well plate reader system may be programmed to automatically calibrate probes on a periodic basis, such as once/day, for example. The controller(s) may be programmed to align an associated calibration standard with a probe, collect measurement data, compare the measurement data to previously stored data for the standard material, and control a corresponding light source wavelength and/or intensity based on the comparison. For example, when the calibration process detects wavelength (X-axis) drift between the measured calibration sample material and known sample material spectrum for one or more peak wavelengths, the controller will automatically correct for this drift by shifting the wavelength of the laser to reduce the difference between the measured peak wavelength with the standard peak wavelength for the material. Similarly, the calibration process may detect intensity drift and may adjust the light source intensity accordingly. Alternatively, the system may suggest a different calibration process using a calibration plate with multiple standard samples that is held by the well plate holder 1606. The calibration plate may be used to calibrate intensity of various light sources relative to one another for throughput matching across different probes.
In the example of
The present inventors have recognized that the intensity of a sharp Raman band for particular materials may be used for intensity drift monitoring to trigger recalibration. Many materials will degrade under laser exposure and are therefore poor candidates for a calibration standard. As such, diamond was selected as a material that is very stable over significant numbers of laser exposures. In addition, hard materials such as diamond and silicon will provide peaks that vary in intensity depending on the Z-axis position of the probes such that calibration using such materials may be used to adjust the Z-axis focus precision of one or more probes.
Liquid wells 1730 can be loaded with a typical liquid/base used in a particular application, or with any of a number of reference liquids. Reference liquids provide more reliable intensity calibration relative to hard material calibration standards because any Z-axis focus imprecision does not significantly affect the intensity. The Raman peak intensities stay relatively stable for a range of Z-axis focus as long as the light is focused under the surface of the liquid. However, liquid calibration standards will evaporate and may require user intervention to refill associated calibration wells.
Use of different calibration standard sample materials may be helpful to identify one or more components needing adjustment or replacement. For example, a Raman peak of a single material (such as diamond in this example) may be insufficient to diagnosis degradation or changes in various other system components in the optical path of the laser, fiber, probe, spectrometer, etc. Calibration standard sample materials having other peaks and/or multiple peaks (silicon wafer, liquids . . . ) may facilitate diagnosis and subsequent compensation for a particular component related to a corresponding portion of the spectrum.
Many components along the optical path other than the laser or other light source may contribute to inaccurate measurements associated with component failure, misalignment, wear, aging, etc. Laser aging compensation is preferably done automatically without user action, as the laser is the most common and frequent source of inaccuracies. Other conditions such as a damaged filter in the probe, a spectrometer/camera shift, a damaged or broken fiber, a positioning error by the positioning mechanism, etc. may all affect resulting measurements. As such, use of multiple calibration standard samples provided by a calibration plate 1700 provides additional diagnosis and calibration flexibility.
A system or method may include simultaneously focusing laser light within at least two samples contained in corresponding wells of a well plate, the at least two samples contained in wells spaced by at least one well without focused laser light as represented at 1802. Light emitted by the at least two samples is simultaneously directed to a spectrometer having a diffraction grating that redirects light at a wavelength-dependent angle to a shared sensor as represented at 1804. Block 1806 represents acquiring Raman spectroscopy data associated with the at least two samples from the shared sensor. Block 1808 represents controlling positioning of the well plate relative to the focused laser light to simultaneously acquire Raman spectroscopy data from at least two different samples contained in previously unexamined wells of the well plate.
Simultaneously focusing laser light as represented by block 1802 may include simultaneously focusing light from a different laser associated with each one of the at least two samples. Block 1802 may also include simultaneously directing light from a source to at least two samples contained in corresponding wells of the well plate, each well spaced by at least one well not illuminated by the focused laser light or light from the source. Block 1804 may also include simultaneously directing light emitted by the samples illuminated by the light from the source to the shared sensor of the spectrometer, while block 1806 may include acquiring fluorescence data from the shared sensor of the spectrometer associated with the at least two samples illuminated by the light from the source.
In at least one embodiment, block 1804 includes simultaneously directing light emitted by the at least two samples by coupling the emitted light to at least one optical fiber for each of the at least two samples, and arranging the optical fibers in a linear array at an input of the spectrometer, the optical fibers associated with each sample grouped together and separated from optical fibers associated with another sample by a distance corresponding to at least one optical fiber diameter.
Various embodiments may also include, by the at least one controller, positioning a calibration standard integrated within a well plate holder to be illuminated by the focused laser light, acquiring Raman spectroscopy data from the calibration standard, and adjusting wavelength of the focused laser light based on the Raman spectroscopy data of the calibration standard compared to a previously stored peak wavelength for the calibration standard.
Alternatively, Raman probes may be aligned along a common axis but focused on different spots within the moving liquid as illustrated in
As described with respect to the previous embodiments, the outputs of the at least two Raman probes are input to an imaging spectrometer and imaged on a shared two-dimensional sensor. Each Raman probe output may be coupled by one or more optical fibers to the spectrometer input with the fibers linearly arranged at the spectrometer input. Optical fibers of one Raman probe may be spaced by a distance corresponding to one or more optical fiber diameters from optical fibers of other Raman probes where required to reduce or eliminate crosstalk between the images on the sensor. In the embodiment of
In general, all embodiments having at least two Raman probes may include a single, higher-power laser with the laser output divided or split between or among two or more probes, a lower-power laser associated with each Raman probe (which may be integrated within the probe housing), lower-power lasers with outputs combined to increase the signal to the input of a single Raman probe, or any combination thereof. The laser configuration, power, etc. may vary based on the particular application requirements and implementation specifics. Similarly, lasers, Raman probes, and the spectrometer may be coupled by one or more optical fibers as described in greater detail herein although free space coupling using associated optical elements of one or more system components may be suitable or acceptable in particular applications or implementations. Similarly, embodiments may include two or more probes focused at the same spot of a sample, or positioned at the same location but focused at different spots within the sample to provide a desired signal or time gain.
As also previously described, a probe positioning mechanism generally indicated by arrows 3050 may be controlled to control positioning of the probes relative to the solid wafer sample 3030. Positioning of probes 3010, 3012, 3014, 3016 may be controlled independently and/or as a unit along one or more orthogonal axes. For example, the probes may have independent z-axis control to adjust the distance between the probes and the sample 3030 while moving together as a unit in x-axis or x,y-axis positioning. Similarly, a sample positioning device may be used to provide rotational positioning and/or positioning along orthogonal x, y, and/or z axes. Probes 3010, 3012, 3014, 3016 may be positioned with outputs perpendicular to the surface of solid wafer sample 3030, such as illustrated in the embodiment of
As illustrated in
While representative embodiments are described above and illustrated in the Figures, it is not intended that these embodiments describe all possible forms encompassed by the teachings of the disclosure and currently claimed subject matter. The words used in the specification are words of description rather than limitation, and it is understood that various changes can be made without departing from the claimed subject matter. As previously described, the features of various representative embodiments can be combined to form further embodiments that may not be explicitly described or illustrated. While various embodiments could have been described as providing advantages or being preferred over other embodiments or prior art implementations with respect to one or more desired characteristics, those of ordinary skill in the art recognize that one or more features or characteristics can be compromised to achieve desired overall system attributes, which depend on the specific application and implementation. These attributes may include, but are not limited to strength, durability, life cycle, marketability, appearance, packaging, size, serviceability, weight, manufacturability, ease of assembly, etc. As such, embodiments described as less desirable than other embodiments or prior art implementations with respect to one or more characteristics are not necessarily outside the scope of the disclosure or claimed subject matter and may be desirable for particular applications.
This application is a continuation-in-part of commonly owned and co-pending U.S. application Ser. No. 18/087,341 filed Dec. 22, 2022, which claims the benefit under 35 U.S.C. § 119 (e) of U.S. App. No. 63/293,733 filed Dec. 24, 2021. This application also claims the benefit under 35 U.S.C. § 119 (e) of U.S. App. No. 63/625,237 filed Jan. 25, 2024. The disclosures of which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63293733 | Dec 2021 | US | |
| 63625237 | Jan 2024 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 18087341 | Dec 2022 | US |
| Child | 19037340 | US |
