Dual-comb photothermal microscopy devices and methods

Abstract

Disclosed herein are dual-comb photothermal microscopy devices and methods. For example, disclosed herein is are hyperspectral imaging devices and methods, comprising simultaneously subjecting a sample to dual-comb spectroscopy and photothermal microscopy.

Description

[0001] 11708-004W01; UHID 2024-064

[0002] DUAL-COMB PHOTOTHERMAL MICROSCOPY DEVICES AND METHODS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 695,535, filed September 17, 2024, which is hereby incorporated herein by reference in its entirety.

[0005] STATEMENT OF GOVERNMENT SUPPORT

[0006] This invention was made with government support under grant number R01DK135870 awarded by the National Institutes of Health and grant number 2019195 awarded by the National Science Foundation. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] Current microscopy approaches lack the combination of subcellular resolution, label-free chemical specificity, and the necessary high speed to characterize complex bio-molecular systems in real-time. Improved devices and methods are therefore needed. The devices, systems, and methods disclosed herein address these and other needs.

[0009] SUMMARY

[0010] In accordance with the purposes of the disclosed devices, methods, and systems as embodied and broadly described herein, the disclosed subject matter relates to dual-comb photothermal microscopy devices and methods.

[0011] For example, disclosed herein is a hyperspectral imaging method, the method comprising simultaneously subjecting a sample to dual -comb spectroscopy and photothermal microscopy.

[0012] In some examples, the method comprises: simultaneously irradiating at least a portion of the sample at a location with a plurality of wavelengths, the plurality of wavelengths each comprising a dual-comb, thereby inducing a photothermal response in the sample, subsequently detecting and analyzing a signal from a continuous wave (cw) laser that simultaneously illuminates the sample, wherein the photothermal response induced by the dual -combs is transferred to the continuous wave laser, which is photodetected as a signal that is analyzed to thereby provide hyperspectral imaging of the sample.

[0013] In some examples, the method further comprises translocating the sample to thereby provide hyperspectral imaging of a plurality of locations of the sample.

[0014] In some examples, the dual -comb spectroscopy is leveraged to provide a multi -frequency modulation for photothermal microscopy, wherein the modulation provides a one-to-one mapping from mid-infrared frequencies to radio frequency signals that are digitized for hyperspectral image reconstruction. 11708-004W01; UHID 2024-064

[0015] In some examples, heterodyning of comb modes leads to chemically-specific thermal excitation of the sample.

[0016] In some examples, hyperspectral information at a single spatial coordinate yields a multifrequency response that can be deconstructed into specific wavelengths and corresponding chemical composition via Fourier transformation.

[0017] In some examples, the plurality of wavelengths includes from 10 to 10,000 wavelengths.

[0018] In some examples, each of the plurality of wavelengths has a wavelength of from 2000 nm to 25,000 nm.

[0019] In some examples, each of the plurality of wavelengths is an infrared wavelength.

[0020] In some examples, each of the plurality of wavelengths has a wavelength of from 5000 to 20000 nm, or from 5000 to 12000 nm.

[0021] In some examples, the continuous wave laser has a wavelength in the ultraviolet, visible, infrared, or other region of the electromagnetic spectrum.

[0022] In some examples, the continuous wave laser has a wavelength in the visible region (e.g., from 400 to 700 nm) of the electromagnetic spectrum.

[0023] In some examples, the sample comprises a biological sample.

[0024] In some examples, the sample comprises tissue.

[0025] In some examples, the sample comprises a microbe.

[0026] In some examples, the sample comprises living cells, and the method is performed in situ on living cells.

[0027] In some examples, the sample comprises condensed phase or solid-state molecular matter.

[0028] In some examples, the method is performed in real time.

[0029] In some examples, the method images at a rate of 1000 cm'1per microsecond or less.

[0030] In some examples, the method is performed in an amount of time of 1 hour or less, 30 minutes or less, 10 minutes or less, 5 minutes or less, or 1 minute or less.

[0031] In some examples, the method is label-free.

[0032] In some examples, the method is substantially free of any added contrast agents.

[0033] In some examples, the method has submicron resolution.

[0034] In some examples, the method has subcellular resolution.

[0035] In some examples, the method has subcellular resolution, label-free chemical specificity, and high speed.

[0036] In some examples, the method is repeatedly performed over time, to thereby provide 4D (two spatial, one wavelength, and one temporal dimension) hyperspectral imaging of the sample. 11708-004W01; UHID 2024-064

[0037] In some examples, the method further comprises use of machine-learning algorithms to further analyze the hyperspectral images.

[0038] Also disclosed herein are devices for performing any of the methods described herein.

[0039] Also disclosed herein are dual -comb photothermal microscopy devices comprising: a photothermal microscope integrated with a dual -comb spectrometer; wherein the device is configured to: simultaneously irradiate at least a portion of a sample at a location with a plurality of wavelengths, the plurality of wavelengths each comprising a dual -comb, thereby inducing a photothermal response in the sample, subsequently detecting and analyzing a signal from a continuous wave (cw) laser that simultaneously illuminates the sample, wherein the photothermal response induced by the dual-combs is transferred to the continuous wave laser, which is photodetected as a signal that is analyzed to thereby provide hyperspectral imaging of the sample.

[0040] In some examples, the device comprises: a dual -comb pump laser; a probe laser; a microscope; and a detector; the dual-comb pump laser being configured to provide a dual-comb pump signal at the plurality of wavelengths; the probe laser being configured to provide a probe signal; the dual -comb pump signal and the probe signal are combined and transmitted to the microscope; the microscope being configured to receive the combined signal and focus the combined signal onto a sample, and subsequently transmit a measured signal to the detector.

[0041] In some examples, the device comprises: a dual -comb pump laser; a probe laser; a single mode fiber; a fiber coupler; a dichroic beam combiner; an all-reflective microscope; and a detector; the dual-comb pump laser being configured to provide a dual -comb pump signal at the plurality of wavelengths; the single mode fiber being configured to receive the dual-comb pump signal from the dual-comb pump laser and transmit the dual -comb pump signal to a dichroic beam combiner; the probe laser being configured to provide a probe signal; the fiber coupler being configured to receive the probe signal from the probe laser and transmit the probe signal to the dichroic beam combiner; the dichroic beam combiner being configured to receive and combine the dual-comb pump signal from the single mode fiber and the probe signal from the fiber coupler, and transmit the combined signal to the all-reflective microscope; the all-reflective microscope being configured to receive the combined signal and focus the combined signal onto a sample, and subsequently transmit a measured signal back through the dichroic beam combiner to the fiber coupler and then to a detector.

[0042] In some examples, the device further comprises one or more focusing elements (e.g., collimator, objective, lens, mirror, etc.).

[0043] In some examples, the device further comprises a movable stage, the sample being 11708-004W01; UHID 2024-064 supported by the movable stage. In some examples, the device further comprises a means for translocating the movable stage, thereby translocating the sample to thereby provide hyperspectral imaging of a plurality of locations of the sample.

[0044] In some examples, the detector comprises a photodiode.

[0045] In some examples, the device further comprises a computing device operably coupled to one or more device components.

[0046] In some examples, the device is as shown in Figure 1C.

[0047] Also disclosed herein are methods of use of any of the devices disclosed herein.

[0048] In some examples, the method comprises using the device to perform any of the methods disclosed herein.

[0049] In some examples, the method comprises in situ imaging for subcellular analysis of tissue and microbial systems, disease diagnosis, plant-based bioenergy research, forensics, art restoration, biomedicine, clinical diagnostics, bioenergy research, studying microbial and biochemical systems, monitoring spatiotemporal molecular dynamics in complex bio-systems, bioenergy-producing microbes, plant root microbiomes, biomedical diagnostics, material science, bio / health science, or a combination thereof.

[0050] In some examples, the method comprises studying microbial colonies in bioenergy production to analyzing nutrient uptake in plant roots.

[0051] In some examples, the method comprises mapping metabolic activity of microbial colonies relevant to the efficient production of bioenergy from biomass; 4D imaging of plantroot microbes in the rhizosphere that help plants with nutrient uptake, improving stress tolerance, disease resistance, and carbon fixation; analyzing spatiotemporal changes in the biochemical secretions of plant microbes such as citric and maleic acids.

[0052] Additional advantages of the disclosed devices, systems, and methods will be set forth in part in the description which follows, and in part will be obvious from the description. The advantages of the disclosed devices, systems, and methods will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosed systems and methods, as claimed.

[0053] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. 11708-004W01; UHID 2024-064 BRIEF DESCRIPTION OF THE FIGURES

[0054] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects of the disclosure, and together with the description, serve to explain the principles of the disclosure.

[0055] Figure 1 A-Figure ID. Overview of the proposed work. Concepts from (Figure 1 A) photothermal microscopy and (Figure IB) dual -frequency comb spectroscopy are combined for a new imaging modality referred to herein as a dual-comb photothermal microscope (DC-PTM). (Figure 1C) A schematic diagram of an example dual-comb photothermal microscope that employs dual mid-infrared (MIR) combs that drive the photothermal response at all wavelengths in parallel. This is subsequently read out with a visible light probe. (Figure ID) The MIR amplitude modulation arises from pairs of comb modes that beat together at Afrand its harmonics. Each beat note nAfridentifies a specific wavelength z„ that introduces a spatially and chemically-dependent photothermal response, as indicated by the different colors. Hyperspectral information at a single spatial coordinate yields a multi -frequency response that can be deconstructed into specific wavelengths and corresponding chemical composition via Fourier transformation.

[0056] Figure 2. 3D MIRSI data. The image in the x-y plane is similar to traditional microscopy, while the third (z-dimension) contains mid-infrared spectra with biochemical information. By extracting the spectrum at each pixel, molecular constituents can be identified, creating maps of molecular composition.

[0057] Figure 3A-Figure 3C. Illustration of the pump-probe measurement mechanism in photothermal microscopy. (Figure 3 A) A mid-IR pump laser is amplitude modulated, combined with a continuous wave (CW) visible probe beam via a dichroic beam combiner. Both are then focused onto a sample. (Figure 3B) In the mid-IR beam’s ’OFF’ state, the sample does not expand. When the modulation state of the mid-IR beam in ’ON’, specific biochemical constituents within the sample absorb this light, based on their unique molecular absorbance characteristics, and expand due to the photothermal effect. The probe beam measures this expansion through changes in the intensity of the backscattered light. The ON-OFF modulation frequency of the mid-IR laser is transferred to the probe beam. By modulating different mid-IR wavelengths at distinct frequencies, multiple mid-IR wavelengths can be multiplexed and faster, parallelized measurements can be obtained. Herein, the multiplexed modulation can be accomplished with a mid-IR dual -comb laser.

[0058] Figure 4 A-Figure 4F. Photothermal MIRSI provides higher image quality than prior diffraction limited MIRSI techniques. Figure 4A. Bone marrow tissue imaged at the Amide I 11708-004W01; UHID 2024-064 peak at 1650 cm'1using prior MIRSI technology. Figure 4B. The same specimen imaged using photothermal MIRSI. The imaging system reveals fine tissue details due to the higher resolution (0.5 pm) evident from the difference in image quality between Figure 4A and Figure 4B. A diagnostically important ~1 pm diameter reticulin fiber that was not visible with the prior state- of-the-art FTIR MIRSI instrumentation in Figure 4C is now clearly visible with photothermal MIRSI in Figure 4D. Cellular structures in ovarian tissue that are heavily pixelated with ~5 pm resolution FTIR in Figure 4E are clearly visible with 0.5 pm resolution photothermal MIRSI in Figure 4F.

[0059] Figure 5A-Figure 5E. (Figure 5A) Setup for dual-comb spectroscopy (DCS). Two frequency combs with slightly different repetition rates Afr=fri- f-2 are heterodyned together on a photodiode. The time domain signal corresponds to an interferogram that is formed as the two pulse trains from the frequency combs walk across each other — similar to the signal from a Fourier Transform Spectrometer, but with no moving parts. In the frequency domain, the signal is comprised of the interference of multiple pairs of optical modes that form a one-to-one mapping of the optical modes to the radio frequency (RF) domain. The Fourier transform of the photodetector signal yields the optical spectrum from which the absorption of an intervening medium can be determined. (Figure 5B) Example spectra of methanol in the fingerprint region. Blue lines are data and red is a model based on HYTRAN. (Figure 5C) and (Figure 5D) show high resolution spectra with the blue points in (Figure 5D) being at the 100 MHz comb mode resolution. (Figure 5E) Broad band DCS spectra of ethanol spanning a 700 cm'1window around 1050 cm'1. The experimental spectrum agrees well with the PNNL reference model in red.

[0060] Figure 6A-Figure 6B. (Figure 6 A) 1 GHz dual comb system with spectrum in the 3-5 pm region. The system uses chirped fiber-integrated chirped pulse amplification and nonlinear temporal compression in nonlinear fiber (AD-HNLF) to produce few-cycle pulses pulse at 1550 nm. These pulses are focused into periodically-poled lithium niobate (PPLN) to generate MIR light via intrapulse DFG. For spectroscopy, the combs are interfered on a detector (MCT) and the resulting interferogram is digitized. (Figure 6B) Spectra of the dual combs taken with a low- resolution spectrometer. Beneath the spectral envelopes there are more than 33,000 pairs of comb teeth separated by nAfr.

[0061] Figure 7A-Figure 7C. (Figure 7A) 1 GHz Dual-comb MIR microscope setup. Two MIR combs are combined, and fiber coupled then sent through an all-reflective microscope before detection with a 1 GHz mercury-cadmium-telluride detector (VIGO) and digitization (GaGe DAQ). The sample is raster- scanned in the focus. (Figure 7B) Hyperspectral image of SU-8 USAF test pattern on a silicon wafer. The spectrum at two different x,y points (red & green) is 11708-004W01; UHID 2024-064 shown. (Figure 7C) Hyperspectral image of ovarian cancer tissue with false color corresponding to the absorbance at 2920 cm'1. The absorbance curves taken on cancer tissue with two second average is shown in green, and 39 ms average in orange. The same data with a commercial Fourier Transform Infrared spectrometer (FTIR) is in red. The bottom right is an equivalent image taken using a commercial FTIR microscope with a focal plane array.

[0062] Figure 8A-Figure 8B. (Figure 8 A) A photograph of a custom-built photothermal MIRSI microscope. (Figure 8B) The photothermal frequency response curve, experimentally measured on a calibration-standard sample with a single-wavelength mid-IR laser that is square wave modulated at 100 kHz with a 5% duty cycle. The data demonstrates that the photothermal response has a bandwidth of >1.5 MHz. This bandwidth indicates that the photothermal response has a time constant on <0.7 ps and enables tailoring of the parameters of the dual-comb lasers accordingly.

[0063] Figure 9A-Figure 9C. The path to high repetition rate MIR frequency combs. (Figure 9A) Basic approach involves using nonlinear optical steps of self-phase modulation in normal dispersion fiber (P2>0), followed by anomalous dispersion (P2<0). (Figure 9B) At 100 MHz and 1 GHz, MIR spectra are generated by focusing the ultrashort pulses into a %(2)nonlinear crystal (X-tal). Shorter wavelengths are generated with PPLN (similar to Figure 6A-Figure 6B) and longer wavelengths with OP-GaP. (Figure 9C). At 10 GHz repetition rate, an electro-optic comb source is used to start with. The nonlinear pulse compression can happen in nanophotonic waveguides, like SiN, which is then followed by intrapulse difference frequency generation (DFG) in OP-GaP. The blue and red spectra are from two different phase matching conditions in the OP-GaP.

[0064] Figure lOA-Figure 10L. Photothermal MIRSI data from the custom-built instrumentation. Figure 10A and Figure 10B depict microscopy data from calibration standard PMMA beads. Figure 10C and Figure 10D showcase corresponding data acquired using our custom-built photothermal MIRSI system. Figure 10E to Figure 10L display biological imaging data from cervical and ovarian tissues, where Figure 10E to Figure 10H were captured using a commercial photothermal MIRSI system, and Figure 101 to Figure 10L feature data from the same samples measured using a custom-built photothermal MIRSI system. The data quality from the custom-built photothermal MIRSI system is comparable to, or surpasses, that of the commercial system.

[0065] Figure 11 A-Figure 1 ID. A demonstration of label -free contrast using photothermal MIRSI. Figure 11 A and Figure 1 IB display data from conventional microscopy with tissue stained (labeled) for collagen. Figure 11C and Figure 1 ID exhibit data from an adjacent tissue 11708-004W01; UHID 2024-064 section analyzed by the photothermal MIRSI system. The purple and blue contrasts from the stains correspond to high values in the MIRSI data.

[0066] Figure 12A-Figure 12D. Demonstration of photothermal MIRSI imaging and spectral analysis in living cell cultures. (Figure 12A) Microscopy image of HeLa cells. (Figure 12B, Figure 12C) Photothermal MIRSI images were captured at various time points. (Figure 12D) Mid-IR spectra of the same cells. These images and data demonstrate the capability of photothermal MIRSI for live cell imaging, a feat not achievable with FTIR or conventional MIRSI technologies.

[0067] Figure 13. Microscopic dual -comb photothermal spectra of a polystyrene bead. The figure shows the comparison of dual comb spectroscopy (blue) and dual-comb photothermal (orange). The insets (black) show independent measurements from existing literature of the spectrum of polystyrene, demonstrating the good agreement with our new dual-comb photothermal spectra.

[0068] Figure 14. Image of a polystyrene bead acquired with dual-comb photothermal microscopy. The grid is 10 x 10 pixels with a spatial resolution of 1 micron per pixel. The false color indicates the strength of the absorption at 3.5 microns.

[0069] Figure 15. Point spectra of the polystyrene bead without (above) and with averaging (below).

[0070] Figure 16. Example spectra with different samples that are relevant for material science and bio / health science.

[0071] Figure 17. Schematic illustration of an example computing device.

[0072] DETAILED DESCRIPTION

[0073] The devices, methods, and systems described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples included therein.

[0074] Before the present devices, methods, and systems are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0075] Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for 11708-004W01; UHID 2024-064 the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0076] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings.

[0077] Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

[0078] As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an agent” includes mixtures of two or more such agents, reference to “the component” includes mixtures of two or more such components, and the like.

[0079] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0080] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. By “about” is meant within 5% of the value, e.g., within 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0081] Values can be expressed herein as an “average” value. “Average” generally refers to the statistical mean value.

[0082] By “substantially” is meant within 5%, e.g., within 4%, 3%, 2%, or 1%.

[0083] “Exemplary” means “an example of’ and is not intended to convey an indication of a preferred or ideal embodiment. “Such as” is not used in a restrictive sense, but for explanatory purposes.

[0084] It is understood that throughout this specification the identifiers “first” and “second” are used solely to aid in distinguishing the various components and steps of the disclosed subject matter. The identifiers “first” and “second” are not intended to imply any particular order, amount, preference, or importance to the components or steps modified by these terms.

[0085] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or 11708-004W01; UHID 2024-064 component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0086] A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0087] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0088] As used herein the term “plurality” means 2 or more (e.g., 3 or more; 4 or more; 5 or more; 10 or more; 15 or more; 20 or more; 25 or more; 30 or more; 40 or more; 50 or more; 75 or more; 100 or more; 150 or more; 200 or more; 250 or more; 300 or more; 400 or more; 500 or more; 750 or more; 1000 or more; 1500 or more; 2000 or more; 2500 or more; 3000 or more; 4000 or more; or 5000 or more).

[0089] The term “artificial intelligence” is defined herein to include any technique that enables one or more computing devices or comping systems (i.e., a machine) to mimic human intelligence. Artificial intelligence (Al) includes, but is not limited to, knowledge bases, back- propagation bases, machine learning, representation learning, and deep learning.

[0090] The term “machine learning” is defined herein to be a subset of Al that enables a machine to acquire knowledge by extracting patterns from raw data. Machine learning techniques include, but are not limited to, logistic regression, support vector machines (SVMs), decision trees, Naive Bayes classifiers, and artificial neural networks. The term “representation learning” is defined herein to be a subset of machine learning that enables a machine to automatically discover representations needed for feature detection, prediction, or classification from raw data. Representation learning techniques include, but are not limited to, autoencoders. The term “deep learning” is defined herein to be a subset of machine learning that that enables a machine to automatically discover representations needed for feature detection, prediction, classification, etc. using layers of processing. Deep learning techniques include, but are not limited to, artificial neural network or multilayer perceptron (MLP). 11708-004W01; UHID 2024-064

[0091] Machine learning models include supervised, semi -supervised, and unsupervised learning models. In a supervised learning model, the model learns a function that maps an input (also known as feature or features) to an output (also known as target or target) during training with a labeled data set (or dataset). In an unsupervised learning model, the model learns a function that maps an input (also known as feature or features) to an output (also known as target or target) during training with an unlabeled data set. In a semi -supervised model, the model learns a function that maps an input (also known as feature or features) to an output (also known as target or target) during training with both labeled and unlabeled data.

[0092] Devices and Methods

[0093] Described herein are dual -comb photothermal microscopy devices and methods.

[0094] For example, disclosed herein are hyperspectral imaging methods, the methods comprising simultaneously subjecting a sample to dual-comb spectroscopy and photothermal microscopy. In some examples, the methods comprise simultaneously irradiating at least a portion of the sample at a location with a plurality of wavelengths, the plurality of wavelengths each comprising a dual-comb, thereby inducing a photothermal response in the sample, subsequently detecting and analyzing a signal from a continuous wave (cw) laser that simultaneously illuminates the sample. The photothermal response induced by the dual-combs is transferred to the continuous wave laser, which is photodetected as a signal that is analyzed to thereby provide hyperspectral imaging of the sample.

[0095] In some examples, the method further comprises translocating the sample to thereby provide hyperspectral imaging of a plurality of locations of the sample.

[0096] In some examples, the dual -comb spectroscopy is leveraged to provide a multi -frequency modulation for photothermal microscopy, wherein the modulation provides a one-to-one mapping from mid-infrared frequencies to radio frequency signals that are digitized for hyperspectral image reconstruction.

[0097] In some examples, heterodyning of comb modes leads to chemically-specific thermal excitation of the sample.

[0098] In some examples, hyperspectral information at a single spatial coordinate yields a multifrequency response that can be deconstructed into specific wavelengths and corresponding chemical composition via Fourier transformation.

[0099] In some examples, the plurality of wavelengths includes 10 or more wavelengths (e.g., 50 or more, 100 or more, 500 or more, 1000 or more, or 5000 or more). In some examples, the plurality of wavelengths includes 10,000 or less wavelengths (e.g., 5000 or less, 1000 or less, 500 or less, 100 or less, or 50 or less). The number of wavelengths can range from any of the 11708-004W01; UHID 2024-064 minimum values described above to any of the maximum values described above. For example, the plurality of wavelengths can include from 10 to 10,000 wavelengths (e.g., from 10 to 1000; from 1000 to 10,000; from 10 to 100; from 100 to 1000; from 1000 to 10,000; from 50 to 10,000; from 10 to 5,000; from 50 to 5,000; or from 100 to 10,000). In some examples, the plurality of wavelengths can include from 100 to 10,000 wavelengths.

[0100] In some examples, each of the plurality of wavelengths has a wavelength of 2000 nanometers (nm) or more (e.g., 3000 nm or more; 4000 nm or more; 5000 nm or more; 6000 nm or more; 7000 nm or more; 8000 nm or more; 9000 nm or more; 10,000 nm or more; 12,000 nm or more; 14,000 nm or more; 16,000 nm or more; 18,000 nm or more; 20,000 nm or more; or 22,000 nm or more). In some examples, each of the plurality of wavelengths has a wavelength of 25,000 nm or less (e.g., 24,000 nm or less; 22,000 nm or less; 20,000 nm or less; 18,000 nm or less; 16,000 nm or less; 14,000 nm or less; 12,000 nm or less; 10,000 nm or less; 9000 nm or less; 8000 nm or less; 7000 nm or less; 6000 nm or less; or 5000 nm or less). The wavelength of each of the plurality of wavelengths can independently range from any of the minimum values described above to any of the maximum values described above. For example, each of the plurality of wavelengths can have a wavelength of from 2000 to 25,000 nm (e.g., from 2000 to 14,000 nm; from 14,000 to 25,000 nm; from 2000 to 24,000 nm; from 3000 to 25,000 nm; from 3000 to 24,000 nm; from 5000 to 20,000 nm; from 3000 to 14,000 nm; or from 5000 to 12000 nm)). In some examples, each of the plurality of wavelengths has a wavelength of from 3000 nm to 14000 nm. In some examples, each of the plurality of wavelengths has a wavelength of from 5000 to 20,000 nm. In some examples, each of the plurality of wavelengths has a wavelength of 5000 to 12000 nm.

[0101] In some examples, each of the plurality of wavelengths is an infrared wavelength.

[0102] The continuous wave laser can have a wavelength in the ultraviolet, visible, infrared, or other region of the electromagnetic spectrum. In some examples, the continuous wave laser can have a wavelength in the visible region (e.g., from 400 to 700 nm) of the electromagnetic spectrum. In some examples, the continuous wave laser can comprise a red or green laser.

[0103] The sample can comprise any sample consistent with the methods and devices described herein. In some examples, the sample comprises a biological sample. In some examples, the sample comprises tissue. In some examples, the sample comprises a microbe. In some examples, the sample comprises living cells, and the method is performed in situ on living cells. In some examples, the sample can comprise condensed phase or solid-state molecular matter.

[0104] In some examples, the method is performed in real time. 11708-004W01; UHID 2024-064

[0105] In some examples, the method images at a rate of 1000 cm'1per microsecond or less (e.g., 900 cm'1per microsecond or less, 800 cm'1per microsecond or less, 700 cm'1per microsecond or less, 600 cm'1per microsecond or less, 500 cm'1per microsecond or less, 400 cm'1per microsecond or less, 300 cm'1per microsecond or less, 200 cm'1per microsecond or less, or 100 cm'1per microsecond or less).

[0106] In some examples, the method is performed in an amount of time of 1 hour or less (e.g., 30 minutes or less, 10 minutes or less, 5 minutes or less, or 1 minute or less).

[0107] In some examples, the method is label-free.

[0108] In some examples, the method is substantially free of any added contrast agents.

[0109] In some examples, the method has submicron resolution.

[0110] In some examples, the method has subcellular resolution.

[0111] In some examples, the method has subcellular resolution, label-free chemical specificity, and high speed.

[0112] In some examples, the method is repeatedly performed over time, to thereby provide 4D (two spatial, one wavelength, and one temporal dimension) hyperspectral imaging of the sample.

[0113] In some examples, the method further comprises use of machine-learning algorithms to further analyze the hyperspectral images.

[0114] Also disclosed herein are devices for performing any of the methods described herein.

[0115] Also disclosed herein are dual-comb photothermal microscopy devices comprising a photothermal microscope integrated with a dual -comb spectrometer. The device is configured to simultaneously irradiate at least a portion of a sample at a location with a plurality of wavelengths, the plurality of wavelengths each comprising a dual -comb, thereby inducing a photothermal response in the sample, subsequently detecting and analyzing a signal from a continuous wave (cw) laser that simultaneously illuminates the sample. The photothermal response induced by the dual -combs is transferred to the continuous wave laser, which is photodetected as a signal that is analyzed to thereby provide hyperspectral imaging of the sample.

[0116] In some examples, the device comprises a dual -comb pump laser, a probe laser, a microscope, and a detector. The dual-comb pump laser is configured to provide a dual-comb pump signal at the plurality of wavelengths. The probe laser is configured to provide a probe signal. The dual -comb pump signal and the probe signal are combined and transmitted to the microscope. The microscope is configured to receive the combined signal and focus the combined signal onto a sample, and subsequently transmit a measured signal to the detector.

[0117] In some examples, the microscope is an all-reflective microscope. 11708-004W01; UHID 2024-064 In some examples, the probe laser is a continuous wave probe laser.

[0118] In some examples, the device further comprises a single mode fiber, a fiber coupler, a dichroic beam combiner, a beam splitter, a beam circulator, a multi-mode fiber, a mirror, or a combination thereof

[0119] In some examples, the device further comprises a single mode fiber, a fiber coupler, a dichroic beam combiner, or a combination thereof.

[0120] In some examples, the device comprises a dual -comb pump laser, a continuous wave probe laser, a single mode fiber, a fiber coupler, a dichroic beam combiner, an all-reflective microscope, and a detector. The dual-comb pump laser is configured to provide a dual-comb pump signal at the plurality of wavelengths. The single mode fiber (e.g., a mid-infrared single mode fiber) is configured to receive the dual -comb pump signal (e.g., light) from the dual-comb pump laser and transmit the dual-comb pump signal to a dichroic beam combiner. The probe laser is configured to provide a probe signal (e.g., light). The fiber coupler is configured to receive the probe signal from the probe laser and transmit the probe signal to the dichroic beam combiner. The dichroic beam combiner is configured to receive and combine the dual -comb pump signal from the single mode fiber and the probe signal from the fiber coupler, and transmit the combined signal to the all-reflective microscope. The all-reflective microscope is configured to receive the combined signal and focus the combined signal onto a sample, and subsequently transmit a measured signal back through the dichroic beam combiner to the fiber coupler and then to a detector.

[0121] In some examples, the device further comprises one or more focusing elements (e.g., a collimator, an objective, a lens, a mirror, etc.).

[0122] In some examples, the device (e.g., the microscope) further comprises a movable stage, the sample being supported by the movable stage. In some examples, the device (e.g., the microscope) further comprises a means for translocating the movable stage, thereby translocating the sample to thereby provide hyperspectral imaging of a plurality of locations of the sample.

[0123] In some examples, the detector comprises a photodiode.

[0124] In some examples, the device further comprises a computing device operably coupled to one or more device components.

[0125] In some examples, the device comprises a device as schematically illustrated in Figure 1C. 11708-004W01; UHID 2024-064 Also disclosed herein are methods of use of any of the devices disclosed herein. In some examples, the method comprises using the device to perform any of the methods disclosed herein.

[0126] In some examples, the method comprises in situ imaging for subcellular analysis of tissue and microbial systems, disease diagnosis, plant-based bioenergy research, forensics, art restoration, biomedicine, clinical diagnostics, bioenergy research, studying microbial and biochemical systems, monitoring spatiotemporal molecular dynamics in complex bio-systems, bioenergy-producing microbes, plant root microbiomes, biomedical diagnostics, material science, bio / health science, or a combination thereof.

[0127] In some examples, the method comprises studying microbial colonies in bioenergy production to analyzing nutrient uptake in plant roots.

[0128] In some examples, the method comprises mapping metabolic activity of microbial colonies relevant to the efficient production of bioenergy from biomass; 4D imaging of plantroot microbes in the rhizosphere that help plants with nutrient uptake, improving stress tolerance, disease resistance, and carbon fixation; analyzing spatiotemporal changes in the biochemical secretions of plant microbes such as citric and maleic acids.

[0129] Figure 17 illustrates an example computing device 240 upon which examples disclosed herein may be implemented. The computing device 240 can include a bus or other communication mechanism for communicating information among various components of the computing device 240. In its most basic configuration, computing device 240 typically includes at least one processing unit 242 (a processor) and system memory 244. Depending on the exact configuration and type of computing device, system memory 244 may be volatile (such as random access memory (RAM)), non-volatile (such as read-only memory (ROM), flash memory, etc.), or some combination of the two. This most basic configuration is illustrated in Figure 17 by a dashed line 246. The processing unit 242 may be a standard programmable processor that performs arithmetic and logic operations necessary for operation of the computing device 240.

[0130] The computing device 240 can have additional features / functionality. For example, computing device 240 may include additional storage such as removable storage 250 and nonremovable storage 252 including, but not limited to, magnetic or optical disks or tapes. The computing device 240 can also contain network connection(s) 258 that allow the device to communicate with other devices. The computing device 240 can also have input device(s) 256 such as a keyboard, mouse, touch screen, antenna or other systems configured to communicate with the camera in the system described above, etc. Output device(s) 254 such as a display, 11708-004W01; UHID 2024-064 speakers, printer, etc. may also be included. The additional devices can be connected to the bus in order to facilitate communication of data among the components of the computing device 240.

[0131] The processing unit 242 can be configured to execute program code encoded in tangible, computer-readable media. Computer-readable media refers to any media that is capable of providing data that causes the computing device 240 (z.e., a machine) to operate in a particular fashion. Various computer-readable media can be utilized to provide instructions to the processing unit 242 for execution. Common forms of computer-readable media include, for example, magnetic media, optical media, physical media, memory chips or cartridges, a carrier wave, or any other medium from which a computer can read. Example computer-readable media can include, but is not limited to, volatile media, non-volatile media and transmission media. Volatile and non-volatile media can be implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data and common forms are discussed in detail below. Transmission media can include coaxial cables, copper wires and / or fiber optic cables, as well as acoustic or light waves, such as those generated during radio-wave and infra-red data communication. Example tangible, computer- readable recording media include, but are not limited to, an integrated circuit (e.g., field- programmable gate array or application-specific IC), a hard disk, an optical disk, a magnetooptical disk, a floppy disk, a magnetic tape, a holographic storage medium, a solid-state device, RAM, ROM, electrically erasable program read-only memory (EEPROM), flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices.

[0132] In an example implementation, the processing unit 242 can execute program code stored in the system memory 244. For example, the bus can carry data to the system memory 244, from which the processing unit 242 receives and executes instructions. The data received by the system memory 244 can optionally be stored on the removable storage 250 or the non-removable storage 252 before or after execution by the processing unit 242.

[0133] The computing device 240 typically includes a variety of computer-readable media. Computer-readable media can be any available media that can be accessed by device 240 and includes both volatile and non-volatile media, removable and non-removable media. Computer storage media include volatile and non-volatile, and removable and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data. System memory 244, removable storage 250, and non-removable storage 252 are all examples of computer storage media. Computer storage media include, but are not limited to, RAM, ROM, electrically erasable 11708-004W01; UHID 2024-064 program read-only memory (EEPROM), flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can be accessed by computing device 240. Any such computer storage media can be part of computing device 240.

[0134] It should be understood that the various techniques described herein can be implemented in connection with hardware or software or, where appropriate, with a combination thereof. Thus, the methods, systems, and associated signal processing of the presently disclosed subject matter, or certain aspects or portions thereof, can take the form of program code (z.e., instructions) embodied in tangible media, such as floppy diskettes, CD-ROMs, hard drives, or any other machine-readable storage medium wherein, when the program code is loaded into and executed by a machine, such as a computing device, the machine becomes an apparatus for practicing the presently disclosed subject matter. In the case of program code execution on programmable computers, the computing device generally includes a processor, a storage medium readable by the processor (including volatile and non-volatile memory and / or storage elements), at least one input device, and at least one output device. One or more programs can implement or utilize the processes described in connection with the presently disclosed subject matter, e.g., through the use of an application programming interface (API), reusable controls, or the like. Such programs can be implemented in a high level procedural or object-oriented programming language to communicate with a computer system. However, the program(s) can be implemented in assembly or machine language, if desired. In any case, the language can be a compiled or interpreted language and it may be combined with hardware implementations.

[0135] In certain examples, the system 200 comprises a computing device 240 comprising a processor 242 and a memory 244 operably coupled to the processor 242, the memory 244 having further computer-executable instructions stored thereon that, when executed by the processor 242, cause the processor 242 to: receive the electromagnetic signal captured by the instrument 230; process the electromagnetic signal to determine a property of the biphasic liquid sample 204; and output the property of the biphasic liquid sample 204.

[0136] The analysis of signals captured by the instrument can be carried out in whole or in part on one or more computing device. For example, the system may comprise one or more additional computing device.

[0137] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. 11708-004W01; UHID 2024-064

[0138] The examples below are intended to further illustrate certain aspects of the systems and methods described herein, and are not intended to limit the scope of the claims.

[0139] EXAMPLES

[0140] The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

[0141] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of measurement conditions, e.g., component concentrations, temperatures, pressures and other measurement ranges and conditions that can be used to optimize the described process.

[0142] Example 1- Dual-comb photothermal spectroscopic imaging

[0143] Current microscopy approaches lack the combination of subcellular resolution, label-free chemical specificity, and the necessary high speed to characterize complex bio-molecular systems in real-time. Described herein is a disruptive multimodal 4D microscopy platform that combines recent MIR dual -comb spectroscopy breakthroughs that enable rapid, label-free chemical identification with fundamental advances in photothermal microscopy that deliver submicron spatial resolution. By parallelizing mid-infrared (MIR) spectral measurements through a dual-comb architecture and utilizing RF modulation transfer via the photothermal effect, lOOx faster spectral data acquisition (up to 1000 cm'Vmicrosecond at 0.3 cm'1resolution) and lOx improvement in spatial resolution (to 0.5 pm) can be achieved. Applications of this new imaging modality include in situ imaging for subcellular analysis of tissue and microbial systems, disease diagnosis, plant-based bioenergy research, and monitoring spatiotemporal molecular dynamics in complex bio-systems.

[0144] Imaging technologies that can provide subcellular resolution, label-free chemical specificity, and high speed are necessary to characterize complex bio-molecular systems in realtime. Current techniques provide one or two of the three (sub-cellular resolution, label-free chemical specificity, high speed) features, but not all three within one system. The technology described herein provides all the three aforementioned features, thereby overcoming challenges with current methods. This technology has a wide array of applications in biomedicine, clinical 11708-004W01; UHID 2024-064 diagnostics, bioenergy research, studying microbial and biochemical systems, and monitoring spatiotemporal molecular dynamics in complex bio-systems, among others.

[0145] The approach described herein is capable of higher speed imaging than current midinfrared photothermal imaging systems. By parallelizing wavelength measurements, lOOx to lOOOx (or greater) spectral data acquisition speed improvement is achieved. This approach provides lOx improvement in spatial resolution (to 0.5 pm) relative to Fourier transform infrared (FTIR) spectroscopic imaging, a widely used mid-infrared imaging technology.

[0146] Here, a new approach is described where multiple wavelengths (100s to 1000s) incident on the sample at the same time and all the wavelengths can be separated using a dual -comb laser and data-processing.

[0147] The concepts central to the technology described herein are shown in Figure 1 A-Figure ID. This work combines recent advances from the field of laser physics and precision spectroscopy with photothermal microscopy. Photothermal microscopy is an emerging technology capable of delivering biochemical contrast without added contrast agents and offering super-resolution capabilities from intrinsic molecular components. Its label-free molecular specificity and subcellular resolution are extremely valuable for a broad range of biological applications, from studying microbial colonies in bioenergy production to analyzing nutrient uptake in plant roots. A challenge limiting its widespread adoption has been its slow data acquisition speed.

[0148] To address this issue, recent innovations in optical frequency combs are leveraged herein to achieve a 100 increase in data acquisition speed, thereby paving the way for its adoption across a wide range of practical applications. Optical frequency combs, which comprise a broad array of phase-coherent frequency modes, have been a revolutionary advance in precision metrology and spectroscopy. The technique of dual-comb spectroscopy (DCS), whereby two frequency combs with slightly offset mode spacing are heterodyned, provides a powerful means for one-to-one mapping of spectroscopic signals from the optical domain to the radio frequency (RF) for convenient detection and digitization. This spectroscopic analog to Fourier transform (FT) spectroscopy has the unique benefits of high-speed (10‘5s) acquisition of broad bandwidth (>1000 cm’1) and high-resolution (0.03 cm’1) frequency accurate spectra, but with no moving parts.

[0149] Combining these two concepts can lead to a disruptive multimodal 4D microscopy platform that unites MIR dual-comb spectroscopy breakthroughs and its rapid, label-free chemical identification with fundamental advances in photothermal microscopy that deliver submicron spatial resolution. 11708-004W01; UHID 2024-064

[0150] The working principle of dual-comb photothermal microscope (DC-PTM) is illustrated in Figure 1 A-Figure ID. In a dual-comb laser, each pair of comb teeth corresponds to a specific mi d-IR wavelength, with the beat frequency of adjacent comb modes producing amplitude- modulated mid-IR emission at that wavelength. This modulation is transferred to the probe laser beam via the photothermal effect, where the amplitude of modulation transfer is proportional to the sample's absorbance at the probe beam's location. Each beat frequency uniquely encodes the absorbance at a specific wavelength. The measured photodiode signal (Figure 1C) is a composite of these probe frequencies (illustrated as the black curve in Figure ID). Through a Fourier transform, this signal is then analyzed to extract the absorbance versus wavelength curve by decomposing and isolating these beat frequencies. Note that the photothermal response time is typically <0.7 ps corresponding to a bandwidth of >1.5 MHz, and the beat frequencies of the dual-comb laser are designed to reside within this bandwidth. This measurement provides the complete mid-IR spectrum for a single pixel. The size of this pixel is determined by the probe laser's wavelength (633 nm), resulting in a resolution of 0.5 pm when using a focusing objective with NA= 0.65. The point is laterally scanned across the sample to generate a three-dimensional (3D, i.e., x-y-wavelength) hyperspectral imaging dataset. Due to the 100 to 1000 speed improvement achieved with parallelized dual-comb measurements, real-time data acquisition is feasible. This capability enables the collection of 3D data of the sample at 1 -minute intervals, yielding four-dimensional (4D) measurements that encompass spatial (x-y), wavelength, and time dimensions.

[0151] Example 2 - A Frequency Comb Photothermal Microscope for Real-Time Hyperspectral Imaging

[0152] Described herein is a disruptive multimodal 4D microscopy platform that combines recent mid-infrared dual-comb spectroscopy breakthroughs that enable rapid, label-free chemical identification with fundamental advances in photothermal microscopy that deliver sub-micron spatial resolution. By parallelizing MIR spectral measurements through a dual -comb architecture and utilizing RF modulation transfer via the photothermal effect, over 100 / faster hyperspectral data acquisition can be achieved compared to current photothermal microscopes and 10 / improvement in spatial resolution can be achieved over direct mid-infrared microscopy.

[0153] A new microscope is described that combines recent advances from the field of laser physics and precision metrology with photothermal microscopy. Photothermal microscopy is an emerging technology capable of delivering biochemical contrast without added contrast agents and offering super-resolution capabilities from intrinsic molecular components. Its label-free molecular specificity and subcellular resolution are extremely valuable for a broad range of 11708-004W01; UHID 2024-064 biological applications, from studying microbial colonies in bioenergy production to analyzing nutrient uptake in plant roots. However, a challenge limiting its widespread adoption has been its slow data acquisition speed. This shortcoming is directly addressed herein by parallelizing the mid-infrared excitation in photothermal microscopy with a new infrared dual -optical frequency comb that spans the entire mid-infrared fingerprint region. An optical frequency comb comprises a broad array of phase-coherent frequency modes, and its use in precision spectroscopy and timekeeping has been revolutionary. Herein, the technique of dual-comb spectroscopy, whereby two frequency combs with slightly offset mode spacing are heterodyned, is leveraged to provide a multi -frequency modulation for photothermal microscopy. This modulation provides a one-to- one mapping from mid-infrared frequencies to radio frequency signals that are conveniently digitized for hyperspectral image reconstruction.

[0154] The devices, systems, and methods described herein can revolutionize in situ quantitative mid-infrared spectroscopic imaging, a pivotal field for label-free biological analysis. By significantly enhancing measurement speed (by over 100*) and spatial resolution (by 10x), barriers to the widespread adoption of mid-infrared spectroscopic imaging can be overcome. This technology can have broad applicability in diverse applications, including (1) mapping the metabolic activity of microbial colonies relevant to the efficient production of bioenergy from biomass, (2) 4D imaging (two spatial, one wavelength and one temporal dimension) of plant-root microbes in the rhizosphere that help plants with nutrient uptake, improving stress tolerance, disease resistance, and carbon fixation, and (3) analyzing spatiotemporal changes in the biochemical secretions of plant microbes such as citric and maleic acids. Furthermore, the technology platform can have applications in disparate fields ranging from forensics to art restoration to early disease diagnosis. This label-free technology allows for the native analysis of biological systems without added stains and the tracking of challenging small molecules such as glucose. Its photothermal microscopy enables surface measurements of thick (>1 mm) plant samples, overcoming limitations of prior methods that required thin (~10 pm) samples, thus simplifying sample preparation and broadening its application range.

[0155] Example 3 - A Frequency Comb Photothermal Microscope for Real-Time Hyperspectral Imaging

[0156] Current microscopy approaches lack the combination of subcellular resolution, label-free chemical specificity, and the necessary high speed to characterize complex bio-molecular systems in real-time. Described herein is a disruptive multimodal 4D microscopy platform that combines recent MIR dual -comb spectroscopy breakthroughs that enable rapid, label-free chemical identification with fundamental advances in photothermal microscopy that deliver sub- 11708-004W01; UHID 2024-064 micron spatial resolution. By parallelizing mid-infrared (MIR) spectral measurements through a dual-comb architecture and utilizing RF modulation transfer via the photothermal effect, 100 / faster spectral data acquisition (up to 1000 crrr' / microsecond at 0.3 cm'1resolution) and 10 / improvement in spatial resolution (to 0.5 pm) can be achieved. Applications of this new imaging modality include in situ imaging for subcellular analysis of microbial systems, plant-based bioenergy research, and monitoring spatiotemporal molecular dynamics in complex bio-systems that are critical to improving bioenergy crops.

[0157] A new multimodal imaging platform that integrates MIR dual frequency combs with photothermal microscopy is described herein. The concepts central to this effort are shown in Figure lA-Figure ID. A new microscope that combines recent advances from the field of laser physics and precision spectroscopy with photothermal microscopy is described herein. Photothermal microscopy is an emerging technology capable of delivering biochemical contrast without added contrast agents and offering super-resolution capabilities from intrinsic molecular components. Its label-free molecular specificity and subcellular resolution are valuable for a broad range of biological applications, from studying microbial colonies in bioenergy production to analyzing nutrient uptake in plant roots. A challenge limiting its widespread adoption has been its slow data acquisition speed.

[0158] To address this issue, recent innovations in optical frequency combs are leveraged herein to achieve a 100 / increase in data acquisition speed, thereby paving the way for its adoption across a wide range of practical applications. Optical frequency combs, which comprise a broad array of phase-coherent frequency modes, have been a revolutionary advance in precision metrology and spectroscopy. The technique of dual-comb spectroscopy (DCS), whereby two frequency combs with slightly offset mode spacing are heterodyned, provides a powerful means for one-to-one mapping of spectroscopic signals from the optical domain to the radio frequency (RF) for convenient detection and digitization. This spectroscopic analog to Fourier transform (FT) spectroscopy has the unique benefits of high-speed (10‘5s) acquisition of broad bandwidth (>1000 cm'1) and high-resolution (0.03 cm'1) frequency accurate spectra, but with no moving parts.

[0159] Combining these two concepts lead to a disruptive multimodal 4D microscopy platform that unites MIR dual-comb spectroscopy breakthroughs and its rapid, label-free chemical identification with fundamental advances in photothermal microscopy that deliver sub-micron spatial resolution. By parallelizing MIR spectral measurements through a dual -comb architecture and utilizing RF modulation transfer via the photothermal effect, over 100 / faster data acquisition (1000 cm'1coverage with 0.3 cm'1resolution) can be achieved compared to existing 11708-004W01; UHID 2024-064 photothermal microscopes and 10x improvement in spatial resolution (0.5 pm) can be achieved. Applications include in situ imaging for subcellular analysis of microbial systems, plant-based bioenergy research, and monitoring spatiotemporal molecular dynamics in complex bio-systems that are critical to improving bioenergy crops.

[0160] Microscopy serves as a fundamental tool in biological and biomedical research, offering insights into the structure, organization, and interactions of cellular systems. Typically, understanding these systems' functions requires introducing contrast agents like dyes, fluorophores, and stains that target specific molecular constituents for the biochemical mapping of selected biomolecules. Beyond requiring time for sample preparation, these agents can alter the environment being studied and come with issues such as photobleaching, phototoxicity, lack of quantification, and dependency on sample preparation protocols.

[0161] Label-free microscopy emerges as a pivotal innovation, addressing these issues by leveraging intrinsic biomolecules for contrast. This approach harnesses the spectroscopic properties of biomolecules to achieve label-free contrast, using technologies that span various optical wavelength ranges from ultraviolet to visible to infrared. In particular, mid-infrared wavelengths are invaluable in studying biological systems because they are sensitive to the fundamental quantum-mechanical vibrational modes of organic molecules. Mid-infrared spectroscopy, a cornerstone technique for fingerprinting isolated organic molecules in chemistry for nearly half a century, utilizes the wavelength range of 5 to 11 pm, known as “fingerprint” wavelengths. Integrating mid-infrared spectroscopy with microscopy resulted in mid-infrared spectroscopic imaging (MIRSI). It represents a recent advance that facilitates the analysis of biochemically complex molecular mixtures. This integration has proven beneficial across a broad range of biological applications, providing detailed maps of biomol ecular composition. While it enables label-free microscopy, conventional MIRSI is limited in spatial resolution to 4 to 10 pm by the diffraction limit of mid-IR light.

[0162] MIRSI delivers three-dimensional (3D) data, where the x-y plane corresponds to an image, akin to traditional microscopy. The third dimension comprises spectra that encode biochemical information. At every x-y pixel of the image, it is possible to extract the spectrum and utilize this to identify molecular constituents, thereby creating maps of microscopic variations in molecular composition (Figure 2). As such, MIRSI has proven to be an invaluable tool for a wide array of applications where spatially resolved microscopic molecular information is critical. This includes analysis of bioenergy -producing microbes, plant root microbiomes, and biomedical diagnostics. 11708-004W01; UHID 2024-064

[0163] The resolution limit of conventional microscopy systems is determined by the diffraction limit, which is given by d=A / (2 NA), where is the wavelength of light, and NA is the numerical aperture of the focusing lens. Since mid-infrared wavelengths in the fingerprint region correspond to 5-11 pm, and NA is <1 (typically 0.3-0.7), the resolution of MIRSI is 4-10 pm. However, most plant cells and microbes are 1-5 pm in diameter, which is smaller than the resolving power of MIRSI systems. This is a fundamental challenge to utilizing MIRSI to identify and track subcellular features that are important in several applications. Therefore, new “super-resolution” MIRSI methods are needed that can deliver subcellular resolutions while retaining the label -free biochemical sensitivity of MIRSI.

[0164] Recent technology innovations in photothermal microscopy have enabled superresolution MIRSI. By utilizing the physics of the photothermal effect and a new instrument architecture based on a pump-probe design, photothermal MIRSI provides label-free chemical contrast at subcellular resolutions (~0.5 pm) facilitating the visualization of previously unobservable biological features and making it possible to study new biological phenomena through direct observation at the microscopic scale. However, current photothermal MIRSI approaches are slow, hindering widespread adoption of this technology. All current photothermal imaging methods depend on commercially available mid-IR lasers. Although these lasers outperform older globar technologies, they tune across the fingerprint wavelengths at a sweep rate of 0.2 to 1 seconds per sweep. Consequently, photothermal methods using these lasers suffer from spectral data acquisition times that are too slow for real-time measurements, limiting their field deployability in many biological applications. For instance, measuring a 100 pm * 100 pm area at 0.5 pm resolution with 100 wavelength points per spectrum would take 2.2 to 6 hours. Reducing this time to 1-2 minutes could vastly increase the adoption of the technology. For example, cell division in bacterial colonies often occurs within 30 to 60 minutes, and tracking their spatiotemporal changes necessitates measurement times of 1-2 minutes. Thus, techniques to enhance photothermal MIRSI measurement speeds by 100* or greater are needed. The devices and methods described herein directly address this issue by leveraging recent advances in dualcomb laser technology to facilitate 100-1000* faster photothermal MIRSI data acquisition.

[0165] The work described herein can revolutionize in situ quantitative MIR spectroscopic imaging (MIRSI), a pivotal field for label-free biological analysis. By significantly enhancing measurement speed (by over 100*) and spatial resolution (by 10*), barriers to the widespread adoption of MIRSI can be overcome. This technology can have broad applicability in diverse applications, including (1) mapping the metabolic activity of microbial colonies relevant to the efficient production of bioenergy from biomass, (2) 4D imaging (two spatial, one wavelength 11708-004W01; UHID 2024-064 and one temporal dimension) of plant-root microbes in the rhizosphere that help plants with nutrient uptake, improving stress tolerance, disease resistance, and carbon fixation, and (3) analyzing spatiotemporal changes in the biochemical secretions of plant microbes such as citric and maleic acids. Furthermore, the technology platform can have applications in disparate fields ranging from forensics to art restoration to early disease diagnosis. This label-free technology allows for the native analysis of biological systems without added stains and the tracking of challenging small molecules such as glucose. Its photothermal microscopy enables surface measurements of thick (>1 mm) plant samples, overcoming limitations of prior methods that required thin (~10 pm) samples, thus simplifying sample preparation and broadening its application range.

[0166] Connecting photothermal microscopy and dual-comb spectroscopy. A dual-comb photothermal microscope (DC-PTM) is described herein. This involves the integration of the powerful techniques of photothermal microscopy and dual-comb spectroscopy with the goal of high-speed and label-free chemical imaging with spatial resolution <1 pm.

[0167] Working principle of photothermal microscopy. As shown in Figure 3 A-Figure 3C, photothermal microscopy utilizes a pump-probe architecture to overcome the diffraction limit of conventional MIRSI systems. Light from a mid-IR laser is modulated and combined with a continuous wave (CW) visible beam through a dichroic beam combiner and made incident on a sample. When the mid-IR beam is 'OFF,' the sample remains unexpanded. However, when the beam is 'ON,' it is absorbed by specific biochemical components within the sample, which expand due to their inherent molecular absorbance properties and the photothermal effect. This expansion causes changes in the backscattered intensity of the visible probe beam proportional to absorbance at the probe location. This absorbance measurement is performed at each pixel location at all mid-IR wavelengths to obtain hyperspectral imaging data.

[0168] How does photothermal MIRSI overcome the diffraction limit of mid-IR light? The resolution of photothermal MIRSI is determined by the spot size of the probe beam, and not by the size of the mid-IR pump beam. Since visible light at 633 nm is used as the probe, the probe resolution is d=Probe / (2NA). For the focusing objective NA=Q.65 andProbe=0.632 pm, the resolution is ~0.5 pm. This is about 10x better than the 4-10 pm diffraction-limited resolution of mid-IR (Figure 4A-Figure 4F).

[0169] Photothermal imaging can deliver molecular insights in complex biological systems. MIRSI has delivered insights into a wide array of complex biological systems. For example, it has facilitated understanding symbiotic associations in the rhizosphere between plants and microorganisms, identifying changes in nutrient distribution that promote plant growth, with 11708-004W01; UHID 2024-064 important implications for bioenergy production. It has also enabled a detailed tracking of carbon dynamics and the heterogeneous distributions of chemical constituents and functional groups of organic carbon at the root / soil interface on a micron scale. In biomedical diagnostics, MIRSI has facilitated the identification of cancer tissue subtypes, as well as the stage and grade of various cancers, including prostate, breast, and colon cancers. While these studies offered significant insights, they relied on Fourier transform infrared (FTIR) imaging — the previous generation of MIRSI technology with diffraction-limited resolution — and faced the aforementioned limitations in spatial and temporal resolution. Recent advances in photothermal MIRSI have overcome the spatial resolution challenge, enabling subcellular-level insights with significant implications. For instance, this technology allows for visualizing diagnostically critical reticulin (type III collagen) fibers in bone marrow tissue, which were invisible using FTIR MIRSI but became evident with photothermal MIRSI, as demonstrated in Figure 4A- Figure 4F.

[0170] Frequency combs and dual-comb spectroscopy . The Nob el -technology of the optical frequency comb is one of the most important developments in laser physics since the invention of the laser itself. Seminal experiments that led to the use of optical frequency combs for precision radio-frequency control of optical waveforms have been carried out. An important aspect of this revolution is the phase-coherent connection a frequency comb provides between RF and optical frequency domains - spanning a gap of 5-orders of magnitude. While frequency combs were first applied to optical clocks, they have also enabled powerful new spectroscopic techniques, including dual-comb spectroscopy (DCS).

[0171] Dual-comb spectroscopy (DCS), also referred to as linear optical sampling, is a technique for reading out spectroscopic information on a frequency comb. The technique uses two frequency combs with slightly different repetition rates (or equivalently mode spacings) f-i and fr2, such that Afr=fri - fr2. The two frequency combs are spatially overlapped and can then traverse a spectroscopic sample before having their interference detected on a photodetector. As shown in Figure 5A-Figure 5E, this multi -heterodyne has a time-domain interpretation analogous to conventional Fourier Transform spectroscopy. The delay between the electric fields from the two combs is repetitively scanned, similar to the mechanical delay between the two arms of a Michelson interferometer. The important difference is that DCS has no moving parts and does not require long delay lines, which allows it to break the size / acquisition-rate / resolution limitations of the Fourier Transform spectrometers. In the frequency domain, each pair of comb modes produce a heterodyne beat at a frequency that is a harmonic of Afr. Because the mode spacing difference of the two combs are overlayed in a Vernier-like pattern, DCS provides a 11708-004W01; UHID 2024-064 one-to-one mapping of the optical spectrum to the radio frequency domain. Straightforward digitization of the superposition of the RF beats at nAfryields an interferogram, and the Fourier transform of that interferogram can be processed to reconstruct the optical spectrum, including absorption by matter in the beam path.

[0172] Advantages of DCS include: (1) Tailored spectral resolution given by the comb tooth spacing, which can be on the order of 100 MHz to 10 GHz; (2) Broad spectral coverage over 10- 100 THz but with a single element detector and no moving parts; (3) Coherent averaging, which enables high sensitivity. Because of these benefits, DCS has been applied to an enormous range of active sensing applications, but most all have been aimed at gas-phase spectroscopy. Herein, DCS is combined with photothermal microscopy. Here, the dual comb heterodyne beats will act as the amplitude modulation of the MIR pump light to induce a wavelength and chemically specific photothermal response in the medium. Thousands of precisely controlled amplitude modulations across 10’s of THz can all occur in parallel, which can be leveraged to significantly increase the speed of photothermal microscopy.

[0173] Combining dual-comb and photothermal microscopy. In photothermal MIR SI, the photothermal effect transfers the modulation from the mid-IR laser to the probe, generating a detector signal at the modulation frequency. Currently, photothermal MIRSI instruments utilize single-wavelength, tunable lasers, leading to slow data acquisition speeds. Dual-comb lasers, however, emit mid-IR light at over 1000 wavelengths simultaneously, each with a unique modulation frequency. This enables the simultaneous measurement of absorbance properties across multiple mid-IR wavelengths by demodulating and extracting wavelength-specific absorbance at 1000 wavelengths, significantly accelerating data acquisition by up to a factor of 1000.

[0174] The working principle of DC-PTM is illustrated in Figure 1 A-Figure ID. In a dual -comb laser, each pair of comb teeth corresponds to a specific mid-IR wavelength, with the beat frequency of adjacent comb modes producing amplitude-modulated mid-IR emission at that wavelength. This modulation is transferred to the probe laser beam via the photothermal effect, where the amplitude of modulation transfer is proportional to the sample's absorbance at the probe beam's location. Each beat frequency uniquely encodes the absorbance at a specific wavelength. The measured photodiode signal (Figure 1C) is a composite of these probe frequencies (illustrated as the black curve in Figure ID). Through a Fourier transform, this signal is then analyzed to extract the absorbance versus wavelength curve by decomposing and isolating these beat frequencies. Note that the photothermal response time is typically <0.7 ps corresponding to a bandwidth of >1.5 MHz, and the beat frequencies of the dual-comb laser are 11708-004W01; UHID 2024-064 designed to reside within this bandwidth. This measurement provides the complete mid-IR spectrum for a single pixel. The size of this pixel is determined by the probe laser's wavelength (633 nm), resulting in a resolution of 0.5 pm when using a focusing objective with NA= 0.65. The point is laterally scanned across the sample to generate a three-dimensional (3D, i.e., x-y- wavelength) hyperspectral imaging dataset. Due to the 100 to 1000* speed improvement achieved with parallelized dual -comb measurements, real-time data acquisition is feasible. This capability enables the collection of 3D data of the sample at 1 -minute intervals, yielding fourdimensional (4D) measurements that encompass spatial (x-y), wavelength, and time dimensions.

[0175] Evaluation with 1 GHz dual-comb system. The DC-PTM concept can be evaluated using an existing 1 GHz dual-comb system. The basic parameter space can be mapped out and proof- of-concept experiments can be conducted with existing frequency combs that operate at 1 GHz in the 3-5 pm spectral region. This 1GHz dual-comb system has also been used recently for microsecond-scale tracking of chemical reaction pathways, and this operational dual-comb system can be interfaced with a transportable photothermal microscope. While these frequency comb parameters are not necessarily optimal in terms of mode spacing and spectral coverage, this work can be conducted to inform the future design of the optimized DC-PTM.

[0176] Figure 6A shows the layout of the existing 1 GHz dual -comb setup that employs robust 1550 nm fiber laser technology and nonlinear optics to generate MIR spectra. Experimental details are presented here as this approach is similar to what can be used for 10 GHz frequency combs in the later parts of the description herein. The power of two frequency-stabilized laser oscillators centered near 1550 nm are increased to approximately 4 W with fiber-based chirped- pulse amplification. The output of the amplifier is coupled into a single-mode optical fiber that is terminated by a few centimeters of anomalous dispersion highly nonlinear fiber. The length of HNLF is designed for the pulse to undergo soliton self-compression, resulting in a bandwidth extending from 1 pm to 2 pm and a pulse with duration as short as 8 fs. This few-cycle pulse is focused into a 1-mm thick fanout periodically poled lithium niobate crystal (PPLN), to generate up to 5 mW of light in the 3-5 pm region via intrapulse difference frequency generation. An example spectrum is shown in Figure 6B, comprising a pair of 33,310 discrete comb teeth across 33 THz (1100 cm’1).

[0177] To begin to evaluate the capabilities of the 1 GHz dual combs and highlight the capabilities, the 1 GHz dual combs were used to perform MIRSI. The results are shown in Figure 7A-Figure 7C. As noted above, MIRSI is a predecessor of photothermal microscopy. With that in mind, these experiments allow establish the basic microscope techniques, including comb generation, sample scanning and data acquisition, to be established. Here, a reflective 11708-004W01; UHID 2024-064 confocal microscope with 0.58 NA was used to image the beam onto the sample. A set of linear translation stages were used to raster scan the sample. The scan speed is limited only by the interferogram acquisition time, which is set by the repetition rate difference of the lasers. The transmitted signal is detected with an MCT detector, whose AC-coupled port is digitized at 1 GS / s. The data is streamed concurrently from the card memory into PC RAM for real-time analysis. Over one thousand interferograms can be directly averaged before phase correction needs to be employed.

[0178] In these studies, two samples were explored. The first is a USAF resolution target composed of SU-8 photoresist patterned onto a 500 pm thick Silicon wafer. Five hundred spectra (39 ms) are averaged at each pixel and apodized to 100 GHz. Point spectra, as shown in the inset of Figure 7 A, are taken at each pixel to generate the hypercube. The second sample is a crosssection of ovarian cancer tissue. As part of the capabilities to validate these results, the results of DCS point scanning microscopy were compared to hyperspectral data taken with a commercial FTIR microscope. Five hundred spectra are again averaged at each pixel and apodized to a resolution of 3.3 cm'1. Point spectra such as the one shown by the orange curve in Figure 7B are collected at each pixel, and the two C-H anti-symmetric stretch bands are visible at 2850 and 2920 cm'1. A DCS spectrum is shown by the green curve, and a comparison spectrum taken using a commercial FTIR (7.6 cm'1frequency resolution) is shown by the red curve. Apart from a broadening of the peak, good agreement is observed between the DCS and FTIR spectra. The images are generated by taking a slice through the hypercube at the peak of the 2920 cm'1band. A zoom-in of the sample is shown in Figure 7B. In these measurements, a spatial resolution of ~5 pm is estimated from the line scans across the SU-8 bars of Figure 7A.

[0179] Integrate photothermal MIR microscope with dual-comb lasers . Multiple photothermal microscopes with unique capabilities tailored for various applications have been designed and built. Current designs utilize a single wavelength, tunable mid-IR laser, which is replaced by a dual-comb laser for the DC-PTM system. Additionally, the current mid-IR laser has a wavelength range of 5-11 pm. The dual-comb laser has an extended range of 3-12 pm, which allows for the identification and tracking of lipids (spectral peaks at ~3 pm) and phosphodiesters (spectral peaks at ~12 pm), both of which are important in studying plant-microbe interactions.

[0180] Proof-of-concept experiments can be conducted with the 1 GHz dual-combs described above and a transportable photothermal MIRSI microscope. This transportable microscope shares properties with a custom-built photothermal microscope that is presented in Figure 8A. Relevant to experiments with dual-comb excitation, the temporal dynamics of the photothermal response have begun to be mapped out. This is an important consideration in designing a DC- 11708-004W01; UHID 2024-064 PTM system because the beating of multiple pairs of frequency comb teeth will be compressed into an RF window that is determined by the photothermal response. Experimental data in Figure 8B demonstrates that the photothermal response can be measured reliably across a bandwidth of >1.5 MHz.

[0181] This photothermal response is an important parameter that will be tied to the total optical bandwidth and data acquisition rate over which DC-PTM can acquire hyperspectral images. In DCS, there is a direct trade-off between the frequency resolution, which is given by the repetition rate frand the size of the optical Nyquist window Av. This relation can be expressed as Av =fr1l2Afr where Afr is the interferogram acquisition rate equal to the difference of the two laser repetition rates. As a concrete example, for fr= 1 GHz, an optical bandwidth of Av =10 THz (333 cm’1) would contain 10,000 pairs of comb teeth (10 THz / 1 GHz). To compress the corresponding 10,000 beat notes into a bandwidth of 1.5 MHz, would imply Afr= .5 MHz / 104= 150 Hz. In order to increase this data acquisition rate, dual frequency combs with higher repetition rate are instead employed, e.g. at - = 10 GHz the acquisition rate would be 10-times faster. Even higher repetition rates, in the range of 100’s of GHz to 1 THz, can be achieved with microresonator combs, however, the spectral coverage of such combs in the fingerprint region has not been established. Instead, as discussed below, dual combs with 10 GHz repetition rate with coverage from 3-12 microns can be used.

[0182] Build dual 10 GHz combs with 3-12 / ini coverage: As described above, the 3-5 pm frequency combs that operate at 1 GHz repetition rate can enable proof of the concept of DC- PTM. However, greater impact for bio-relevant imaging needs frequency comb spectra in the MIR fingerprint region. In addition, a mode spacing of 1 GHz (0.03 cm’1) provides a resolution that is excessive for biological soft-matter samples. To address these issues, new dual frequency combs in the MIR can be developed that are tailored to photothermal microscopy. Specifically, a dual comb system can be built with combs operating at 10 GHz with spectral coverage from 3- 12 pm. Electro-optic comb generation in the 1550 nm region can be utilized, which offers full control of the mode spacing and the offset frequency. To extend the spectral coverage to the MIR, efforts can build on recent work that has shown a compelling path to generating extremely broad bandwidth infrared frequency combs at high repetition rate.

[0183] Central to this approach is the use of robust 1550 nm fiber laser technology (electro-optic modulation) and waveguide nonlinear optics to first generate ultrashort pulses around 1550 nm, and then use those pulses to efficiently drive nonlinear frequency conversion (difference frequency generation) into the MIR. The basic idea for generating ultrashort pulses is shown in Figure 9A, and a significant challenge to overcome at 10 GHz repetition rate is the 11708-004W01; UHID 2024-064 corresponding lower pulse energy — 10’s of pJ at 10 GHz instead of 1 nJ at 100 MHz. To address this, the frequency comb can first be amplified to ~5W and then spectrally broadened in normal dispersion fiber (P2>0). This spectrum can then be re-compressed temporally in anomalous dispersion fiber (P2<0) to yield a pulse with higher peak power. Depending on its nonlinearity, in this second fiber the pulse can also undergo soliton self-compression to lead extremely short few-cycle pulses — as was initially demonstrated in work with 100 MHz and 1 GHz frequency combs. To create the MIR comb, this short pulse subsequently drives intrapulse difference frequency generation (DFG) in a %(2)nonlinear optical crystal, as shown in Figure 9B. Here, orientation-patterned gallium phosphide was used as the nonlinear crystal to generate light out to 12 microns. Shorter wavelengths are generated in lithium niobate.

[0184] At 10 GHz, as shown in Figure 9C, instead of optical fiber, nonlinear propagation in a highly-nonlinear SiN waveguide can be used to compress the 10 GHz pulses to approximately 10 fs before going through DFG in OP-GaP. While the data of Figure 9C demonstrate conceptual feasibility, it will be important to increase the generated MIR power to the 10-20 mW level, while still further increasing the bandwidth. Towards these goals, in-house nonlinear optical modeling can guide the design and fabrication of unique combinations of SiN waveguides and orientation patterned GaP and AlGaAs.

[0185] Capabilities of dual-comb photothermal microscope. As described above, an infrared microscope built around the dual-comb concept is being explored. This concept can be expanded and verified through the full integration of a 10 GHz dual comb system and a photothermal microscope. As shown in Figure 1 A-Figure IB, the heterodyning of comb modes leads to chemically-specific thermal excitation of the sample, which is then read out with a diffractionlimited visible wavelength probe. This new concept combines the benefits of MIR label -free chemical specificity with visible wavelength sub-micron spatial resolution — all with wavelength accuracy, high-speed acquisition, and implementation with robust telecom fiber technologies. In this section, some of the potential capabilities of the DC-PTM are described. Here, this is done by describing current photothermal imaging that has been realized. The DC-PTM can permit these imaging capabilities with 100* faster acquisition.

[0186] Furthermore, expertise in artificial intelligence (Al) informed machine-learning algorithms can be utilized to identify subcellular components such as mitochondria, nucleus, and lipid droplets and bio-molecular constituents such as amino acids, glucose, RNA, and protein secondary structures. The subcellular distributions of these cellular components are important across a wide range of applications, including monitoring the growth of microbial colonies, energy production in plant systems, and the early detection of diseases, including breast and 11708-004W01; UHID 2024-064 ovarian cancers. Further, the instrumentation described herein can be validated against the gold- standard commercial instrument for MIR spectroscopic imaging (Agilent Cary 620 / 670) and the state-of-the-art in photothermal imaging (mIRage, Photothermal Spec. Corp).

[0187] Figure lOA-Figure 10L showcases an example of the imaging capabilities with custom- built instrumentation designed to obtain photothermal MIRSI data on unstained, thick biological samples. Microscopy data from calibration standard PMMA beads are illustrated in Figure 10A and Figure 10B, while Figure 10C and Figure 10D present corresponding data obtained with a photothermal MIRSI system. Biological imaging data, focusing on cervical and ovarian tissue, are displayed in Figure 10E to Figure 10L. Figure 10E through Figure 10H were captured with a commercial photothermal MIRSI system (mIRage), whereas Figure 101 to Figure 10L show data from the same samples but measured using the custom-built photothermal MIRSI system. The quality of data produced by the custom-built photothermal MIRSI system is on par with, if not superior to, that of the commercial system, showcasing the effectiveness of the custom-built photothermal microscopy instrumentation.

[0188] Figure 11 A-Figure 1 ID showcases the ability of the custom-built photothermal MIRSI instrument to provide label-free imaging contrast. In Figure 11 A and Figure 1 IB, data from traditional microscopy is observed, where tissue has been stained to highlight collagen, demonstrating conventional labeled imaging techniques. Conversely, Figure 11C and Figure 1 ID present analysis from an adjacent tissue section using the custom photothermal MIRSI system, which employs no such stains. The areas that appear purple and blue in the stained images correlate to regions of high values in the MIRSI data, illustrating the system's ability to provide detailed contrast and information without the need for labeling.

[0189] Figure 12 A-Figure 12D presents an illustrative example of photothermal MIRSI imaging and spectral analysis in living cell cultures. A microscopy image of HeLa cells is shown in Figure 12A, while Figure 12B and Figure 12C feature photothermal MIRSI images taken at different time points to capture dynamic changes. Figure 12D provides the mid-IR spectra associated with these observations. Together, these images and data highlight the unique ability of photothermal MIRSI to conduct live cell imaging, surpassing the capabilities of traditional FTIR and conventional MIRSI technologies in capturing the intricate details of cellular processes as they occur.

[0190] The super-resolution capabilities of photothermal MIRSI and its advantages over diffraction-limited FTIR MIRSI are illustrated in Figure 4A-Figure 4F. For example, bone marrow tissue imaged at the Amide I spectral peak (1650 cm’1) using older technology appears in Figure 4A, while the same tissue imaged with photothermal MIRSI is shown in Figure 4B, 11708-004W01; UHID 2024-064 highlighting finer tissue details due to the superior 0.5 pm resolution. This enhancement enables the clear visualization of previously indiscernible features, such as a ~1 pm diameter reticulin fiber, visible with photothermal MIRSI but not with FTIR MIRSI. Similarly, cellular structures in ovarian tissue, pixelated at ~5 pm resolution with FTIR, are distinctly visible at 0.5 pm resolution with photothermal MIRSI, demonstrating its advanced capability in detailed imaging.

[0191] System Performance and Validation. A 100 pm * 100 pm area can be imaged with 0.5 pm resolution, capturing >100 wavelength points per spectrum, and hyperspectral data can be acquired within 1 minute at an SNR of >20 dB. Validation of the DC-PTM system performance can encompass three tiers of verification:

[0192] Calibration Reference Standards'. The DC-PTM system can be assessed by evaluating its resolution, contrast, SNR, spectral fidelity, and other parameters against previously reported methods. National Institute of Standards and Technology (NIST) standard polystyrene and polymethyl methacrylate (PMMA) beads, with well-documented spectroscopic properties and sizes from 0.3 to 3 pm, can be employed to confirm the imaging performance based on established metrics.

[0193] Validation by direct comparison to state-of-the-art commercial instrumentation'. The DC- PTM system performance can be benchmarked against the industry-standard commercial instrument for conventional MIRSI, namely the FTIR imaging system (Agilent Cary 620 / 670). This evaluation can utilize both calibration standards and biological tissue samples. Additionally, the system can be compared to the state-of-the-art instrumentation for photothermal MIRSI (mIRage, Photothermal Spec. Corp.).

[0194] Characterizing live-cell imaging performance'. Given that conventional FTIR imaging is unsuitable for live cell imaging, the live cell imaging capabilities of the DC-PTM system can be characterized through comparison to brightfield microscopy, applying recognized metrics. The Nikon TI2E microscope with live cell features can serve as a benchmark.

[0195] Applications of the technology in biological and environmental research (BER). The DC-PTM system's label-free biochemical specificity, subcellular resolution, and real-time data acquisition capabilities make it suitable for a broad spectrum of biological and environmental research applications. A distinctive feature of MIRSI is its ability to detect small molecules like glucose, which are difficult to monitor with traditional tagging methods involving molecular tags, dyes, or fluorophores. Moreover, Al-driven machine-learning algorithms can be employed to pinpoint subcellular structures, such as mitochondria, nuclei, and lipid droplets, as well as crucial biomolecules like amino acids, glucose, RNA, and protein secondary structures. Machine learning has previously been employed for molecular identification from MIRSI data. These 11708-004W01; UHID 2024-064 capabilities are important in several applications involving metabolic byproducts in plant cells and microbial colonies.

[0196] Mapping microbial metabolic activity for bioenergy production from biomass. Microbial pretreatment of lignocellulosic biomass is a promising route to eco-friendly biofuel production, a sustainable alternative to fossil fuels. Optimizing these bioconversion processes is important for scaling up and enhancing biofuel efficiency. DC-PTM, with its 0.5 pm spatial resolution, allows for detailed visualization of microbes and quantitatively assesses byproducts of microbial activity, including biofuels.

[0197] 4 ) imaging (two spatial, one wavelength, and one temporal dimension) with the DC- PTM system provides insights into the rhizosphere's plant-root microbes. These microbes assist in nutrient uptake, and their interactions with plant roots play a vital role in enhancing plant growth, stress tolerance, disease resistance, and carbon fixation. Understanding nutrient flows is essential for studying nutrient transport mechanisms and exploiting natural nutrient cycles for sustainable agriculture. However, detecting small, inorganic nutrients like nitrate, sulfate, and phosphate spatially and temporally in the rhizosphere remains challenging due to the limitations of current detection techniques. There is a demand for advanced chemical visualization methods to track changes in nutrient levels accurately. While FTIR imaging has contributed to this field, its lack of subcellular resolution limits detailed biochemical analysis. High-resolution DC-PTM, offering 4D imaging that delivers spatiotemporal and biochemical specificity, provides deeper insights into the interactions between plant roots and microbes. This technology can enhance the understanding of how symbiotic associations facilitate nutrient uptake, enriching the knowledge of nutrient cycling's spatial and temporal dynamics.

[0198] Imaging subcellular structures in situ: The DC-PTM system, with its 0.5 pm superresolution and live-cell imaging capabilities (Figure 12A-Figure 12D), allows for the analysis of spatiotemporal changes in biochemical secretions from plant microbes. This technology facilitates the monitoring of plant growth interactions with microbes in a label-free manner during imaging, providing insights into root exudation and carbon mobilization from soil. Metabolites produced by both microbes and plant roots, such as phenolics, organic acids (including citric and maleic acids), and antimicrobial peptides, play crucial roles in combating infection processes. This technology can be instrumental in identifying how microbes support plant cells in these processes. It enables the detailed exploration of cellular and subcellular interactions between plants and microbes, as well as the mapping of chemical pathways and their temporal variations. Understanding these pivotal metabolic processes is vital for ecological sustainability and enhancing human food production. The proposed technology's ability to image 11708-004W01; UHID 2024-064 without labels ensures that these complex biological interactions can be studied in their natural state, paving the way for breakthroughs in ecological research and agronomy.

[0199] Example 4

[0200] Photothermal Microscopy + Dual-Comb Spectroscopy. Concepts from photothermal microscopy and dual -frequency comb spectroscopy are combined herein for a new imaging modality referred to herein as a dual -comb photothermal microscope (Figure 1 A-Figure 1C). This new microscope (Figure 1C) employs dual mid-infrared (MIR) combs that drive the photothermal response at all wavelengths in parallel in contrast to serial excitation with a tunable continuous wave laser. The photothermal response from all infrared wavelengths is subsequently read out in parallel with a visible light probe. The MIR amplitude modulation arises from pairs of comb modes that beat together at and its harmonics. Each beat note nAfridentifies a specific wavelength znthat introduces a spatially and chemically-dependent photothermal response, as indicated by the different colors in Figure ID. Hyperspectral information at a single spatial coordinate yields a multi-frequency response that can be deconstructed into specific wavelengths and corresponding chemical composition via Fourier transformation.

[0201] Combining these two concepts can lead to a disruptive multimodal 4D microscopy platform that unites MIR dual-comb spectroscopy breakthroughs and its rapid, label-free chemical identification with fundamental advances in photothermal microscopy that deliver submicron spatial resolution. By parallelizing MIR spectral measurements through a dual-comb architecture and utilizing RF modulation transfer via the photothermal effect, over 100X faster data acquisition (1000 cm'1coverage with 0.3 cm'1resolution) compared to existing photothermal microscopes and 10X improvement in spatial resolution (0.5 pm) can be achieved. Applications include in situ imaging for subcellular analysis of microbial systems, plant-based bioenergy research, and monitoring spatiotemporal molecular dynamics in complex bio-systems that are critical to improving bioenergy crops.

[0202] Figure 13 shows a microscopic dual -comb photothermal spectra of a polystyrene bead. The figure shows the comparison of dual comb spectroscopy (blue) and dual -comb photothermal (orange). The modulation in the index of refraction of the polystyrene bead from the interfering mid-infrared frequency combs (at around 150 kHz) is detected with a visible light probe. The wavelength dependence of the mid-infrared absorption is one-to-one mapped to the modulation of the visible light that is detected in the time domain. Fourier transformation yields the dualcomb photothermal spectrum, which shows up as a background free spectra that is measured with the spatial resolution of the visible wavelength probe. The insets (black) show independent measurements from existing literature of the spectrum of polystyrene, demonstrating the good 11708-004W01; UHID 2024-064 agreement with our new dual-comb photothermal spectra.

[0203] Figure 14 shows an image of a polystyrene bead acquired with dual-comb photothermal microscopy. The grid is 10 x 10 pixels with a spatial resolution of 1 micron per pixel. The false color indicates the strength of the absorption at 3.5 microns. Figure 15 shows point spectra of the polystyrene bead without (above) and with averaging (below). Figure 16 shows example spectra with different samples that are relevant for material science and bio / health science.

[0204] EXEMPLARY ASPECTS

[0205] In view of the described compositions, devices, systems, and methods, herein below are described certain more particularly described aspects of the inventions. The particularly recited aspects should not, however, be interpreted to have any limiting effect on any different claims containing different or more general teachings described herein or that the “particular” aspects are somehow limited in some way other than the inherent meanings of the language and formulas literally used therein.

[0206] Example 1 : A hyperspectral imaging method, the method comprising simultaneously subjecting a sample to dual -comb spectroscopy and photothermal microscopy.

[0207] Example 2: The method of any examples herein, particularly example 1, the method comprising: simultaneously irradiating at least a portion of the sample at a location with a plurality of wavelengths, the plurality of wavelengths each comprising a dual -comb, thereby inducing a photothermal response in the sample, subsequently detecting and analyzing a signal from a continuous wave (cw) laser that simultaneously illuminates the sample, wherein the photothermal response induced by the dual-combs is transferred to the continuous wave laser, which is photodetected as a signal that is analyzed to thereby provide hyperspectral imaging of the sample.

[0208] Example 3 : The method of any examples herein, particularly example 2, wherein the method further comprises translocating the sample to thereby provide hyperspectral imaging of a plurality of locations of the sample.

[0209] Example 4: The method of any examples herein, particularly examples 1-3, wherein the dual-comb spectroscopy is leveraged to provide a multi -frequency modulation for photothermal microscopy, wherein the modulation provides a one-to-one mapping from mid-infrared frequencies to radio frequency signals that are digitized for hyperspectral image reconstruction.

[0210] Example 5: The method of any examples herein, particularly examples 1-4, wherein heterodyning of comb modes leads to chemically-specific thermal excitation of the sample.

[0211] Example 6: The method of any examples herein, particularly examples 1-5, wherein 11708-004W01; UHID 2024-064 hyperspectral information at a single spatial coordinate yields a multi -frequency response that can be deconstructed into specific wavelengths and corresponding chemical composition via Fourier transformation.

[0212] Example 7: The method of any examples herein, particularly examples 2-6, wherein the plurality of wavelengths includes from 10 to 10,000 wavelengths.

[0213] Example 8: The method of any examples herein, particularly examples 2-7, wherein each of the plurality of wavelengths has a wavelength of from 2000 nm to 25,000 nm.

[0214] Example 9: The method of any examples herein, particularly examples 2-8, wherein each of the plurality of wavelengths is an infrared wavelength.

[0215] Example 10: The method of any examples herein, particularly examples 2-9, , wherein each of the plurality of wavelengths has a wavelength of from 5000 to 20000 nm, or from 5000 to 12000 nm.

[0216] Example 11 : The method of any examples herein, particularly examples 2-10, wherein the continuous wave laser has a wavelength in the ultraviolet, visible, infrared, or other region of the electromagnetic spectrum

[0217] Example 12: The method of any examples herein, particularly examples 2-11, wherein the continuous wave laser has a wavelength in the visible region (e.g., from 400 to 700 nm) of the electromagnetic spectrum.

[0218] Example 13: The method of any examples herein, particularly examples 1-12, wherein the sample comprises a biological sample.

[0219] Example 14: The method of any examples herein, particularly examples 1-13, wherein the sample comprises tissue.

[0220] Example 15: The method of any examples herein, particularly examples 1-14, wherein the sample comprises a microbe.

[0221] Example 16: The method of any examples herein, particularly examples 1-15, wherein the sample comprises living cells, and the method is performed in situ on living cells.

[0222] Example 17: The method of any examples herein, particularly examples 1-16, wherein the sample comprises condensed phase or solid-state molecular matter.

[0223] Example 18: The method of any examples herein, particularly examples 1-17, wherein the method is performed in real time.

[0224] Example 19: The method of any examples herein, particularly examples 1-18, wherein the method images at a rate of 1000 cm’1per microsecond or less.

[0225] Example 20: The method of any examples herein, particularly examples 1-19, wherein the method is performed in an amount of time of 1 hour or less, 30 minutes or less, 10 minutes or 11708-004W01; UHID 2024-064 less, 5 minutes or less, or 1 minute or less.

[0226] Example 21: The method of any examples herein, particularly examples 1-20, wherein the method is label-free.

[0227] Example 22: The method of any examples herein, particularly examples 1-20, wherein the method is substantially free of any added contrast agents.

[0228] Example 23: The method of any examples herein, particularly examples 1-22, wherein the method has submicron resolution.

[0229] Example 24: The method of any examples herein, particularly examples 1-23, wherein the method has subcellular resolution.

[0230] Example 25: The method of any examples herein, particularly examples 1-24, wherein the method has subcellular resolution, label-free chemical specificity, and high speed.

[0231] Example 26: The method of any examples herein, particularly examples 1-25, wherein the method is repeatedly performed over time, to thereby provide 4D (two spatial, one wavelength, and one temporal dimension) hyperspectral imaging of the sample.

[0232] Example 27: The method of any examples herein, particularly examples 1-26, wherein the method further comprises use of machine-learning algorithms to further analyze the hyperspectral images.

[0233] Example 28: A device for performing the methods of any examples herein, particularly examples 1-27.

[0234] Example 29: A dual-comb photothermal microscopy device comprising: a photothermal microscope integrated with a dual -comb spectrometer; wherein the device is configured to: simultaneously irradiate at least a portion of a sample at a location with a plurality of wavelengths, the plurality of wavelengths each comprising a dual -comb, thereby inducing a photothermal response in the sample, subsequently detecting and analyzing a signal from a continuous wave (cw) laser that simultaneously illuminates the sample, wherein the photothermal response induced by the dual-combs is transferred to the continuous wave laser, which is photodetected as a signal that is analyzed to thereby provide hyperspectral imaging of the sample.

[0235] Example 30: The device of any examples herein, particularly example 28 or example 29, wherein the device comprises: a dual -comb pump laser; a probe laser; a microscope; and a detector; the dual-comb pump laser being configured to provide a dual -comb pump signal at the plurality of wavelengths; the probe laser being configured to provide a probe signal; the dualcomb pump signal and the probe signal are combined and transmitted to the microscope; the microscope being configured to receive the combined signal and focus the combined signal onto 11708-004W01; UHID 2024-064 a sample, and subsequently transmit a measured signal to the detector.

[0236] Example 31 : The device of any examples herein, particularly examples 28-30, wherein the device comprises: a dual -comb pump laser; a probe laser; a single mode fiber; a fiber coupler; a dichroic beam combiner; an all-reflective microscope; and a detector; the dual-comb pump laser being configured to provide a dual-comb pump signal at the plurality of wavelengths; the single mode fiber being configured to receive the dual -comb pump signal from the dualcomb pump laser and transmit the dual-comb pump signal to a dichroic beam combiner; the probe laser being configured to provide a probe signal; the fiber coupler being configured to receive the probe signal from the probe laser and transmit the probe signal to the dichroic beam combiner; the dichroic beam combiner being configured to receive and combine the dual-comb pump signal from the single mode fiber and the probe signal from the fiber coupler, and transmit the combined signal to the all-reflective microscope; the all-reflective microscope being configured to receive the combined signal and focus the combined signal onto a sample, and subsequently transmit a measured signal back through the dichroic beam combiner to the fiber coupler and then to a detector.

[0237] Example 32: The device of any examples herein, particularly examples 28-31, further comprising one or more focusing elements (e.g., collimator, objective, lens, mirror, etc.).

[0238] Example 33: The device of any examples herein, particularly examples 28-32, further comprising a movable stage, the sample being supported by the movable stage.

[0239] Example 34: The device of any examples herein, particularly example 33, further comprising a means for translocating the movable stage, thereby translocating the sample to thereby provide hyperspectral imaging of a plurality of locations of the sample.

[0240] Example 35: The device of any examples herein, particularly examples 28-34, wherein the detector comprises a photodiode.

[0241] Example 36: The device of any examples herein, particularly examples 28-35, further comprising a computing device operably coupled to one or more device components.

[0242] Example 37: The device of any examples herein, particularly examples 28-36, the device being as shown in Figure 1C.

[0243] Example 38: A method of use of the device of any examples herein, particularly examples 28-37.

[0244] Example 39: The method of any examples herein, particularly example 38, wherein the method comprises using the device to perform the methods of any examples herein, particularly examples 1-27.

[0245] Example 40: The method of any examples herein, particularly examples 1-27 or 38-39, 11708-004W01; UHID 2024-064 wherein the method comprises in situ imaging for subcellular analysis of tissue and microbial systems, disease diagnosis, plant-based bioenergy research, forensics, art restoration, biomedicine, clinical diagnostics, bioenergy research, studying microbial and biochemical systems, monitoring spatiotemporal molecular dynamics in complex bio-systems, bioenergyproducing microbes, plant root microbiomes, biomedical diagnostics, material science, bio / health science, or a combination thereof.

[0246] Example 41 : The method of any examples herein, particularly examples 1-27 or 38-40, wherein the method comprises studying microbial colonies in bioenergy production to analyzing nutrient uptake in plant roots.

[0247] Example 42: The method of any examples herein, particularly examples 1-27 or 38-41, wherein the method comprises mapping metabolic activity of microbial colonies relevant to the efficient production of bioenergy from biomass; 4D imaging of plant-root microbes in the rhizosphere that help plants with nutrient uptake, improving stress tolerance, disease resistance, and carbon fixation; analyzing spatiotemporal changes in the biochemical secretions of plant microbes such as citric and maleic acids.

[0248] Other advantages which are obvious and which are inherent to the invention will be evident to one skilled in the art. It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

[0249] The devices and methods of the appended claims are not limited in scope by the specific devices and methods described herein, which are intended as illustrations of a few aspects of the claims and any devices and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the devices and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative method steps disclosed herein are specifically described, other combinations of the method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

Claims

11708-004W01; UHID 2024-064CLAIMSWhat is claimed is:

1. A hyperspectral imaging method, the method comprising simultaneously subjecting a sample to dual -comb spectroscopy and photothermal microscopy.

2. The method of claim 1, the method comprising: simultaneously irradiating at least a portion of the sample at a location with a plurality of wavelengths, the plurality of wavelengths each comprising a dual -comb, thereby inducing a photothermal response in the sample, subsequently detecting and analyzing a signal from a continuous wave (cw) laser that simultaneously illuminates the sample, wherein the photothermal response induced by the dual -combs is transferred to the continuous wave laser, which is photodetected as a signal that is analyzed to thereby provide hyperspectral imaging of the sample.

3. The method of claim 2, wherein the method further comprises translocating the sample to thereby provide hyperspectral imaging of a plurality of locations of the sample.

4. The method of claim 1, wherein the dual -comb spectroscopy is leveraged to provide a multi -frequency modulation for photothermal microscopy, wherein the modulation provides a one-to-one mapping from mid-infrared frequencies to radio frequency signals that are digitized for hyperspectral image reconstruction.

5. The method of claim 1, wherein heterodyning of comb modes leads to chemically- specific thermal excitation of the sample.

6. The method of claim 1, wherein hyperspectral information at a single spatial coordinate yields a multi -frequency response that can be deconstructed into specific wavelengths and corresponding chemical composition via Fourier transformation.

7. The method of claim 2, wherein the plurality of wavelengths includes from 10 to 10,000 wavelengths; wherein each of the plurality of wavelengths has a wavelength of from 2000 nm to 25,000 nm; or a combination thereof.

8. The method of claim 2, wherein each of the plurality of wavelengths is an infrared wavelength.11708-004W01; UHID 2024-0649. The method of claim 1, wherein the sample comprises a biological sample.

10. The method of claim 1, wherein the sample comprises living cells, and the method is performed in situ on living cells.

11. The method of claim 1, wherein the method is performed in real time.

12. The method of claim 1, wherein the method is label-free.

13. The method of claim 1, wherein the method is substantially free of any added contrast agents.

14. The method of claim 1, wherein the method has submicron resolution.

15. A device for performing the methods of any one of claims 1-14.

16. A dual -comb photothermal microscopy device comprising: a photothermal microscope integrated with a dual-comb spectrometer; wherein the device is configured to: simultaneously irradiate at least a portion of a sample at a location with a plurality of wavelengths, the plurality of wavelengths each comprising a dual -comb, thereby inducing a photothermal response in the sample, subsequently detecting and analyzing a signal from a continuous wave (cw) laser that simultaneously illuminates the sample, wherein the photothermal response induced by the dual -combs is transferred to the continuous wave laser, which is photodetected as a signal that is analyzed to thereby provide hyperspectral imaging of the sample.

17. The device of claim 15, wherein the device comprises: a dual -comb pump laser; a probe laser; a microscope; and a detector; the dual -comb pump laser being configured to provide a dual-comb pump signal at the plurality of wavelengths; the probe laser being configured to provide a probe signal; the dual -comb pump signal and the probe signal are combined and transmitted to the microscope;11708-004W01; UHID 2024-064 the microscope being configured to receive the combined signal and focus the combined signal onto a sample, and subsequently transmit a measured signal to the detector.

18. The device of claim 15, wherein the device comprises: a dual -comb pump laser; a probe laser; a single mode fiber; a fiber coupler; a dichroic beam combiner; an all-reflective microscope; and a detector; the dual-comb pump laser being configured to provide a dual-comb pump signal at the plurality of wavelengths; the single mode fiber being configured to receive the dual -comb pump signal from the dual-comb pump laser and transmit the dual -comb pump signal to a dichroic beam combiner; the probe laser being configured to provide a probe signal; the fiber coupler being configured to receive the probe signal from the probe laser and transmit the probe signal to the dichroic beam combiner; the dichroic beam combiner being configured to receive and combine the dual -comb pump signal from the single mode fiber and the probe signal from the fiber coupler, and transmit the combined signal to the all-reflective microscope; the all-reflective microscope being configured to receive the combined signal and focus the combined signal onto a sample, and subsequently transmit a measured signal back through the dichroic beam combiner to the fiber coupler and then to a detector.

19. The device of claim 15, the device being as shown in Figure 1C.

20. A method of use of the device of any one of claims 16-19.

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