Stimulated emission depletion for raman spectroscopy

Abstract

Systems and methods for conducting optical material analysis, such as Raman spectroscopy, using two lasers. A sample location is simultaneously irradiated with two laser beams along different optical axes. A detector acquires signals by detecting scattered photons from the sample location. The acquired signals include Raman signal. The fluorescence signal in the acquired signals is suppressed due to stimulated emission depletion.

Description

PCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001STIMULATED EMISSION DEPLETION FOR RAMAN SPECTROSCOPYRELATED APPLICATIONS

[0001] This application is a continuation of U.S. Patent Application No. 63 / 683,444, filed August 15, 2024, the entire content of which is hereby incorporated by reference.FIELD

[0002] Examples described herein generally relate to systems and methods for conducting optical material analysis, such as Raman spectroscopy.BACKGROUND

[0003] Raman spectroscopy is an effective tool for identifying and characterizing various sample compounds and substances. In Raman spectroscopy, light, typically from a laser and of a known wavelength (also sometimes referred to as a Raman pump light), is directed at a sample compound or substance (referred to herein as a “sample”). The laser photons inelastically scatter, or “Raman scatter,” off the molecules in the sample and experience wavelength shifting to new frequencies given by bond vibrational frequencies present in the molecules of the sample. The precise nature of this wavelength shifting from the Raman pump light depends upon the materials present in the sample. The sample specific Raman scattering photons can be acquired with a spectrometer as a Raman spectrum (or Raman signal). This unique Raman spectrum permits the sample to be identified and characterized.SUMMARY

[0004] In one aspect, there is provided a method for Raman spectroscopy. The method includes simultaneously irradiating a sample location of a sample with a first laser beam along a first optical axis and a second laser beam along a second optical axis. The method includes acquiring signals by detecting at least a portion of photons emitted from the sample location responsive to the irradiation, wherein the detected photons include Raman scattered photons, and analyzing characteristics of the sample based on the acquired signal.

[0005] In another aspect, there is provided a Raman spectroscopic system comprising a first laser to generate a first laser beam, a second laser to generate a second laser beam, optical components for directing the first laser beam and the second laser beam towards a sample location of a sample from different angles of incidence, and a detector for detecting at least aPCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 portion of photons emitted from the sample location, the detected photons including Raman scattered photons. The system includes a controller including an electronic processor and a memory for storing a computer readable program. By executing the computer readable program in the electronic processor, the controller is configured to control the first laser and the second laser to simultaneously irradiate the sample location, acquire, with the detector, signals emitted from the sample location responsive to the irradiation, and analyze characteristics of the sample based on the acquired signals.

[0006] There is no specific requirement that a system, method, or technique relating to Raman spectroscopy include all of the details characterized herein to obtain some benefit according to the present disclosure. Thus, the specific examples characterized herein are meant to be example applications of the techniques described and alternatives are possible.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Features and advantages of the present technology will become more apparent from the following detailed description of examples thereof taken in conjunction with the accompanying drawings in which:

[0008] FIG. 1 is a block diagram of an analyzer in accordance with some aspects of the present disclosure.

[0009] FIG. 2 is a block diagram of a spectroscopic system included in the analyzer of FIG. 1 in accordance with some aspects of the present disclosure.

[0010] FIG. 3 is an illustrative example of an implementation of an optical architecture comprising optical components of the spectrometer of FIG. 1 in accordance with some aspects of the present disclosure.

[0011] FIG. 4A, FIG. 4B, and FIG. 4C are illustrative examples of an optical system in accordance with some aspects of the present disclosure.

[0012] FIG. 5 is a block diagram of a method performed by the spectroscopic system of FIG.2 in accordance with some aspects of the present disclosure.

[0013] While the present technology is susceptible to various modifications and alternative forms, specific aspects have been shown by way of example in the drawings and will be described in detail herein. It should be understood, however, that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is to cover allPCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.DETAILED DESCRIPTION

[0014] Raman spectroscopy may be used to identify characteristics of compounds and substances of samples. Performing Raman spectroscopy includes exciting the sample with a laser and obtaining a Raman spectrum (or Raman signal) of the scattered light. The acquired Raman spectrum is compared to a library of known Raman spectra to identify characteristics of the sample. Statistical matching algorithms, such as classical least square, may be used for the comparison. For example, Raman spectroscopy may be used to measure a glucose concentration of a sample. The glucose concentration may be estimated using the obtained Raman spectrum with a trained machine learning model or a chemometric model.

[0015] The photons emitted from the sample responsive to laser irradiation also includes fluorescence. The fluorescence generated by most samples is many orders of magnitude greater than the Raman signal itself, and may interfere with the sample composition analysis. In many cases, the shot noise of the fluorescence may completely overwhelm the Raman signals. Therefore, there is a need to suppress or reduce the fluorescence for Raman spectroscopy.

[0016] One method to reduce the fluorescence is to perform Raman spectroscopy using a longer excitation wavelength. However, the scattering efficiency reduces quartically with increased wavelength. Therefore, excitation at longer wavelength leads to reduced Raman scattering. Another method is to filter or remove the fluorescence from the acquired signals. However, since the fluorescence dominates the limited dynamic range of the detector, the signal to noise ratio (SNR) of the detected Raman signal cannot be further increased.

[0017] To address the above issues, a sample is irradiated by a first laser and a second laser simultaneously from different angles of incidence. The first laser is also referred to herein as an excitation laser that emits an excitation beam (first laser beam) onto a sample location, which causes Raman shift of scattered photons (i.e., Raman scattered photons) emitted from the sample. The second laser is also referred to herein as a stimulated emission depletion (STED) laser, that delivers a STED beam (second laser beam) onto the same sample location. The STED beam causes stimulated emission depletion that can manipulate and potentially suppress the fluorescence generated responsive to the irradiation of the excitation beam. Light emitted / scattered from the sample is acquired by a detector to generate the acquired signal. ThePCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 photons collected by the detector includes Raman scattered photons. The detector may be a spectrograph such that the acquired signal is a spectrum. The acquired signal may be a Raman spectrum that can be used to determine sample characteristics. In some examples, Raman signal can be extracted from the acquired signals for determining sample characteristics.

[0018] The excitation beam travels along a first optical axis towards the sample and the STED beam travels along a second, different, optical axis towards the sample. The detector may be arranged along the first optical axis for acquiring the Raman signal. A beam stop may be positioned along the second optical axis to capture the stimulated emission. The angle between the first and second optical axes is sufficient large so that the STED signal (emitted along the second optical axis) is outside the numerical aperture of the photon collection optics for acquiring the signals. In this way, the STED signal is not collected by the detector. In one example, the angle between the first and second optical axes is greater than half of the acceptance angle of the photon collection optics for collecting at least a portion of the photons emitted from the sample.

[0019] When the excitation beam interrogates the sample, electrons are excited from the ground state to an exited state. Spontaneous fluorescence is generated when the excited electrons spontaneously relax to the ground state. Spontaneous fluorescence or emission is isotropic, emitting in all directions from the sample. The STED beam irradiates the same location of the sample as the excitation beam. Stimulated emission differs from spontaneous emission in that it travels in the same direction as the input STED beam. The STED beam has a longer wavelength than the excitation beam. The STED beam transitions the excited electrons from their excited states to their ground states along with a stimulated emission photons. The stimulated emission process scales with the number of photons in the STED beam. The stimulated emission will compete with the spontaneous fluorescence. If the intensity of the STED beam is sufficient, the stimulated emission dominates the spontaneous fluorescence. By reducing the spontaneous fluorescence reaching the detector through STED, most of the dynamic range of the detector can be used for acquiring the Raman signal, therefore increasing the SNR of the Raman signal. Further, reduced spontaneous fluorescence signal allows shorter excitation wavelengths to be used for Raman excitation to further enhance the Raman scattering.

[0020] The detector is arranged along an optical axis different from the optical axis of the STED beam to acquire the Raman signal. Unlike the spontaneous fluorescence and RamanPCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 scattering, wherein the photons scatter in all directions, the stimulated emission’s wavevector points only in the direction of the input field. That is, the stimulated emission’s wavevector points in the direction of travel of the STED beam (along the second optical axis). By collecting the scattered photons along a different optical axis from the STED beam, the stimulated emission is directed away from the detector for acquiring the Raman signal, therefore removing the interference of the stimulated emission from the Raman signal acquisition.

[0021] The excitation beam has a wavelength centered in the visible to near infrared portion of the EM spectrum. The wavelength range of the STED beam is within or overlaps with the wavelength range of the fluorescence generated from the sample. For example, the excitation and STED beams will be paired as such (excitation / STED) and may have wavelength centered at (405 / 455) nm, (455 / 532) nm, (532 / 633) nm, (633 / 785) nm, or (785 / 1064) nm.

[0022] In some examples, both the first and second lasers are continuous wave (CW) lasers. In some example, at one or both of the first and second lasers is a pulsed laser.

[0023] In some examples, the STED laser is a CW laser. The STED laser may continuously emit the STED beam while the excitation laser may be controlled to emit the excitation beam when interrogation of the sample is initiated. In other examples, the depletion laser is controlled to emit the STED beam synchronized with the excitation laser and emission of excitation beam.

[0024] In one example, the detector acquires the Raman scattered photons in the transmission mode. That is, the detector detects photons from an opposite side of the sample excitation beam and along the optical bath of the excitation beam. In another example, the detector acquires the scattered photons in a backscattered geometry. That is, the detector detects signals from the same side of the sample and along the same optical path as the excitation beam.

[0025] In some examples, the polarization and / or power of the first and / or second laser may be adjusted based on the signals acquired by the detector. The power and / or polarization of the second STED laser may be adjusted based on the amount of non-Raman signal acquired at the detector. For example, the power of the second laser may be increased to enhance the STED responsive to the amplitude of acquired fluorescence signal exceeding a threshold fluorescence amplitude. In another example, the polarization of the second laser may be adjusted to minimize the fluorescence signal in the acquired signal. In another example, the power of the first and / or second laser may be adjusted based on the SNR of the Raman signal. Responsive toPCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 SNR of the Raman signal lower than a threshold SNR, the power of the first and second laser may be increased.

[0026] In some implementations, a wavelength of the fluorescence emitted by the sample is first measured using a sensor or estimated from the scattered photos acquired by the detector. The wavelength of the STED beam emitted by the STED laser is then set based on the measured wavelength. For example, the wavelength of the STED beam may be set to at least overlap the wavelength range of the fluorescence.

[0027] An optical analysis system for performing Raman spectroscopy includes an excitation laser generating an excitation beam of the first wavelength range and a STED laser generating a STED beam of the second wavelength range, wherein the center wavelength of the second wavelength range is longer than the center wavelength of the first wavelength range. In some examples, first wavelength range does not overlap with the second wavelength range. The system includes at least one detector for acquiring a Raman signal. The system includes a controller including an electronic processor and a memory for storing computer readable program, wherein by executing the computer readable programs in the electronic processor, the controller is configured to control the excitation laser and the depletion laser to simultaneously irradiate a sample location of a sample from different angles of incidence, and detect at least a portion of photons emitted from the sample location via the detector. The detected photons include Raman scattered photons. The detector may be a part of a spectrograph that acquires the Raman signals in the form of spectra.

[0028] In one example, the excitation beam is delivered to the sample and the scattered photons reflected from the sample are collected from the sample via the same objective. The STED beam may be delivered to the sample via the same or different objectives. The collected photons are directed to the detector. In one example, when the STED beam is delivered to the sample via the same objective as the excitation beam, the excitation beam and the STED beam irradiate different portions of a back aperture of the objective when entering the objective. In another example, the STED beam and the excitation beam irradiate the sample from different angles of incidence via a parabolic mirror.

[0029] In another example, the excitation beam and the STED beam irradiates from one side of the sample, and the detector is positioned to acquire scattered photons transmitted through the sample.PCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001

[0030] Methods and systems are provided for suppressing fluorescence while performing Raman spectroscopy. Although some examples provided herein may reference a particular type or configuration of a Raman spectrometer device, it should be understood that the methods and systems described herein may be performed using various types of Raman spectrometer, such as, for example, a benchtop or a portable Raman spectrometer device.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Example methods and systems are described below, although methods and systems similar or equivalent to those described herein can be used in practice or testing of the present disclosure. The systems, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0032] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9- 1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0033] As used herein, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or.” That is, unless specified otherwise, or clear from context, “X employs A or B” is intended to mean any of the natural inclusive permutations. That is, if X employs A, X employs B, or X employs both A and B, then “X employs A or B” is satisfied under any of the foregoing instances. Moreover, articles “a” and “an” as used in the subject specification and annexed drawings should generally be construed to mean “one or more” unless specified otherwise or clear from context to be directed to a singular form.

[0034] The present disclosure is now described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout. In the following description, for purposes of explanation, numbers of specific details are set forth in order to provide an improved understanding of the present disclosure. It may be evident, however, that the systems and methods of the present disclosure may be practiced without one or more of these specificPCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 details. In other instances, well-known structures and devices are shown in block diagram form in order to facilitate describing the systems and methods of the present disclosure.

[0035] FIG. 1 illustrates an implementation of an analyzer 100 that includes a spectroscopic system 110 communicatively connected to a computing device 120 via network 130. It will be appreciated that, in some implementations, at least a portion of the computing device 120 may be located separate from the spectroscopic system 110, providing the opportunity for increased computing power at a central location or across multiple locations. One skilled in the art can envision various interconnections, both physical and wireless, between the components of the analyzer 100. It will further be appreciated that, in some implementations, the spectroscopic system 110 and the computing device 120 may be communicatively connected physically without network 130. Alternatively, some implementations of the analyzer 100 and / or the spectroscopic system 110 may not require the resources of the computing device 120, instead utilizing the resources of a controller 111 and / or an electronic processor 113 optionally included in the spectroscopic system 110. Thus, the computing device 120 may not be necessary for operation of the analyzer 100 and / or the spectroscopic system 110, and the example of FIG. 1 should not be considered as limiting.

[0036] It should be understood that, in some implementations, the components of the analyzer 100 and / or the spectroscopic system 110 may be included in a common housing forming an analytical instrument that may include a benchtop or a portable Raman spectrometer device (e.g., a handheld device). However, in other implementations, one or more components of the analyzer 100 and / or the spectroscopic system 110 may be contained in separate housings or devices and may be coupled (e g., optically, communicatively, electrically, mechanically, or the like) as needed to carry out the methods described herein. Also, in some implementations, the operations described herein as being performed by the components of the analyzer 100 and / or the spectroscopic system 110 may be combined and distributed in various ways. For example, in some implementations, the electronic processor 113 may be part of the controller 111, where the controller I l l is configured to perform the operations of the electronic processor 113 as described herein. Furthermore, the operations described herein as being performed by the controller 111 may be distributed among multiple controllers. In the same or alternative example, the operations described herein as being performed by the controller 111 may be distributed among one or more computing devices (e.g., the electronic processor 113 and / or the computing device 120).PCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001

[0037] The analyzer 100 and / or the spectroscopic system 110 may also include additional components (such as power components), a user interface 114 (such as display 112 and / or input / output (I / O) devices 109 such as a keyboard, a mouse, a touch screen, and the like), and optical components 115 (e.g., mirrors, lenses, fiber optic cables, gratings, and / or filters). Spectroscopic system 110 may also include a spectrometer 140 that includes optical components 145, a detector 147 (e.g., a CCD detector, a PMT detector, or other detector known in the art), a first laser source 149 (e.g., a first laser light source, a first laser source) to provide an excitation beam (e.g., an LED, excitation laser, or other source providing light with a wavelength range that may include 532 nm, 785 nm or 1064 nm wavelengths of light), and a second laser source 151 (e.g., a second laser light source, a second laser source) to provide a STED beam (e.g., a stimulated emission depletion laser or other source providing light with a wavelength range that may include 633 nm wavelength of light). In some instances, both the first laser source 149 and the second laser source 151 are continuous wave (CW) lasers. In some example, at one or both of the first laser source 149 and the second laser source 151 is a pulsed laser.

[0038] As described above, in some implementations, the analyzer 100 and / or the spectroscopic system 110 comprises a fully integrated portable system operated by a user on battery power to take Raman spectroscopy measurements in a variety of environments that include a laboratory setting, a manufacturing (e.g., bioreactor based) setting, a remote setting, etc. Also, in the same or alternative implementations, elements of spectroscopic system 110 may be utilized as separated systems communicatively connected (e.g., optically, wirelessly, electrically, mechanically, and the like) and operated on battery power and / or power outlets connected to a central power source to take Raman spectroscopy measurements in the variety of environments described.

[0039] Referring now to FIG. 2, the spectroscopic system 110 may include the electronic processor 113, data storage device(s) 202, and the user interface 114, in addition to the first laser source 149, the second laser source 151, and the detector 147 that form the spectrometer 140. However, it should be understood that the spectroscopic system 110 may have additional or fewer components.

[0040] The spectroscopic system 110 is suitable for the application and setting, and can include, for example, multiple electronic processors, multiple I / O interfaces, multiple data storage devices, or combinations thereof. In some implementations, some or all of thePCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 components included in the spectroscopic system 110 may be attached to one or more mother boards and enclosed in a housing (e.g., including plastic, metal and / or other materials). In some implementations, some of these components may be fabricated onto a single system-on-a-chip, or SoC (e.g., an SoC may include one or more processing devices and one or more storage devices). Additionally, one or more of these components may be situated in a separate housing. For example, the electronic processor 113 may be situated in a first housing, while the data storage device(s) 302 are situated in a second housing communicatively coupled to the first housing.

[0041] As used herein, “processors” or “electronic processor” refers to any device(s) or portion(s) of a device that process electronic data from registers and / or memory to transform that electronic data that may be stored in registers and / or memory. The electronic processor 113 may include one or more digital signal processors (DSPs), application-specific integrated circuits (ASICs), central processing units (CPUs), graphics processing units (GPUs), cryptoprocessors (specialized processors that execute cryptographic algorithms within hardware), server processors, or any other suitable processing devices.

[0042] The data storage device 202 may include one or more local or remote memory devise such as random-access memory (RAM) devices (e.g., static RAM (SRAM) devices, magnetic RAM (MRAM) devices, dynamic RAM (DRAM) devices, resistive RAM (RRAM) devices, or conductive-bridging RAM (CBRAM) devices), hard drive-based memory devices, solid-state memory devices, networked drives, cloud drives, or any combination of memory devices. In some implementations, the data storage device 202 may include memory that shares a die with a processor. In such an implementation, the memory may be used as a cache memory and may include embedded dynamic random-access memory (eDRAM) or spin transfer torque magnetic random-access memory (STT-MRAM), for example. In some implementations, the data storage device 202 may include non-transitory computer readable media having instructions thereon that, when executed by one or more processors (e.g., the electronic processor 113), causes the spectroscopic system 110 to store various applications and data for performing one or more of the methods described herein or portions described herein. It should be understood that each method described herein may be implemented via one application or multiple applications and, in some examples, the data storage device 202 stores additional data in various configurations.

[0043] The user interface 114 (e.g., the I / O 109) of spectroscopic system 110 may include one or more communication chips, connectors, and / or other hardware and software to governPCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 communications between the spectroscopic system 110 and other components. For example, the I / O 109 may include circuitry for managing wireless communications for the transfer of data to and from the spectroscopic system 110. In some implementations, the I / O 109 may include one or more antennas (e.g., one or more antenna arrays) for receipt and / or transmission of wire communications. As noted above, the display 112 may include a screen, such as a touch screen, providing a graphical user interface indicating operational and analytical characteristics related to the spectroscopic system 110. In some instances, rather than the display 112, data may be communicated (e.g., via the network 130) to one or more other devices for output.

[0044] FIG. 3 provides another illustrative example of an implementation of an optical architecture comprising optical components of the spectrometer 140 (shown in FIG. 1) that are otherwise collectively referred to as optical system 300. It will be appreciated that different optical architectures of Raman spectrometers are known in the art and thus the example of FIG. 3, similar to the example of FIG. 1, should not be considered as limiting.

[0045] The example of FIG. 3 illustrates one implementation of the first laser source 149 comprising a laser source that produces a beam of light that travels along a first beam path 305 (the direction of the travel of the light beam indicated by arrows) to the sample 330. It will be appreciated that the sample 330 may include any type of sample of interest to a user which may include substantially dry samples (e g., a powder, a solid material), substantially fluid samples (e.g., a liquid, a gas), or some combination thereof (e g., a gel). In response to the light from first laser source 149, the sample 330 produces scattered light (e.g., comprising a Raman portion and a fluorescence portion of scattered light). A portion of the scattered light is directed along a scattered beam path 310 and is directed by a selective element 311 to a collecting beam path 320, as discussed below in more detail.

[0046] In some implementations, the first laser source 149 may produce laser power as needed for an application, for example, including or between a range of about 250 mW to about 1050 mW, including various subranges therebetween such as the non-limiting subranges described above for the first laser source 149. It will also be appreciated that in some implementations, the laser power affects the values of the base value and the bright-max intensity values when sample 330 is scanned.

[0047] In some instances, excitation light (e.g., laser light) that is projected by the first laser source 149 onto the sample 330 causes the sample 330, in reaction to the excitation light, to emit a Raman signal light (e.g., effectively “glow”). In some instances, light emitted by thePCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 sample 330 (also referred to as Raman shifted light or emitted light or Raman signal light) is directed toward the detector 147. In other words, light emitted from the first laser source 149 initiates an analytical interrogation of the sample 330 (e.g., exciting atomic bonds of molecules in the sample 330) such that a Raman spectrum can be captured by the detector 147 as a response to the interrogation of the sample 330.

[0048] The Raman spectrum can be analyzed, for example, by the detector 147 or by a separate component included in the spectroscopic system 110 or remote from the spectroscopic system 110. For example, the analysis of the Raman spectrum can be based on reference Raman spectra stored at spectroscopic system 110 or remote to but accessible by the spectroscopic system 110.

[0049] The example of FIG. 3 also illustrates an implementation of the second laser source 151 comprising a laser source that produces a beam of light that travels along a second beam path 306 (the direction of travel of the light beam indicated by arrows) to the sample 330. The beam of light emitted by the second laser source 151 may be, for example a STED beam that causes STED emission.

[0050] A first waveplate 307 may be provided along the first beam path 305 to alter the polarization of the excitation beam emitted by the first laser source 149. Additionally, a second waveplate 309 may be provided along the second beam path 306 to alter the polarization of the STED beam emitted by the second laser source 151.

[0051] FIG. 3 illustrates an architecture that, in some implementations, directionally controls the first beam path 305, the second beam path 306, the scattered beam path 310, and / or the collecting beam path 320. In some implementations, beam paths 305, 306, 310, 320 can be controlled using one or more of turning mirrors, waveguide phase scramblers, various lenses, broadband filters, or selective elements (e.g., mirrors, notch filters, or other elements with substantially reflective characteristics to the wavelength(s) of the beam from first laser source 149 and / or substantially transmissive characteristics to a wavelength or wavelength range associated with Raman scattered light from sample 330). In the described example, the selective element 311 (e.g., a beam splitter 311 such as a dichroic mirror) is transmissive to the laser wavelengths emitted from first laser source 149, allowing the first beam path 305 and / or the second beam path 306 to be directed to lens 308 that focuses the beam to sample 330. In the described example, lens 308 may include any type of lens known in the art such as an objective lens or lens architecture that focuses the beam on to sample 330.PCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001

[0052] Some implementations of the lens 308 include special configurations and characteristics that provides advantages for different types of samples. For example, lens 308 can collect a portion of the light emitted from sample 330 in response to the beam from the first laser source 149. The scattered light collected by lens 308 is directed back from the surface of the sample 330 travels along the scattered beam path 310 to the selective element 311 which directs the scattered light along the collecting beam path 320. In some implementations, the selective element 311 is substantially reflective to the wavelengths of the Raman scattered light, allowing light traveling along the collecting beam path 320 to be directed to additional optical elements that further adjusts the path and conditions the characteristics of the beam traveling along the collecting beam path 320. Other optical arrangements are also contemplated for selective element 411 for directing the scattered light along the collecting beam path 320.

[0053] A wavelength selection element 315 receives the scattered light from the collecting beam path 320. The wavelength selection element 315 may be situated within a microscope 312. The wavelength selection element 315 selectively transmits a wavelength range of scattered light to the detector 147. The wavelength selection element 315 may include one or more of a filter, a monochromator, and a spectrometer. The detector 147 may be, for example, a camera integrated within a spectrograph or connected to a spectrograph. In some examples, the wavelength selection element 315 and the detector 147 may be integrated into a detector. An electrical signal is transmitted from the detector 147 to the electrical signal processor 113. In some implementations, the electrical signal processor 113 may be a suitably programmed microprocessor or application specific integrated circuit including a read-only or read-write memory of any known type which holds instructions and data for spectrometer operation as described herein.

[0054] As described above, it will be appreciated that a variety of implementations of lens 308 are available that provide different focusing and light collection characteristics.

[0055] Accordingly, embodiments described herein introduce a STED beam (e.g., beam generated from a depletion laser) to a sample at approximately the same location as the excitation beam. FIG. 4A illustrates an example optical system 400 introducing such a STED beam. The optical system 400 includes a first lens 406 (for example, a focusing lens) that receives an excitation laser beam 402 emitted by the first laser source 149 (shown in FIG. 1) and a STED beam 404 emitted by the second laser source 151 (shown in FIG. 1). In thePCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 example optical system 400, the excitation laser beam 402 and the STED beam 404 travel along separate parallel axes prior to receipt by the first lens 406 such that the excitation laser beam 402 and the STED beam 404 are received by the first lens 406 at different portions of the first lens 406. For example, the excitation laser beam 402 is received by a first portion of the back aperture of an objective and the STED beam 404 is received by a second, different, portion of the back aperture of the same objective. In some examples, the first and second portions may overlap.

[0056] The first lens 406 focuses the excitation laser beam 402 and the STED beam 404 onto the sample 330. The sample 330 may be situated at a focal length of the first lens 406. In the example optical system 400, the first lens 406 focuses the excitation laser beam 402 along a first axis 420 and focuses the STED beam 404 along a second axis 422 that is offset from the first axis 420 by an angle $. Interrogation of the sample 330 by the excitation laser beam 402 results in the sample emitting a Raman light 412. While the Raman light 412 is illustrated as being directed toward a second lens 408 (e.g., a collimating lens), the Raman light 412 may be emitted by the sample in all directions with a portion of the Raman light 412 being received by the second lens 408. The second lens 408 directs the Raman light 412 towards the detector 147. In some instances, the second lens 408 collimates the Raman light 412. Interrogation of the sample by the STED beam 404 causes STED emission. The STED emission 414 travels along the second axis 422 and toward a light dump 410. Accordingly, the STED emission 414 does not reach the second lens 408.

[0057] In another implementation, in a reflective mode, Raman light 424 that is emitted back towards the first lens 406 may be captured. For example, as shown in FIG. 4B, the optical system 401 may include a dichroic mirror 430 that transmits the excitation laser beam 402 traveling towards the first lens 406. Raman light 424 that is emitted along the same path as the excitation laser beam 402 (traveling in the opposite direction) is directed by the first lens 406 towards the dichroic mirror 430. The dichroic mirror 430 reflects the Raman light 424 towards detector 147.

[0058] In yet another implementation, a mirror 434 may be positioned along first axis 420 of FIG. 4B to reflect Raman light 412 along the first axis 420 and direct the Raman light 412 back toward the first lens 406. The first lens 406 redirects the Raman light 424 first along the same optical path as the excitation laser beam 402 and then to the detector 147. By including mirror 434, more Raman scattered photons may be collected. FIG. 4C illustrates an example opticalPCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 system 450, which is part of the system 401 of FIG. 4B. The optical system 450 may be part of a Raman probe placed into a liquid sample 330. In the example of FIG. 4C, the excitation laser beam 402 and the STED beam 404 are directed towards sample located in the opening 452 between the first lens 406 and a reflective surface (i.e., mirror) 454. The opening 452 facilitates a free flow (or a turbulent flow) of the sample 330 between the first lens 406 and a reflective surface 454. The reflective surface 454 is supported by a holder 456 positioned at a distance from the first lens 406. The reflective surface 454 is positioned along a path (such as the first axis 420 of FIG. 4B) of the excitation light emitted from the first lens 406. When the Raman light is emitted from the sample 330 situated within the opening 452, Raman light traveling towards the first lens 406, as well as the Raman light reflected back towards to first lens by the reflective surface 454, are captured by the detector 147.

[0059] FIG. 5 illustrates a block diagram of an example method 500 for interrogating a sample using Raman spectroscopy. The method 500 may be performed by the electronic processor 113, by the controller 111, or a combination thereof. The steps of the method 500 are described in iterative manner for descriptive purposes. Various steps described herein with respect to the method 500 are capable of being executed simultaneously, in parallel, or in an order that differs from the illustrated serial and iterative manner of execution.

[0060] At block 502, the method 500 includes positioning a sample. For example, a user positions the sample 330 for analysis by the analyzer 100. At block 504, the method 500 includes identifying a location of the sample. For example, the user interacts with the user interface 114 to navigate over the sample to identify a region of interest of the sample 330 to be analyzed. The user may navigate the sample 330 based on optical images, such as the white light image.

[0061] At block 506, the method 500 includes simultaneously irradiating the sample location identified at block 504 using the first light beam and the second light beam. For example, the electronic processor 113 controls the first laser source 149 to emit an excitation beam onto the sample 330. The first laser source 149 may have a wavelength of, for example, approximately 532nm. Responsive to the excitation beam, the sample 330 emits both Raman and fluorescence photons. Simultaneously, the electronic processor 113 controls the second laser source 151 to emit a STED beam onto the sample 330. The second laser source 151 may have a longer wavelength than the wavelength of the first laser source 149. For example, the second laser source 151 may have a wavelength of approximately 633nm. Additionally, the wavelength ofPCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 second laser source 151 may overlap a wavelength of the fluorescent light. The STED beam causes STED, which suppresses the fluorescence and generates stimulated emission photons. The Raman signal detected by the detector 147 is less affected by fluorescence compared to when only the excitation beam interrogates the sample 214.

[0062] At block 508, the method 500 includes acquiring signals emitted by the sample. For example, the electronic processor 113 obtains, from the detector 147, a portion of photons emitted by the sample 330 in response to the excitation beam.

[0063] At block 510, the method 500 includes adjusting beam polarization and / or laser power based on the acquired signal. For example, the electronic processor 113 may adjust a polarization of the first laser source 149 by controlling the first waveplate 307, may adjust a polarization of the second laser source 151 by controlling the lens 308, may adjust a power of the first laser source 149, and / or may adjust a power of the second laser source 151. The beam polarization and / or laser power may be adjusted to minimize the fluorescence signal in the acquired signals. For example, fluorescence signal intensity may be observed from the acquired signal, and the STED laser's intensity and polarization may be adjusted to reduce the observed fluorescence signal intensity.

[0064] At block 512, the method 500 includes optionally imaging another location of the sample. For example, the electronic processor 113 images another location of the sample 330 identified in a region of interest.

[0065] At block 514, the method 500 includes processing the acquired signals to determine the sample characteristics. The sample characteristics may include one or more of sample composition, chemical structure, polymorphism, crystallinity, molecular interactions. For example, the electronic processor 113 extracts the Raman signal from the acquired signals and processes the Raman signal to characterize the sample 330 at the imaged sample location.

[0066] At block 514, the method 500 includes displaying the sample image including the sample characteristics. For example, the captured image of the sample 330 and the identified composition may be output by the electronic processor 113 via the display 112 (e.g., as part of a graphical user interface) and / or communicated to another device or computing system.

[0067] As described above in the detailed description, reference is made to the accompanying drawings that form a part hereof wherein like numerals designate like parts throughout, and in which is shown, by way of illustration, implementations that may be practiced. It is to be understood that other implementations may be utilized, and structured or logical changes mayPCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 be made, without departing from the scope of the present disclosure. Therefore, the detailed description as described above is not to be taken in a limiting sense.

[0068] Various operations may be described as multiple discrete actions or operations in turn, in a manner that is most helpful in understanding the subject matter disclosed herein. However, the order of description should be construed as to imply that these operations are necessarily order dependent. In particular, these operations may not be performed in the order of presentation. Operations described may be performed in a different order from the described implementation. Various additional operations may be performed, and / or described operations may be omitted in additional implementations.Clauses

[0069] Implementations of the present disclosure are disclosed in the following clauses:

[0070] Clause 1 : A method for Raman spectroscopy, comprising: simultaneously irradiating a sample location of a sample with a first laser beam along a first optical axis and a second laser beam along a second optical axis; acquiring signals by detecting at least a portion of photons emitted from the sample location responsive to the irradiation, wherein the detected photons include Raman scattered photons; and analyzing characteristics of the sample based on the acquired signals.

[0071] Clause 2: The method of clause 1, wherein the second laser beam has a longer wavelength than the first laser beam.

[0072] Clause 3: The method of any one of clause 1 to clause 2, wherein detecting at least a portion of the photons emitted from the sample location includes collecting the at least a portion of the photons along an optical axis different from the second optical axis.

[0073] Clause 4: The method of any one of clause 1 to clause 2, wherein detecting at least a portion of the photons emitted from the sample location includes collecting the at least a portion of the photons along the first optical axis.

[0074] Clause 5: The method of any one of clause 1 to clause 4, wherein the detected photons include fluorescence, and wherein the acquired signals include a fluorescence signal.

[0075] Clause 6: The method of clause 5, further comprising adjusting the first laser beam and / or the second laser beam based on the acquired signals.PCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001

[0076] Clause 7: The method of clause 6, wherein adjusting the first laser beam and / or the second laser beam based on the acquired signals includes adjusting an intensity and / or polarization of the first laser beam and / or the second laser beam based on the acquired signals.

[0077] Clause 8: The method of clause 6, wherein adjusting the first laser beam and / or the second laser beam based on the acquired signals includes adjusting the first laser beam and / or the second laser beam to minimize the fluorescence signal of the acquired signals.

[0078] Clause 9: The method of any one of clause 5 to clause 8, wherein a wavelength of the second laser beam is within a wavelength range of the fluorescence.

[0079] Clause 10: The method of any one of clause 5 to clause 9, wherein the detected Raman scattered photons forms a Raman signal in the acquired signals, and the method further comprising removing the fluorescence signal from the acquired signals to obtain the Raman signal, and wherein analyzing characteristics of the sample based on the acquired signals includes analyzing the characteristics of the sample based on the Raman signal.

[0080] Clause 11 : The method of any one of clause 1 to clause 10, wherein the first laser beam has a wavelength centered in the visible to near infrared portion of the electromagnetic spectrum.

[0081] Clause 12: The method of any one of clause 1 to clause 11, wherein the first laser beam excites the sample location to generate the Raman scattered photons and wherein the second laser beam causes stimulated emission depletion.

[0082] Clause 13 : The method of any one of clause 1 to clause 12, wherein the signal is acquired via photon collection optics, and wherein an angle between the first optical axis and the second optical axis is greater than half of an acceptance angle of the photon collection optics.

[0083] Clause 14: A Raman spectroscopic system comprising: a first laser to generate a first laser beam; a second laser to generate a second laser beam; optical components for directing the first laser beam and the second laser beam towards a sample location of a sample from different angles of incidence; a detector for detecting at least a portion of photons emitted from the sample location, wherein the detected photons include Raman scattered photons; and a controller including an electronic processor and a memory for storing a computer readable program, wherein by executing the computer readable program in the electronic processor, the controller is configured to: control the first laser and the second laser to simultaneously irradiate the sample location; acquire, with the detector, signals emitted from the samplePCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 location responsive to the irradiation; and analyze characteristics of the sample based on the acquired signals.

[0084] Clause 15 : The Raman spectroscopic system of clause 14, wherein the first laser and the second laser are both continuous wave lasers.

[0085] Clause 16: The Raman spectroscopic system of clause 14, wherein at least one of the first and second lasers is a pulsed laser.

[0086] Clause 17: The Raman spectroscopic system of clause 14, wherein the first laser beam has a first wavelength range and the second laser beam has a second, longer wavelength range, and wherein the first and second wavelength ranges do not overlap.

[0087] Clause 18: The Raman spectroscopic system of clause 17, wherein the detected photons from the sample location includes fluorescence, and wherein the second wavelength range overlaps with the wavelength of the fluorescence.

[0088] Clause 19: The Raman spectroscopic system of clause 18, wherein the controller is further configured to: analyze an amplitude of the fluorescence in the acquired signals; and adjust a power of the first and / or second laser based on the amplitude.

[0089] Clause 20: The Raman spectroscopic system of any of clause 14 to clause 19, wherein the optical components include an objective, and wherein the first laser beam and the second laser beam are directed towards the sample location via the objective.

[0090] Clause 21 : The Raman spectroscopic system of clause 20, wherein the first laser beam and the second laser beam irradiate different portions of a back aperture of the objective when entering the objective.

[0091] Clause 22: The Raman spectroscopic system of any of clause 14 to clause 21, wherein the optical components include a parabolic mirror, and wherein the first laser beam and the second laser beam are directed towards the sample location via the parabolic mirror.

[0092] Clause 23 : The Raman spectroscopic system of any of clause 14 to clause 21, wherein the optical components include a first objective to direct the first laser beam along a first optical axis towards the sample location, and a second objective to direct the second laser beam along a second optical axis towards the sample location, wherein the first optical axis is different from the second optical axis.

[0093] Clause 24: The Raman spectroscopic system of any of clause 14 to clause 23, wherein the optical components include at least a waveplate positioned along a beam path of the firstPCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 laser beam or the second laser beam, and wherein the controller is further configured to adjust, via the at least one waveplate, a polarization of the first laser beam relative to a polarization of the second laser beam based on the acquired signals.

Claims

PCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W001 CLAIMSWhat is claimed is:

1. A method for Raman spectroscopy, comprising: simultaneously irradiating a sample location of a sample with a first laser beam along a first optical axis and a second laser beam along a second optical axis; acquiring signals by detecting at least a portion of photons emitted from the sample location responsive to the irradiation, wherein the detected photons include Raman scattered photons; and analyzing characteristics of the sample based on the acquired signals.

2. The method of claim 1, wherein the second laser beam has a longer wavelength than a wavelength of the first laser beam.

3. The method of claim 1, wherein detecting at least a portion of the photons emitted from the sample location includes collecting the at least a portion of the photons along an optical axis different from the second optical axis.

4. The method of claim 1, wherein detecting at least a portion of the photons emitted from the sample location includes collecting the at least a portion of the photons along the first optical axis.

5. The method of claim 1, wherein the detected photons include fluorescence, and wherein the acquired signals include a fluorescence signal.

6. The method of claim 5, further comprising adjusting the first laser beam and / or the second laser beam based on the acquired signals.

7. The method of claim 6, wherein adjusting the first laser beam and / or the second laser beam based on the acquired signals includes adjusting an intensity and / or polarization of the first laser beam and / or the second laser beam based on the acquired signals.

8. The method of claim 6, wherein adjusting the first laser beam and / or the second laser beam based on the acquired signals includes adjusting the first laser beam and / or the second laser beam to minimize the fluorescence signal of the acquired signals.PCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W0019. The method of claim 5, wherein a wavelength of the second laser beam is within a wavelength range of the fluorescence.

10. The method of claim 5, wherein the detected Raman scattered photons forms a Raman signal in the acquired signals, and the method further comprising removing the fluorescence signal from the acquired signals to obtain the Raman signal, and wherein analyzing characteristics of the sample based on the acquired signals includes analyzing the characteristics of the sample based on the Raman signal.

11. The method of claim 1, wherein the first laser beam has a wavelength centered in the visible to near infrared portion of the electromagnetic spectrum.

12. The method of claim 1, wherein the first laser beam excites the sample location to generate Raman scattered photons and wherein the second laser beam causes stimulated emission depletion.

13. The method of claim 1, wherein the signal is acquired via photon collection optics, and wherein an angle between the first optical axis and the second optical axis is greater than half of an acceptance angle of the photon collection optics.

14. A Raman spectroscopic system comprising: a first laser to generate a first laser beam; a second laser to generate a second laser beam; optical components for directing the first laser beam and the second laser beam towards a sample location of a sample from different angles of incidence; a detector for detecting at least a portion of photons emitted from the sample location, wherein the detected photons include Raman scattered photons; and a controller including an electronic processor and a memory for storing a computer readable program, wherein by executing the computer readable program in the electronic processor, the controller is configured to: control the first laser and the second laser to simultaneously irradiate the sample location; acquire, with the detector, signals emitted from the sample location responsive to the irradiation; and analyze characteristics of the sample based on the acquired signals.PCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W00115. The Raman spectroscopic system of claim 14, wherein the first laser and second laser are both continuous wave lasers.

16. The Raman spectroscopic system of claim 14, wherein at least one of the first and second lasers is a pulsed laser.

17. The Raman spectroscopic system of claim 14, wherein the first laser beam has a first wavelength range, and the second laser beam has a second, longer wavelength range, and wherein the first and second wavelength ranges do not overlap.

18. The Raman spectroscopic system of claim 17, wherein the detected photons from the sample location includes fluorescence, and wherein the second wavelength range overlaps with the wavelength of the fluorescence.

19. The Raman spectroscopic system of claim 18, wherein the controller is further configured to analyze an amplitude of the fluorescence in the acquired signals, and adjust a power of the first and / or second laser based on the amplitude.

20. The Raman spectroscopic system of claim 14, wherein the optical components include an objective, and wherein the first laser beam and the second laser beam are directed towards the sample location via the objective.

21. The Raman spectroscopic system of claim 20, wherein the first laser beam and the second laser beam irradiate different portions of a back aperture of the objective when entering the objective.

22. The Raman spectroscopic system of claim 14, wherein the optical components include a parabolic mirror, and wherein the first laser beam and the second laser beam are directed towards the sample location via the parabolic mirror.

23. The Raman spectroscopic system of claim 14, wherein the optical components include a first objective to direct the first laser beam along a first optical axis towards the sample location, and a second objective to direct the second laser beam along a second optical axis towards the sample location, wherein the first optical axis is different from the second optical axis.PCT / US25 / 39898 30 July 2025 (30.07.2025)Docket No. TP386078W01-0154-W00124. The Raman spectroscopic system of claim 14, wherein the optical components include at least a waveplate positioned along a beam path of the first laser beam or the second laser beam, and wherein the controller is further configured to adjust, via the at least one waveplate, a polarization of the first laser beam relative to a polarization of the second laser beam based on the acquired signals.

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