PARS imaging method

Abstract

1. A method for visualizing details of a sample, the method comprising: directing an excitation beam to an excitation location below the surface of the sample to generate a signal in the sample; directing an interrogation beam toward the excitation location on the sample; directing a signal-enhancing beam to the sample to increase the temperature of a portion of the sample by 5 Kelvin or less compared to the temperature of the portion of the sample in the absence of the signal-enhancing beam; and detecting a portion of the interrogation beam returning from the sample, the portion representing the generated signal.

Description

[Technical Field]

[0001] The present invention relates to the field of optical imaging, and in particular to laser-based methods and systems for non-contact imaging of samples such as engineered materials or biological tissues in vivo, ex vivo, or in vitro. [Background technology]

[0002] Photoacoustic imaging techniques are a powerful family of modalities capable of visualizing the inherent intrinsic optical absorption contrast within light-scattering media. In a typical photoacoustic architecture, nanosecond or picosecond laser pulses are directed toward a sample to generate thermoelastically induced acoustic waves, which are then observed and reconstructed to form an image of the optical absorption distribution. By carefully selecting the wavelength of the excitation light source, the absorption contrast of specific biomolecules can be targeted. For example, 532 nm is a commonly used wavelength for targeting hemoglobin. These systems have proven effective in recovering clinically relevant biological structures from within living tissues. Some examples include vasculature from macrovasculature to microvasculature, cellular structures, and lipid-rich plaques, along with functional imaging, including visualization of blood oxygen saturation.

[0003] Photoacoustic imaging can be divided into two main categories: photoacoustic tomography (PAT) uses reconstruction-based imaging, and photoacoustic microscopy (PAM) uses focusing-based imaging. In PAT, an unfocused light beam excites a region of interest, and an array of transducers measures the generated ultrasound at multiple locations. PAM uses raster scanning of optical and acoustic foci to directly form images from the recorded depth-resolved signals. PAM is further classified into optically resolved PAM (OR-PAM), where the optical focusing is much tighter than the acoustic focusing, and acoustically resolved PAM (AR-PAM), where the acoustic focusing is tighter. In all three embodiments, the acoustic signal is typically collected via an acoustically coupled transducer or other acoustic or acousto-optical resonator. In all cases, the photoacoustic signal (generally associated with pressure generation and temperature in the sample) can be recorded to form an image representing the optical absorption of the sample at the excitation wavelength, with the amplitude of the various recorded peaks suggesting localized optical absorption.

[0004] However, conventional photoacoustic techniques require physical coupling to the sample, making them unsuitable for a wide variety of clinical applications, such as ophthalmic imaging, intraoperative imaging, wound healing monitoring, and many endoscopic procedures.

[0005] A recently reported photoacoustic technique known as photoacoustic remote sensing (PARS) microscopy (Patent Documents 1 and 2) overcomes many of these sensitivity issues through a novel detection mechanism. PARS allows for direct detection of the excited photoacoustic field, rather than detecting the acoustic pressure at an external surface after it has propagated from its source. This detection is achieved by monitoring changes in the optical properties of the material that occur simultaneously with photoacoustic excitation. These changes then encode various salient material properties, such as optical absorption, physical target dimensions, and constituent chromophores. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US Patent Application Publication No. 2016 / 0113507 [Patent Document 2] US Patent Application Publication No. 2017 / 0215738 Summary of the Invention

[0007] According to one aspect, a thermally enhanced photoacoustic remote sensing (TE-PARS) system is provided for imaging subsurface structures in a sample that provide absorption contrast within the sample. A TE-PARS system comprises an excitation beam or collection of beams configured to generate a PARS signal in the sample at an excitation location or collection of locations; a signal-enhancing beam or collection of beams configured to modify the observation or generation of temperature and pressure signals incident on the sample at the excitation location or collection of locations or an interrogation location or collection of locations; an interrogation beam or collection of beams incident on the sample at the interrogation location or collection of locations; an optical system or collection of systems that focuses or directs the excitation beam or collection of beams to a first focal point or collection of foci, the signal-enhancing beam at a second focal point or collection of foci, the interrogation beam at a third focal point or collection of foci, and the first, second, and third focal points or collections of foci are below the surface of the sample; a photodetector or collection of photodetectors for detecting a portion of the interrogation and / or signal-enhancing beam returning from the sample indicative of the generated PARS signal; and a processing unit for interpreting the collected results.

[0008] An embodiment of TE-PARS may include several collections of PARS signal enhancement pathways that can also function as detection pathways. According to another aspect, a temperature-sensitive photoacoustic remote sensing (TS-PARS) system is provided for detecting the temperature of subsurface structures in a sample.

[0009] The TS-PARS system comprises an excitation beam or collection of beams configured to generate a PARS signal in the sample at an excitation location or collection of locations; an interrogation beam or collection of beams incident on the sample at an interrogation location or collection of locations; an optical system or collection of systems that focuses or directs the excitation beam or collection of beams to a first focal point or collection of foci and the interrogation beam to a second focal point or collection of foci, the first and second focal points or collections of foci being below the surface of the sample; a photodetector or collection of photodetectors for detecting a return portion of the interrogation beam and / or the signal-enhancing beam from the sample; and a thermal processing unit for interpreting the collected results.

[0010] According to another aspect, a super-resolution photoacoustic remote sensing (SR-PARS) system is provided that images subsurface structures in a sample at a resolution greater than that defined by the optical diffraction limit by exploiting optical absorption contrast in the sample.

[0011] An SR-PARS system comprises an excitation beam or collection of beams configured to generate a PARS signal in the sample at an excitation location or collection of locations; an interrogation beam or collection of beams incident on the sample at an interrogation location or collection of locations; an optical system or collection of systems that focuses or directs the excitation beam or collection of beams to a first focal point or collection of foci and the interrogation beam to a second focal point or collection of foci, the first and second focal points or collections of foci being below the surface of the sample; a photodetector or collection of photodetectors for detecting a portion of the interrogation beam or collection of beams returning from the sample indicative of the generated PARS signal; and a super-resolution processing unit for interpreting the collected results.

[0012] According to another aspect, a spectrally enhanced photoacoustic remote sensing (SE-PARS) system for imaging subsurface structures in a sample is provided that utilizes color effects and spatial filtering methods to encode spatial information in the sample.

[0013] The SE-PARS system comprises an excitation beam or collection of beams configured to generate a PARS signal in the sample at an excitation location or collection of locations; an interrogation beam or collection of beams incident on the sample at an interrogation location or collection of locations; an optical system or collection of optical systems that disperses the interrogation beam based on its wavelength or spatial positioning; an optical system or collection of systems that focuses or directs the excitation beam or collection of beams to a first focal point or collection of focal points and the interrogation beam to a second focal point or collection of focal points, the first and second focal points or collection of focal points being below the surface of the sample; a portion of the interrogation beam or collection of beams returning from the sample indicative of the generated PARS signal; an optical system or collection of optical systems that recombines the interrogation beams based on their wavelength or spatial positioning; a photodetector or collection of photodetectors for detecting the returning portion of the interrogation beam; and a processing unit for interpreting the collected results.

[0014] According to another aspect, a smart detection photoacoustic remote sensing (SD-PARS) system for imaging subsurface structures in a sample is provided that exploits wavelength-specific absorption to encode or suppress spatial information in the sample.

[0015] An SD-PARS system includes an excitation beam or set of beams configured to generate a PARS signal in the sample at an excitation location or set of locations; an interrogation beam or set of beams incident on the sample at an interrogation location or set of locations; an optical system or set of systems that focuses or directs the excitation beam or set of beams to a first focal point or set of foci and the interrogation beam to a second focal point or set of foci, where the first and second focal points or sets of foci are below the surface of the sample; a portion of the interrogation beam or set of beams returning from the sample that represents the generated PARS signal; and a processing unit for interpreting the collected results. SD-PARS differs from standard PARS devices in that it can purposefully select detection wavelengths to suppress photoacoustic or PARS signals originating from specific regions. For example, if a desired target is located immediately adjacent to a large blood vessel (which could otherwise overwhelm the signal from the desired target), the detection wavelength may be selected to suppress the signal from the vessel by populating the absorbed energy level prior to detection. A suppressed signal may be suppressed by about 1% to about 100% compared to an unsuppressed signal. In other examples, a suppressed signal may be suppressed by about 5% to about 95%, about 10% to about 90%, about 25% to about 75%, or any other suitable percentage compared to an unsuppressed signal.

[0016] Embodiments of TA-PARS, TE-PARS, TS-PARS, SE-PARS, SD-PARS and SR-PARS may comprise several collections of detection paths for PARS, TE-PARS, SE-PARS, SD-PARS, TA-PARS, TS-PARS and SR-PARS.

[0017] PARS pathways may include, but are not limited to, conventional PARS as described in U.S. Pat. No. 10,117,583, non-coherent PARS as described in U.S. Pat. No. 10,327,646, camera-based PARS as described in U.S. Pat. No. 10,627,338, coherence-gated PARS as described in International Publication No. WO2019 / 145764, single-source PARS as described in International Patent Application No. PCT / IB2020 / 051804, filed March 3, 2020, and PARS extensions as described in International Patent Application No. PCT / IB2019 / 061131, filed December 19, 2019, each of which is incorporated herein by reference in its entirety.

[0018] According to another aspect, a dual-modality photoacoustic remote sensing combined with optical coherence tomography (PARS-OCT) system is provided for imaging subsurface structures in a sample, providing tissue absorption and scattering contrast.

[0019] The PARS subsystem of PARS-OCT comprises an excitation beam or collection of beams configured to generate pressure and temperature signals in the sample at an excitation location or collection of locations; an interrogation beam or collection of beams incident on the sample at an interrogation location or collection of locations; an optical system or collection of systems that focuses or directs the excitation beam or collection of beams to a first focal point or collection of foci and the interrogation beam to a second focal point or collection of foci, the first and second focal points or collections of foci being below the surface of the sample; a photodetector or collection of photodetectors for detecting a portion of the interrogation beam or collection of interrogation beams returning from the sample indicative of the generated pressure and temperature signals; and a processing unit for interpreting the collected results.

[0020] The OCT subsystem of PARS-OCT comprises a light source or collection of light sources, an interferometer or collection of interferometers each having a single or multiple sample arm and a reference arm, where the sample arm directs a sample portion of the beam or collection of beams to a third focus and the reference arm directs a reference portion of the beam or collection of beams into a path of known length, a photodetector or collection of photodetectors for detecting a portion of light returning from the sample arm indicative of scattering collected by the sample arm, a portion of light returning from the reference arm indicative of scattering collected by the reference arm, and the portions returning from the sample arm and the reference arm, and a processing unit for interpreting the collected results.

[0021] According to another aspect, an endoscopic photoacoustic remote sensing combined with optical coherence tomography (EPARS-OCT) device is provided that provides absorption and scattering information of a sample.

[0022] The PARS subsystem of the PARS-OCT comprises an excitation beam or collection of beams configured to generate pressure and temperature signals in the sample at an excitation location or collection of locations; an interrogation beam or collection of beams incident on the sample at an interrogation location or collection of locations; a fiber optic cable or collection of cables having an input end and a detection end; an optical system or collection of systems that focuses or directs the excitation beam or collection of beams to a first focal point or collection of foci and the interrogation beam to a second focal point or collection of foci, the first and second focal points or collections of foci being below the surface of the sample; a photodetector or collection of photodetectors for detecting portions of the interrogation beam or collection of interrogation beams returning from the sample indicative of the generated pressure and temperature signals; and a processing unit for interpreting the collected results.

[0023] The OCT subsystem of PARS-OCT includes a light source or collection of light sources, an interferometer or collection of interferometers each having a single or multiple sample arm and a reference arm, where the sample arm directs a sample portion of the beam or collection of beams through a fiber optic cable or collection of cables having an input end and a detection end to a third focus, and the reference arm directs a reference portion of the beam or collection of beams through a path of known length, a photodetector or collection of photodetectors for detecting a portion of the light returning from the sample arm indicative of the scatter collected by the sample arm, a portion of the light returning from the reference arm indicative of the scatter collected by the reference arm, and the portions returning from the sample arm and the reference arm, and a processing unit for interpreting the collected results.

[0024] An embodiment of PARS-OCT may comprise some collection of PARS and OCT detection paths. An embodiment of EPARS-OCT may comprise some collection of PARS and OCT detection paths.

[0025] PARS detection pathways include conventional PARS, as described in U.S. Patent No. 10,117,583, issued November 6, 2018; non-coherent PARS, as described in U.S. Patent No. 10,327,646, issued June 25, 2019; camera-based PARS, as described in U.S. Patent No. 10,627,338, issued April 21, 2020; and coherent PARS, as described in U.S. Patent Application Publication No. 2020 / 0359903, published November 19, 2020. These may include, but are not limited to, Sense-Gate PARS, Single-Source PARS as described in International Publication No. WO2020 / 188386 published September 24, 2020, PARS extensions as described in International Patent Application No. PCT / IB2019 / 061131 filed December 19, 2019, TA-PARS, TE-PARS, TS-PARS, SR-PARS, SE-PARS, and SD-PARS. All of the patent applications and patents mentioned herein are incorporated by reference in their entirety.

[0026] The OCT detection path can include, but is not limited to, known implementations of TD-OCT, SS-OCT, SD-OCT, or other OCT embodiments. For example, a TD-OCT system with a broadband light source, a scanning reference path delay, and a photodetector. In another example, an SS-OCT system with a tunable narrowband light source, a fixed reference path delay, and a photodetector. In yet another example, an SD-OCT system with a broadband light source, a fixed reference path delay, and a spectrometer.

[0027] Any combination of the PARS or OCT pathways listed above can be envisioned, such as a specific PARS-OCT, but any such combination or obvious extension can also be fabricated.

[0028] Novel PARS signal extraction algorithms can exploit a variety of absorption-induced modulation effects, including but not limited to modulation of material reflectivity, scattering, polarization, phase accumulation, nonlinear absorption, and nonlinear scattering. These algorithms can be used in multiple acquisitions to unmix constituent chromophores from within a sample by varying excitation, detection, and signal-enhancement beam properties, including but not limited to, variations in wavelength, pulse width, power, energy, coherence length, repetition rate, and exposure time. These properties can take any value appropriate to the task. Typical ranges can include wavelength (nanometers to micrometers (microns)), pulse width (attoseconds to milliseconds), power (attowatts to watts), pulse energy (attojoules to joules), coherence length (nanometers to kilometers), and repetition rate (continuous wave to gigahertz). In contrast to the signal-enhancing beam, which may only need to produce thermal perturbations and therefore can be implemented using relatively long pulse widths (nanoseconds or longer), the excitation beam can generally be implemented using short pulse widths (nanoseconds and subnanoseconds) intended to generate a PARS signal impulse response. For example, the excitation beam pulse width can be greater than or less than 1 ns, while the signal-enhancing beam pulse width can be much longer. In a given system architecture, the excitation, detection, and signal-enhancing wavelengths can be implemented using different wavelengths or polarization states to provide a means of optical differentiation between the respective paths.

[0029] Other novel PARS signal extraction algorithms can exploit unique features of the collected time-domain behavior to improve signal fidelity, enhance image contrast, recover information about the shape, size, and dimensions of the sample, or perform multiplexed / functional imaging. Processing techniques can include, but are not limited to, lock-in amplification (both software- and hardware-based implementations), machine learning methods, extensive feature extraction, multidimensional decomposition, and frequency content-based feature extraction and signal processing methods.

[0030] PARS can be used to unmix the composition of multiple targets based on the targets' absorption, temperature, polarization, frequency, phase, nonlinear absorption, structure, velocity, fluorescence, nonlinear scattering, and scattering content.

[0031] PARS can also be used to unmix the size, shape, features, and dimensions of multiple targets based on their absorption, temperature, polarization, frequency, phase, nonlinear absorption, nonlinear scattering, and scattering content.

[0032] PARS signals can be used to unmix targets with their absorption content, scattering content, fluorescence, polarization content, frequency content, and phase content by utilizing different wavelengths, different pulse widths, different coherence lengths, repetition rates, laser exposure times, and laser fluences.

[0033] The PARS signal can be dominated by the generated pressure and can also be analyzed based on the PARS signal amplitude / intensity, frequency content, content related to polarization changes, fluorescence, second harmonic generation, and phase fluctuations to derive information.

[0034] PARS signals can be governed by the temperature at which they are generated and can also be analyzed to derive information based on their amplitude / intensity, fluorescence, frequency content, second harmonic generation, content related to polarization changes, and phase fluctuations.

[0035] The PARS system can be configured to capture any light absorption-induced fluctuations in the sample, which may include, but are not limited to, pressure signals, temperature signals, ultrasound signals, and autofluorescence signals.

[0036] Portions of the interrogation, signal enhancement, excitation, or autofluorescence from the sample can be collected to form an image. These signals can be used to unmix the size, shape, features, dimensions, properties, and composition of the sample.

[0037] In a given architecture, any portion of the light returning from the sample can be collected, such as the detection beam, excitation beam, or thermally enhanced beam. The returning light can be analyzed based on wavelength, phase, polarization, etc. to capture any absorption-induced signals, including pressure, temperature, and optical emission. In this way, PARS can simultaneously capture, for example, the scattering, autofluorescence, and polarization contrast resulting from each detection, excitation, and thermally enhanced light source. Furthermore, the PARS laser source can be specifically chosen to emphasize these different contrast mechanisms.

[0038] Other aspects will become apparent from the following description and claims. As used herein, the word "comprising" is used in its open-ended sense to mean that the items that follow the word are included, but do not exclude items not specifically mentioned. Referring to an element by the indefinite article "a" does not require that there be one, and only one, of that element.

[0039] The scope of the following claims should not be limited by the preferred embodiments shown in the above examples and drawings, but should be accorded the broadest interpretation consistent with the specification as a whole. [Brief explanation of the drawings]

[0040] [Figure 1] Schematic diagram of the TE-PARS system. [Figure 2] Schematic of the TE-PARS system including PARS excitation and PARS detection. [Figure 3] Schematic diagram of the SR-TE-PARS system. [Figure 4] FIG. 1 shows a possible implementation of TE-PARS combined with other modalities. [Figure 5] FIG. 1 is a diagram showing the signal processing path of a TE-PARS signal. [Figure 6] FIG. 1 illustrates an embodiment of multiple PARS pump lasers. [Figure 7] FIG. 1 illustrates an embodiment of multiple PARS detection lasers. [Figure 8] FIG. 1 illustrates an embodiment of multiple PARS signal enhancement lasers. [Figure 9] FIG. 1 illustrates an embodiment of multiple excitation lasers, multiple detection lasers, and multiple signal enhancement lasers. [Figure 10] Diagram showing an example of a TE-PARS system layout. [Figure 11] Figure showing yet another example of a TE-PARS system layout. [Figure 12] Figure showing yet another example of a TE-PARS system layout. [Figure 13] Diagram showing an example of combining TE-PARS with other modalities. [Figure 14] FIG. 1 shows an example of a TE-PARS imaging method using sequential thermal enhancement. [Figure 15] An example of TE-PARS imaging using pulsed thermal enhancement. [Figure 16] FIG. 1 illustrates signal generation in a PARS system. [Figure 17] 1 illustrates the TE-PARS signal unmixing method. [Figure 18] Diagram showing the signal flow for super-resolution imaging. [Figures 19a-19k] 10A and 10B are diagrams showing different spot arrangements. [Figure 20] Diagram showing an example of a TE-PARS-based functional imaging method. [Figure 21] FIG. 1 shows an example of a PARS excitation sequence and the resulting PARS signal. [Figure 22] FIG. 1 illustrates an embodiment of the SE-PARS system. [Figure 23] Schematic diagram of the PARS-OCT system. [Figure 24a-24b]Figure 1 shows an embodiment of the imaging arm of the PARS-OCT system. [Figure 25] FIG. 1 illustrates an embodiment of a PARS-OCT system. [Figures 26a-26c] Diagram showing the mechanism of the OCT imaging system. [Figure 27] Schematic diagram of the OCT signal data processing pathway. [Figure 28] FIG. 1 shows an exemplary OCT image (B-scan) of a human retina. [Figure 29a-29b] Diagram showing the PARS imaging system setup. [Figure 29c] Diagram showing the PARS imaging system setup. [Figure 30] Schematic diagram of the PARS signal data processing pathway. [Figure 31a-31b] FIG. 1 shows an exemplary PARS image. [Figure 32] Diagram showing an exemplary system layout for PARS-OCT. [Figure 33] Diagram showing another exemplary system layout for EPARS-OCT. [Figure 34] Diagram showing yet another exemplary system layout for PARS-OCT. [Figure 35] Diagram showing an exemplary system layout for multi-modal PARS-OCT. [Figure 36] Diagram showing yet another exemplary system layout for PARS-OCT. [Figure 37] Schematic diagram of TS-PARS. [Figure 38] FIG. 1 illustrates exemplary combinations between TE-PARS, TS-PARS, SR-PARS, and other modalities. [Figure 39] Diagram showing PARS-OCT featuring a thermally enhanced light source. [Figure 40] Schematic showing a PARS system in which the optical subsystem is mechanically scanned around the sample. [Figure 41] FIG. 10 is a diagram showing an example of TS-PARS detection processing. [Figure 42]FIG. 10 is a diagram showing a scatter subtraction processing path. [Figure 43a-43b] 10A-10C show examples of various constituent light beams demonstrating that some paths can be performed in transmission mode. [Figures 44a-44k] Diagram showing different spot placements for the PARS-OCT system. [Figure 45] FIG. 10 is a diagram showing another example of a scatter subtraction processing path. [Figure 46] Figure showing an example of SD-PARS combined scattering and absorption contrast visualization. [Figure 47] Figure 1 shows an example of the different contrasts obtained by changing the wavelength of the SD-PARS detection light source. [Figure 48] Diagram showing an example of an autofluorescence-sensitive total absorption PARS (TA-PARS) architecture. [Figure 49] Diagram showing an example of visualization afforded by the autofluorescence-sensitive total absorption PARS (TA-PARS) architecture. [Figure 50] A diagram showing an example of TA-PARS. DETAILED DESCRIPTION OF THE INVENTION

[0041] Reference will now be made in detail to examples of the present disclosure, which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. In the following description, relative terms such as "about," "substantially," and "approximately" are used to indicate possible variations in stated numerical values.

[0042] Because the PARS device utilized two optical beams capable of confocal configuration, the spatial resolution of the imaging technique may be defined as excitation-defined (ED) or interrogation-defined (ID), depending on which beam forms a tighter focus at the sample. This aspect can also facilitate imaging deeper targets beyond the limitations of conventional contact-based OR-PAM devices. This imaging can be achieved by utilizing deep-penetrating (long transport mean free path) detection wavelengths, such as shortwave infrared (e.g., 1310 nm or 1700 nm), which can achieve spatial resolution to depths superior to those achieved with a given excitation (e.g., 532 nm) in highly scattering media such as biological tissue. It is worth noting that when more than two beams are used, such that the system consists of more than two foci at the sample, a clear expansion of these components is expected. For example, if an additional beam is added that amplifies the signal within the beam's focal region, that beam can also contribute to defining the expected resolution of the system.

[0043] The PARS systems described herein are fundamentally different from previously described PARS systems. These devices utilize novel physical discoveries to significantly improve the capabilities of previous reports. These PARS systems utilize the temperature dependence of materials to enhance the absorption contrast available for PARS acquisition (by approximately an order of magnitude, depending on the material being tested) and improve sensitivity. Material saturation effects are exploited to surpass the resolution achievable with diffraction-limited optics alone. Modern spatial-spectral coding techniques are integrated, enabling orders of magnitude improvements in acquisition efficiency and imaging speed. This speed improvement results from reducing the scanning dimensionality, enabling two-dimensional scans to be completed in as little as one dimension, or three-dimensional scans to be completed in as little as two dimensions. Similarly, novel processing techniques are described for use during multiplexed acquisition, such as separating chromophores using various beam characteristics (wavelength, pulse width, power, coherence length, repetition rate, exposure time, signal frequency content, and optical saturation, scattering, polarization, and phase effects, to name a few), and for extracting additional information from time-domain signals.

[0044] Possible mechanisms include pressure-induced refractive index modulation, thermally induced refractive index modulation, surface vibration, and scatterer position modulation due to restricted thermal expansion. Changes in refractive index due to increased temperature and pressure can affect the scattering of light. In some cases, the detected PARS signal may be dominated by the pressure and / or temperature at which it is generated.

[0045] Many of these novel aspects utilize disparate physical effects, and so these additions are emphasized. First, in a highly simplified abstraction, the pressure p0 produced by a sufficiently short light pulse of fluence φ can be defined by the following relation:

[0046] p0=Γμ a Φ where μ arepresents the optical absorption in the sample, and Γ is known as the Grueneisen parameter, which indicates the ratio of material properties. However, this pressure increase is accompanied by a temperature increase due to exposure to multiple beams in the PARS system. This temperature increase is related to the Grueneisen parameter Γ and the optical absorption μ a This affects pressure generation by changing both the pressure and the temperature. This implies that the efficiency of pressure generation can be varied with temperature, T, in this case. In PARS devices, the pressure increase p0 is typically measured as a change in scattering or reflectance from the excitation region. This change in light scattering is measured as a change in the local refractive index due to pressure p0, a quantity δn that follows the relationship for a given detection wavelength λ: eo This may be caused by an elasto-optic effect that is modulated only by

[0047]

number

[0048] S PARS (μ a ,φ,λ,T)∝δn eo +δn T Thus, intensity-modulated PARS signals retain their dependence not only on optical absorption and incident excitation fluence, but also on detection laser wavelength, fluence, and sample temperature. PARS signals can also arise from other phenomena, such as scatterer position modulation and surface vibration. Similar analogs may exist for PARS devices that utilize other modulating optical properties, such as intensity, polarization, frequency, phase, fluorescence, nonlinear scattering, and nonlinear absorption.

[0049] Because material properties depend on ambient temperature, there is a corresponding temperature dependence in the PARS signal. These temperature dependences can facilitate temperature sensing with a PARS system. Temperature dependence may also facilitate thermally enhanced photoacoustic remote sensing (TE-PARS) techniques. TE-PARS systems may use a signal-enhancing light source in addition to the PARS excitation and detection light sources. The signal-enhancing light source deposits light energy that alters local material properties and thus the induced pressure modulation.

[0050] At some intensity levels, further saturation effects can also be exploited. For example, the light absorption μ a is the intrinsic saturation intensity I sat It saturates at an intensity level I0 approaching .

[0051]

number

[0052] The above mechanisms suggest important sources of scattering position modulation or scattering cross-section modulation, which can be easily measured when the probe beam is focused to detect a restricted excitation volume. However, these large local signals are not the only potential source of the PARS signal. Modulation of the PARS signal can also be caused by acoustic signals propagating to the sample surface. These acoustic signals can also generate surface vibrations that result in phase modulation of the PARS signal.

[0053] These generated signals can be intentionally controlled or caused by secondary physical effects, such as vibration, temperature, stress, surface roughness, mechanical bending, and the like. For example, a temperature can be introduced into the sample, thereby increasing the generated PARS signal compared to that generated without the introduction of this additional temperature. Another example is the introduction of mechanical stress into the sample (such as bending), which affects the density of the sample and thereby perturbs the generated PARS signal compared to that generated without the introduction of this mechanical stress.

[0054] Additional contrast agents can be added to the sample to increase the PARS signal produced, including but not limited to dyes, proteins, specially designed cells, fluids, and optical agents or windows. Targets can be optically altered for optimal results.

[0055] Temperature changes directly affect the PARS signal measured by the detection laser at the excitation location. The most direct effect can be described by the following equation:

[0056] p0=Γη th μ a F where η th is the fraction of light converted to heat, and F is the local light fluence (J / cm 2 ) and the dimensionless Gruneisen parameter Γ is defined as

[0057]

number

[0058] The signal-enhancing beam deposits heat, altering the temperature-dependent Gruneisen parameters and other mechanical properties of the material at or adjacent to the focal point of the detection beam and / or the focal point of the signal-enhancing beam. This results in a higher p0, according to the aforementioned relationship, and therefore an order of magnitude increase in the photoacoustic and PARS signals, depending on the material being tested. For example, the signal-enhancing beam can increase p0 by at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold compared to the value of p0 without the signal-enhancing beam. In other examples, the signal-enhancing beam can increase p0 by at least 5%, at least 10%, at least 25%, or at least 50%.

[0059] Another thermal enhancement effect focuses on the detection beam. The backreflection of the detection beam depends on the local refractive index of the material. The reflectivity of the material is also temperature-dependent. Therefore, a signal-enhancing light source can be used to deposit heat, thereby altering the optical properties of the material by a few percent compared to the optical properties of the unaltered material. This causes the amplitude of the observed PARS signal to vary approximately as the square of the optical property change. For example, using PARS detection, which is sensitive to intensity-reflectivity perturbations, a given refractive index modulation can produce an increase in PARS amplitude by the square of the difference due to the relationship between the backreflection intensity and the refractive index of the material being tested.

[0060] These limited examples highlight some of the more direct signal-enhancing effects. However, many additional material properties are temperature dependent. Any of these material properties can also be targeted with a signal-enhancing light source, which can alter the amplitude, frequency content, etc. of the observed PARS signal.

[0061] A suitable temperature range for the target exists. For example, a biological sample should be heated by several degrees. For example, the temperature rise of the sample at the focus of the signal-enhancing beam may be 0.1-1 Kelvin, 0.1-2 Kelvin, 0.1-5 Kelvin, or 0.1-10 Kelvin, although other suitable temperature rises are also contemplated.

[0062] Another aspect exploited by these new disclosures centers on the scattering, polarization, frequency, and phase content of the generated PARS signal. Excitation events occur over short time periods, e.g., less than 100 ns, during which the monitored modulations in the detected signal contain a wealth of information. For example, traditional PARS techniques that simply monitored reflected intensity could extract the amplitude of these time-domain signals. However, additional information can be extracted from the time-varying aspects of the signal. For example, some of the scattering, polarization, frequency, and phase content associated with a PARS signal may be attributed to the size, shape, features, and dimensions of the region from which the signal was generated. This allows for the encoding of additional, unique, and orthogonal information that can be useful for improving the final image fidelity, classifying sample regions, sizing constituent chromophores, and so on. Such techniques can generate independent data sets for the same interrogation region, allowing the data sets to be combined and compared with each other. For example, frequency information can reveal microscopic structures in a sample, which can be combined with traditional PARS techniques that use scattering modulation to highlight regions that are both absorbing and of a particular size.

[0063] The final aspect of the disclosure herein centers around the combination of a PARS device with optical coherence tomography (OCT). OCT is a complementary imaging modality to the PARS device. While the PARS technique visualizes optical absorption contrast, OCT imaging devices visualize optical scattering contrast. Each technique captures an independent set of information about the sample. For example, PARS provides high-contrast vascular information with high specificity, while OCT provides high-contrast information about surrounding tissues, such as the adjacent dermal layer.

[0064] OCT measurements can be performed using a variety of techniques in the form of time-domain optical coherence tomography (TD-OCT) or frequency-domain optical coherence tomography (FD-OCT), as described in U.S. Patent Application Publication Nos. 2010 / 0265511 and 2014 / 0125952.

[0065] In TD-OCT, a laser is passed through an interferometer, with one arm (the reference arm) incident on a movable mirror and the other arm (the sample arm) incident on the sample. Scattering information is typically extracted by scanning the reference path length and recording the resulting interferogram pattern corresponding to the path length on a photodetector such as a photodiode. The envelope of this pattern, commonly called an A-scan, represents a map of reflectivity in the sample versus depth, with the depth resolution given by the coherence length of the source laser.

[0066] FD-OCT is also commonly performed using an interferometer, a sample arm, and a reference arm. FD-OCT is generally divided into two distinct methods. The first, spectral-domain optical coherence tomography (SD-OCT), or spectrometer-based OCT, uses a continuous-wave broadband light source and achieves spectral discrimination through a dispersive spectrometer in the detector arm. The second, called swept-source optical coherence tomography (SS-OCT), time-codes wavenumber reflectance by rapidly tuning a narrowband light source through a wide optical bandwidth. Both techniques can enable dramatic SNR improvements of up to 15.0–20.0 dB compared to TD-OCT.

[0067] In OCT systems, multiple A-scans are typically acquired while the sample beam is scanned laterally across the tissue surface to construct a two-dimensional map of reflectivity versus depth and lateral extent, commonly referred to as a B-scan. The lateral resolution of the B-scan is approximated by the confocal resolution of the sample arm optics, which is typically given by the size of the focused light spot in the tissue.

[0068] In the field of OCT, a large body of research has been devoted to achieving quantitative optical absorption measurements. This research is of particular interest within the ophthalmic imaging community, which requires oxygen saturation measurements near the fundus. While there has been some notable research on this topic, current techniques still cannot directly measure optical absorption (unlike the PARS modality). Rather, optical absorption must be inferred by using a visible probe light source, which can significantly limit the penetration depth into the sample. Providing an improved optical absorption modality would be highly beneficial to the biomedical imaging community.

[0069] Given these complementary characteristics between PARS and OCT, there should be clear benefits to augmenting PARS with OCT. Here, we discuss the technical details of a novel dual-modality PARS-OCT system.

[0070] Figure 1 shows a high-level diagram of the TE-PARS system. The system consists of a PARS system (101), an optical combiner (102), a signal enhancement system (103), and an imaging head (104). The optical combiner is used to combine the beams from the PARS system (101) and the signal enhancement system (103).

[0071] Figure 2 shows a high-level diagram of the PARS excitation (202), PARS detection (204), and optical combiner (203), which are coupled to the signal enhancement system (201) and imaging head (205).

[0072] Figure 3 shows a high-level diagram of the SR-PARS system, which consists of a super-resolution processing unit (301), a PARS system (302), an optical combiner (303), a signal enhancement system (304), and an imaging head (305).

[0073] Figure 4 shows a high-level embodiment of a TE-PARS system coupled with other modalities (405). The system consists of a PARS system (401), an optical coupler (402), a signal enhancement system (403), and an imaging head (404). These can be coupled with a variety of other modalities, including brightfield microscopy, scanning laser ophthalmoscopes, ultrasound imaging, stimulated Raman microscopy, fluorescence microscopy, two-photon and confocal fluorescence microscopy, coherent anti-Raman-Stokes microscopy, Raman microscopy, other PARS, photoacoustic, and ultrasound systems, among others.

[0074] Figure 5 shows the signal processing path, which consists of a photodetector (501), a signal processing unit (502), a digitizer (503), a digital signal processing unit (504), and a signal extraction unit (505).

[0075] FIG. 6 shows an embodiment consisting of multiple pump lasers combined with an optical combiner. FIG. 7 shows an embodiment consisting of multiple detection lasers combined with an optical combiner. FIG. 8 shows an embodiment consisting of multiple signal-enhancing lasers combined with an optical combiner.

[0076] FIG. 9 shows an embodiment consisting of multiple excitation lasers, multiple detection lasers, and multiple signal enhancement lasers combined with an optical combiner. Figure 10 shows one embodiment of TE-PARS. A multi-wavelength fiber excitation laser (1012) is used to generate a PARS signal. The excitation beam (1017) passes through a multi-wavelength unit (1040) and a lens system (1042) to adjust its focus on the sample (1018). The optical subsystem used to adjust the focus can be constructed with components known to those skilled in the art, including, but not limited to, beam expanders, adjustable beam expanders, adjustable collimators, adjustable reflective expanders, telescope systems, and the like. The acoustic signature is interrogated using either a short-coherence or long-coherence length probe beam (1016) from a detection laser (1014), which is confocalized and co-aligned with the excitation spot (1018) on the sample. The interrogation / probe beam (1016) passes through a lens system (1043), a polarizing beam splitter (1044), and a quarter-wave plate (1056) to direct the reflected light (1020) from the sample (1018) to the photodiode (1046). However, this architecture is not limited to the inclusion of the polarizing beam splitter (1044) and the quarter-wave plate (1056). The aforementioned components may be replaced with equivalent fiber-based components, such as nonreciprocal elements such as circulators, couplers, WDMs, and / or double-clad fibers. Such elements can receive light from a first path but then redirect it to a second path. A signal-enhancing laser (1061) is used to enhance the PARS signal using the signal-enhancing beam (1060). The signal-enhanced beam (1060) passes through a lens system (1045) and its focus is adjusted on the sample (1018). The signal-enhanced beam (1060) is combined with the interrogation beam (1016) using a beam combiner (1031). The combined signal-enhanced beam (1060) and interrogation beam (1016) is further combined with the excitation beam using another beam combiner (1030). The combined beam (1021) is scanned by a scanning unit (1019). This beam passes through an objective lens (1055) and is focused onto the sample (1018).The reflected beam (1020) returns along the same path and is reflected by a polarizing beam splitter (1044) toward the signal collection / analysis path. This path consists of a photodiode (1046), an amplifier (1048), a high-speed data acquisition card (1050), and a computer (1052). In some embodiments, the signal-enhanced beam does not necessarily need to be tightly focused on the sample at the excitation location and therefore does not need to be directed along the same path as the other beams. The signal-enhanced beam may include any one or more of the parameters of the excitation beam or the interrogation beam. The signal-enhanced beam can also be directed from any angle using separate optics. The signal-enhanced beam can be focused or unfocused. The signal-enhanced beam can be pulsed or continuous and can be of any wavelength depending on the sample.

[0077] The beam characteristics of the signal-enhancing beam can be selected to achieve the desired enhancement. The wavelength may be selected within the same type range as the excitation, based on what is appropriate for the desired contrast. The intensity may be similarly low compared to the other two beams, but still within a similar type range.

[0078] Figure 11 shows another embodiment of TE-PARS. This implementation is similar to that shown in Figure 10, but instead of scanning the interrogation spot around the sample, a scanning unit (1111) is used to move the sample relative to the interrogation spot. Components labeled similarly to Figure 10 serve similar purposes in this architecture.

[0079] Figure 12 shows yet another embodiment of TE-PARS. This implementation is similar to that shown in Figure 10, but adds components for collecting and analyzing the signal-enhanced beam (1210) reflected from the sample. The signal-enhanced beam (1260) passes through a lens system (1245), a polarizing beam splitter (1259), and a quarter-wave plate (1257). This beam is confocalized with the interrogation beam (1216) and excitation beam (1217) on the sample. The reflected signal-enhanced beam (1210) is reflected toward a signal collection path consisting of a photodiode (1258), an amplifier (1268), a data acquisition card (1269), and a computer (1270). Note that while this particular example emphasizes non-interferometric detection, signal-enhanced detection can take the form of any of the PARS detection paths previously shown, including interferometric designs. Components labeled similarly to those in Figure 10 serve similar purposes in this architecture.

[0080] Figure 13 shows yet another embodiment of a multimodal TE-PARS system. This implementation is similar to Figure 10 but adds brightfield detection, where a beam combiner (1373) directs light through a tube lens (1371) to a camera (1372). Additional modalities may be added in this manner, including brightfield microscopy, scanning laser ophthalmoscopy, ultrasound imaging, stimulated Raman microscopy, fluorescence microscopy, two-photon confocal fluorescence microscopy, coherent anti-Raman-Stokes microscopy, Raman microscopy, other PARS, photoacoustic, and ultrasound systems, among others. Components labeled similarly to Figure 10 serve similar purposes in this architecture. Such integrated additional pathways may need to operate in narrow wavelength bands that remain open along a unique path toward the sample. This operation may require careful selection of operating wavelengths between modalities. A potential advantage of such an approach is that a single, integrated device could potentially offer a broad complement of different modalities, each with its own advantages. For example, in Figure 13, the addition of a camera results in a conventional brightfield microscope that can give different contrast and achieve different imaging speeds as opposed to the fused PARS device.

[0081] Figure 14 shows a comparison of a standard PARS acquisition (left) and a TE-PARS acquisition (right). In TE-PARS acquisitions, the additional heat generated by the signal-enhancing laser (from 1 millikelvin to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) improves the photoacoustic conversion efficiency, resulting in modulation roughly equivalent to the reflected intensity from that region. This additional signal can be used simply to increase overall fidelity or to enhance the contrast of wavelengths different from the excitation wavelength. In some instances, TE-PARS can improve the signal-to-noise ratio by at least 5%. In some instances, TE-PARS can achieve photoacoustic conversion efficiencies of up to 1000%. For example, the excitation here uses wavelength "a" to capture a baseline PARS signal. Then, another acquisition is performed using the same excitation wavelength, but this time using a signal-enhancing laser emitting wavelength "b." In this case, the difference between these two signals can be directly attributed to absorption at that signal enhancement wavelength.

[0082] Figure 15 shows an example of the signal acquisition process used by TE-PARS using a pulsed signal-enhanced beam. In this example, a mid-infrared (MIR) enhanced beam is used between two standard PARS acquisitions. One of these PARS acquisitions immediately follows the MIR pulse, allowing the MIR pulse to encounter additional PARS excitation resulting from the temperature increase introduced by the MIR excitation.

[0083] An example of the signal acquisition process used in a standard PARS acquisition is shown in Figure 16. Signal generation is based solely on the absorption of the excitation pulse. Figure 17 shows an example of the signal acquisition process used by TE-PARS with multiple acquisition. In conventional photoacoustics, multiple acquisition is performed by using multiple excitation wavelengths. However, in TE-PARS, a single excitation wavelength can be used with separate signal-enhancing wavelengths. This approach can then be used to separate multiple chromophores based on their optical absorption at the signal-enhancing wavelengths. This allows for easier visualization of independent constituents (e.g., chromophores) despite the individual original data sets resulting in the superposition of these components. In some embodiments, the system can unmix different chromophores, such as hemoglobin, DNA, and lipids, with each other (as they may be mixed in complex tissues). In this case, the chromophores can be given separate color maps and displayed in a single image. Furthermore, this allows for easy differentiation of chromophores / targets by viewing the image.

[0084] Figure 18 shows an example of the signal acquisition process used by SR-PARS. Multiple standard PARS acquisitions are performed at various known excitation energies. Due to saturation effects, the observed output PARS signal may exhibit a nonlinear relationship to these excitation energies. With such a system, resolution finer than the optical diffraction limit can be achieved by exploiting nonlinear optical absorption contrast effects in the sample, such as light intensity-induced optical absorption decay (sometimes called photobleaching), and the nonlinear thermal dependence of material properties, such as the thermal expansion coefficient. The algorithm uses several scans of the sample as input, allowing for nonlinear PARS signal generation between acquisitions and the application of Vandermonde matrix-based processing to isolate N-th order power relationships. These nonlinear effects can be exploited by the PARS super-resolution processing algorithm to extract higher spatial frequencies, resulting in improved resolution that can reach beyond the optical diffraction limit.

[0085] FIG. 19 shows an example of the positioning of several localized spots. Each beam (1901), (1902), and (1903) can be either an excitation beam, a detection beam, or a signal-enhanced beam. FIG. 19a highlights an orientation in which one of the excitation, detection, and signal-enhanced beams forms a smaller focal spot compared to the other two beams. Similarly, FIG. 19b highlights another case in which two of the excitation, detection, and signal-enhanced beams form a smaller focal spot than the third beam. FIG. 19c illustrates a third case in which the excitation, detection, and signal-enhanced beams each form a nearly equal focal spot. FIGS. 19d and 19e show focusing situations in which the constituent beam spots do not completely overlap at the focal spot but are laterally offset. In the first case (FIG. 19d), only one beam is offset, while the other two remain overlapping, and in the second case (FIG. 19e), all three beams are offset from one another. Similar to Figures 19d and 19e, Figures 19f and 19g illustrate focal situations where the constituent beam spots do not overlap at the focal spot but are axially offset. In the first case (Figure 19f), only one beam is offset while the other two beams remain overlapping. In the second case (Figure 19g), all three beams are offset from one another. These offsets can be any reasonable value depending on the requirements of the imaging session. Figures 19h and 19i highlight situations where the central beam axes are at an angle between themselves and the sample; this angle can generally range from 5 to 90 degrees relative to the sample surface. Figure 19h shows the case where two beams remain coaligned while the angle of the third beam is varied. Figure 19i shows the case where each beam maintains an individual angle relative to the others. Finally, Figures 19j and 19k show two different cases for scanning the sample (1910). In Figure 19k, the sample is placed directly in the path of the beam, or in Figure 19j, there is some scattering medium or optical window (1911) placed in the beam path before the sample; an example of a common medium in the beam path before the sample is a glass slide or cover slip to contain the sample.This illustration is not limiting; obvious enhancements exist for systems with more than three beams. These various states can be controlled by adjusting the beam alignment to one or more focusing optics. For example, the excitation can be directed to one side relative to the other beams, creating a state similar to Figure 19d. Similarly, the excitation focus can be moved axially relative to the other two beams, creating a state similar to Figure 19f. Intentionally shifting these focuses may be desirable in some instances, such as high-speed optical scanning, where it is desirable to have the interrogation point track the excitation point to compensate for the fast focus scan. By angling the beams relative to each other (Figures 19h and 19i), improvements in detection sensitivity can be realized due to the dominance of side light scattering as opposed to back light scattering. Another advantage may be achieved by utilizing the tight lateral focus of one beam to compensate for the relatively poor axial focus of the other beam by overlapping the beams at approximately 90 degrees to each other.

[0086] Figure 20 shows another example of signal processing used by TE-PARS to perform multiplexed acquisition. Here, thermal effects are used to measure the ratio of two constituent chromophores. In one example (Sample 1), the absorption of detection wavelength "c" is greater than that of "a," resulting in a smaller return signal for wavelength "c" and a larger return signal for wavelength "a." Similarly, in Sample 2, the absorption of detection wavelength "a" is greater than that of "c," resulting in a larger return signal for wavelength "c" and a smaller return signal for wavelength "a." This difference in return amplitude is primarily due to the difference in the target's optical absorption for wavelengths "a" and "c," respectively. Furthermore, the ratio of Sample 1 to Sample 2 can be determined based on the proportionality of the return signal for wavelength "a" compared to wavelength "c." By exploiting the thermal enhancement effect in this way, it is possible to perform chromophore unmixing using only a single excitation light source.

[0087] Figure 21 shows an example of a TE-PARS system that utilizes customized excitation pulse trains of varying pulse width, energy, wavelength, and pulse timing to induce specific thermal and pressure effects within a sample. These pulse trains can be used to shape and design specific, customized PARS signals. Furthermore, such excitation pulse trains can be utilized to enhance signals from specific chromophores, suppress signals from specific chromophores, or generate signals of specific shapes and frequencies to aid in signal extraction. For example, a second pulse can be timed relative to the first so that the relaxation period of the first signal coincides with the peak of the second signal to further increase the overall signal amplitude.

[0088] Figure 22 shows an embodiment of an SE-PARS detection system. This embodiment utilizes a PARS excitation and delivery system similar to those shown in Figures 10-13, but adds components for encoding information related to the spatial distribution of foci at the focal plane within the spectral range of the detection beam (2270). This eliminates the requirement for optical or mechanical scanning of a small field of view and also facilitates encoding spatial information through a single fiber, where the spatial distribution is encoded within the spectral distribution. The detection beam (2216) is passed through a lens system (2243), a polarizing beam splitter (2244), and a quarter-wave plate (2256). The beam then passes through a diffractive optic (2232), which laterally expands the detection beam based on the wavelength of light. The expanded beam then passes through a scanning unit (2219) and an objective lens (2255) to be confocalized with the excitation beam (2221) on the sample (2218). The reflected signal detection beam (2220) is reflected towards a signal collection path consisting of another diffractive optic (2233) that laterally expands the detection beam based on the wavelength of the light, a lens (2257) to focus the light onto a photodiode array, a photodiode array (2246), an amplifier (2248), a data acquisition card (2250), and a computer (2252). Components labeled similarly to those in Figure 10 serve similar purposes in this architecture.

[0089] TE-PARS, TS-PARS, TA-PARS, SE-PARS, SD-PARS, or SR-PARS may also be envisioned, which use a single light source for all constituent paths or collections of paths: PARS excitation, PARS detection, and signal enhancement. In any of these modalities, one or more of the beam paths may be oriented in transmission mode, meaning that the focusing optics are positioned on the opposite side of the sample from the optics that direct the light onto the sample.

[0090] Figure 23 shows a high-level schematic of a PARS-OCT system, which consists of a PARS imaging system (2301), an OCT imaging system (2303), an optical combiner (2302), and an imaging head (2304) that focuses the beam path onto the sample (2305).

[0091] Figure 24 shows embodiments of a PARS imaging subsystem (2301) and an OCT imaging subsystem (2303). Figure 24(a) shows one embodiment of a PARS subsystem (2301), which is comprised of a PARS system (2401) consisting of one or more PARS system configurations (1, 2, ..., N), which may include, but are not limited to, single-source, dual-source, pulse detection, etc. In this case, the outputs of the systems are combined via an optical combiner (2402), which may be implemented by devices such as a free-space beam combiner, a free-space dichroic mirror, a fiber-based interferometer, or a fiber-based coupler. 24(b) illustrates one embodiment of an OCT subsystem (2303) comprised of OCT systems (2404) of one or more OCT system configurations (1, 2, ..., M), which may include, but are not limited to, spectral-domain OCT (SD-OCT), swept-source OCT (SS-OCT), time-domain OCT (TD-OCT), full-field OCT (FF-OCT), line-field OCT (LF-OCT), polarization-sensitive OCT (PS-OCT), Gabor-domain OCT (GD-OCT), etc. In this case, the outputs of the systems are combined via an optical combiner (2403), which may be implemented by devices such as a free-space beam combiner, a free-space dichroic mirror, a fiber-based interferometer, a fiber-based coupler, or the like.

[0092] Figure 25 shows a combination of the PARS subsystem (2501) and OCT subsystem (2502) shown in Figure 24 with an additional alternative imaging subsystem (2503), which can be, but is not limited to, a bright-field microscope, a scanning laser ophthalmoscope, ultrasound imaging, stimulated Raman microscopy, a fluorescence microscope, a two-photon confocal fluorescence microscope, a coherent anti-Raman-Stokes microscope, a Raman microscope, another PARS, photoacoustic, or ultrasound system, etc. In this case, these subsystems (2501, 2502, 2503) are combined via an optical combiner (2504), which can be implemented by devices such as a free-space beam combiner, a free-space dichroic mirror, a fiber-based interferometer, or a fiber-based coupler.

[0093] Figure 26 illustrates how OCT signals are generated and captured. Figure 26(a) shows a representative example of a time-domain interferogram for a particular depth. Figure 26(b) shows a representative example of a Fourier-domain interferogram of an OCT system. Figure 26(c) shows a representative example of an A-scan of an OCT system attempting to capture scattering contrast with depth.

[0094] FIG. 27 illustrates the signal processing path of an exemplary OCT system. The OCT photodetector signal (2701) can be captured using devices including, but not limited to, photodiodes, avalanche photodiodes, phototubes, photomultipliers, CMOS cameras, CCD cameras (including EM-CCDs, intensified CCDs, and backside thin-film cooled CCDs), spectrometers, and the like. The signal can then undergo analog signal processing (2702), including, but not limited to, low-pass filtering, high-pass filtering, amplification, attenuation, and the like. Additionally, the signal can also be routed through alternative paths (2703) to emphasize different signal characteristics, with or without utilizing alternative analog processing. The processed and / or unprocessed OCT signal then undergoes signal digitization (2704). The digital signal then undergoes digital signal processing (2705), which can include, but is not limited to, low-pass filtering, high-pass filtering, Hilbert transformation, Fourier transform, and the like. From this fully processed signal, in some embodiments, key features can be extracted (2706) to generate an OCT image, which may include techniques such as, but not limited to, absolute maximum projection.

[0095] Figure 28 shows a representative OCT image (B-scan) of a human retina, captured by the SS-OCT system, showing significant scattering contrast that allows for detailed analysis of retinal physiology.

[0096] Figure 29 illustrates how a PARS signal is generated and captured. Figure 29(a) shows representative examples of the PARS signal, excitation laser activation signal, and interrogation laser activation signal over time during an exemplary imaging session. In this case, the exemplary PARS system is implemented using a CW interrogation laser and a pulsed excitation laser. Several time points are highlighted in Figure 29(a) to help illustrate the functionality of this PARS system. Here, the interrogation beam is active throughout. At time t1, the excitation laser is inactive, and the measured PARS signal remains stationary with a constant (DC) offset. At time t2, the excitation laser delivers a short pulse, and the sample is now excited, so measurable AC waveforms can be seen in the photothermal and photoacoustic signals. At time t3, enough time has passed since the excitation pulse that the PARS signal has now returned to its stationary state. Figure 29(b) illustrates the pressure and refractive index changes with depth through the boundary layer transition from a non-absorbing to an absorbing medium. This diagram represents the sample characteristics at times t1 and t3 in Figure 29(a). In this example, there is a constant increase in refractive index after entering the absorbing medium, and because the sample is stationary, there is no pressure change upon entering the absorbing medium. Figure 29(c) shows the sample pressure gradient and refractive index with respect to depth through the boundary layer transition moving from a non-absorbing to an absorbing medium. This figure represents the sample properties at time t2 in Figure 29(a). At this point, the sample has just been pulsed by the excitation laser, which has created a large pressure gradient with respect to depth in the absorbing medium. This large pressure increase changes the refractive index of the sample, which in turn causes a measurable change in the return intensity of the interrogation laser. This change can be seen shown as the AC waveform of the PARS signal in Figure 29(a).

[0097] Figure 30 shows the signal processing path of an exemplary PARS system. The PARS photodetector signal (3001) can be captured using devices including, but not limited to, photodiodes, avalanche photodiodes, phototubes, photomultipliers, CMOS cameras, CCD cameras (including EM-CCDs, intensified CCDs, and backside thin-film cooled CCDs), spectrometers, etc. The signal can then undergo analog signal processing (3002), which can include, but is not limited to, low-pass filtering, high-pass filtering, amplification, attenuation, etc. Additionally, the signal can also be routed through alternative paths (3003) to emphasize different signal characteristics. The processed and / or unprocessed PARS signal then undergoes signal digitization (3004). The digital signal can then undergo digital signal processing (3005), which can include, but is not limited to, low-pass filtering, high-pass filtering, Hilbert transforms, Fourier transforms, PARS signal discrimination methods, extraction of polarization, phase, and frequency content, etc. From this fully processed signal, in some embodiments, key features can be extracted (3006) to generate a PARS image, which may include, but is not limited to, techniques such as absolute maximum projection, peak frequency, etc.

[0098] Figure 31 shows two PARS images using different excitation wavelengths to target specific absorbers. Figure 31(a) shows an in vivo PARS image of mouse ear vasculature, exploiting the absorption contrast of green light (532 nm) to target hemoglobin. Figure 31(b) shows an ex vivo PARS image of human tissue, exploiting the absorption contrast of ultraviolet light (266 nm) to target DNA.

[0099] Figure 32 highlights one implementation of PARS-OCT. In this example, the beam from the interrogation light source (3222) passes through an appropriate collimator (3208), is directed to a polarizing beam splitter (3214), a quarter-wave plate (3215), and is further directed to an appropriate dichroic mirror (3216) before being directed into the sample path along with the excitation beam (3228) from a pulsed laser (3230) and an appropriate collimator (3212). In the OCT subsystem, the beam from the broadband light source (3225) is directed to a free-space / fiber coupler beam splitter (3202) with an appropriate split ratio before being split into a reference beam (3227) and a sample beam (3226). These systems may require beams capable of achieving broad-spectrum illumination, such as broadband CW sources or swept sources. A polarization controller (3204) can also be used to maximize interference efficiency. The reference beam is collimated by an appropriate collimator (3203), passes through a dispersion compensation unit (3205), and is focused onto a reference mirror (3207) using an appropriate lens (3206). The sample beam is directed towards an appropriate dichroic mirror (3223) and combined with the PARS excitation beam (3228) and the interrogation beam (3229). The entire beam is then directed towards the sample (3220). In this case, a galvo scanner mirror (3217) is used in conjunction with a pair of telecentric lenses (3218) and an objective lens (3219). The return beam is directed towards the dichroic mirror (3223) and split into an OCT interrogation beam and a PARS interrogation beam. The OCT sample beam interferes with the reference beam, and the resulting mixture is detected by an appropriate detector (3201), and the signal is directed towards the corresponding processor (3224). The PARS interrogation beam is directed towards an appropriate filter (3209) to filter out the interrogation beam spectral range and focused onto a detector (3211) using an appropriate lens (3210), and the signal is post-processed in a corresponding processor (3221).

[0100] Figure 33 highlights one embodiment of EPARS-OCT, which is similar to that of Figure 32, except that the combined beams are delivered to the sample through an endoscope (3331), which includes, but is not limited to, a collimator (3332) along with appropriate imaging optics (3333 and 3334).

[0101] Figure 34 highlights another novel implementation of PARS-OCT in which the PARS excitation and OCT light source (3436) is shared. The light source can be, but is not limited to, a nanosecond-pulsed supercontinuum laser that is directed toward appropriate dichroic mirrors (3435) to separate the PARS excitation beam (3437) and the OCT illumination beam (3438). When the light source (3436) is shared among multiple paths in this manner, the light source may require a broadband output that can be filtered to the required subsections (PARS, OCT, etc.) or tuned to address each requirement separately. Other than broadband, the light source may not require any special characteristics beyond those inherent to the individual beam sources in other systems. Such a device layout can offer significant advantages over architectures that use separate light sources for each path. Some of these advantages include overall device cost, size, and ease of maintenance (including items such as alignment). For example, system alignment may be easier since multiple paths can share the same wavelength reducing undesirable color effects. In this example, the remainder of the system embodiment is similar to FIG.

[0102] Figure 35 highlights yet another embodiment of a multimodal PARS-OCT system. This embodiment is similar to that of Figure 32. However, instead of a single combiner, two beam combiners (3542) are used to combine the PARS beam, the OCT beam, and the brightfield microscope beam (3541). Here, the brightfield detector is implemented as a tube lens (3540) and a camera (3539). Additional modalities such as fluorescence microscopy, scanning laser ophthalmoscope, and ultrasound imaging may be added in this manner.

[0103] Figure 36 highlights another novel implementation of PARS-OCT in which the PARS survey and OCT light source (3643) is shared, which can be, but is not limited to, a continuous wave laser that is directed toward an appropriate beam combiner (3644) to separate the PARS survey beam (3646) and the OCT illumination beam (3645). Such a device layout can offer significant advantages over architectures that use separate light sources for each path. Some of these advantages may include overall device cost, size, and ease of maintenance (including items such as alignment). For example, system alignment may be easier because multiple paths can share the same wavelength, which reduces undesirable chromatic effects. In this example, the remainder of the system implementation is similar to that of Figure 32.

[0104] Similarly, one can envision a similar system combination using a single (ie, exactly one) laser source for all three of the PARS excitation beam, the PARS interrogation beam, and the OCT beam.

[0105] Figure 37 highlights a block diagram of a TS-PARS system. This system can be implemented as a conventional PARS system (3702) coupled to (3703) and directed onto a sample through an imaging head (3704). Here, at least one notable difference includes the addition of a temperature sensing unit (3701) that can decode the information contained in the PARS signal and interpret that information to generate a measurement of the absolute or relative temperature of the sample. Individual PARS measurements can be compared to a known signal for a given sample at a given temperature. Similarly, multiple PARS measurements can be compared to one another for a given sample to generate a relative change in temperature between these multiple measurements. In this case, the sensing unit and / or controller converts these relative changes in the PARS signal into a measurement of relative or absolute temperature.

[0106] Figure 38 highlights a block diagram of the combination between multiple described systems, in this case, TS-PARS (3809), SR-PARS (3801), TE-PARS (3808), and a collection of other modalities (3806). These individual systems are combined (3802, 3803, 3804, 3807) and directed to a single imaging head (3805) before being directed onto the sample. Other combinations of the systems described herein can be combined as well.

[0107] Figure 39 highlights yet another PARS-OCT embodiment featuring a thermally enhanced light source. A signal-enhancing laser (3947) is used to enhance the PARS signal with a signal-enhancing beam (3949). The enhanced beam from the laser passes through a suitable collimator (3948) before being combined with the PARS interrogation beam (3929) using a suitable beam combiner (3950). In this example, the remainder of the system embodiment is similar to that of Figure 32.

[0108] Figure 40 highlights an anno-OCT embodiment in which the optical subsystem is mechanically scanned around the sample. In this example, the imaging head is mounted on a mechanical scanning stage (4051) unit that allows for lateral, axial, and rotational scanning. In this example, the remainder of the system embodiment is similar to that of Figure 32.

[0109] Figure 41 presents a high-level description of the temperature sensing process that can exist in TS-PARS. As the sample temperature changes (in this case, from 25° to 35°), the efficiency of photoacoustic pressure generation also changes. In this case, for a constant excitation energy level (pulses a, b, c, and d), the output signal increases with increasing temperature. Such temperature modulations can then be recorded and fitted to an expected temperature-dependent model to extract relative or absolute changes in temperature in the sample.

[0110] Figure 42 shows a high-level schematic of the scattering compensation method. The PARS signal has a strong dependence on the unperturbed backscattered light and, therefore, on the local scattering efficiency of the sample. To decouple the PARS signal amplitude from the local scattering efficiency, the backscattered amplitude is extracted and removed from the PARS signal using a controller specific for the task, which may be the same or a different controller as either the controllers described above or below. The controller subtracts the collected scattering contrast from the PARS signal; this subtraction helps reduce background noise due to randomly scattered photons and increases the signal-to-noise ratio.

[0111] In any of the TE-PARS, TS-PARS, SE-PARS, SD-PARS, TA-PARS, SR-PARS, PARS, or OCT-PARS modalities, one or more of the beam paths can be oriented in transmission mode, meaning that beam-focusing optics are located opposite the optics directing light toward the sample. Figure 43 illustrates this case with an example TE-PARS system, where beams (4301), (4302), and (4303) each represent one of the TE-PARS excitation, detection, and signal-enhancement beams. In each case, one or more of these beams may be pointing in the same or opposite direction as the other beams. Furthermore, these orientations are not limiting and can be applied to any of the example beam overlap situations shown in Figure 21 or any other logical beam positioning. Some potential advantages of doing so may arise when thin samples (<1 mm) are being imaged, since forward scattering tends to be more significant compared to backscatter. Similarly, such an embodiment can greatly improve multiplexing capabilities, helping to support the simultaneous use of multiple modalities by not requiring all modalities to reach the sample through the same objective.

[0112] Figure 44 shows an example of the positioning of several local spots in PARS-OCT. Each beam (4401), (4402), and (4403) can be either an excitation beam, a detection beam, a signal-enhancing beam, or an OCT beam. Figure 44a highlights an orientation in which one of the PARS or OCT excitation, detection, and signal-enhancing beams forms a smaller focal spot compared to the other two beams. Similarly, Figure 44b highlights another case in which two of the excitation, detection, and signal-enhancing beams form a smaller focal spot than the third beam. Figure 44b illustrates a third case in which the PARS or OCT excitation, detection, and signal-enhancing beams each form approximately equal focal spots. Figures 44d and 44e show focusing situations in which the constituent beam spots do not completely overlap at the focal spot but are laterally offset. In the first case (Figure 44d), only one beam is misaligned while the other two remain overlapping; in the second case (Figure 44e), all three beams are misaligned with respect to each other. Similar to Figures 44d and 44e, Figures 44f and 44g show focusing situations where the constituent beam spots do not overlap at the focal spot but are misaligned axially. In the first case (Figure 44f), only one beam is misaligned while the other two remain overlapping; in the second case (Figure 44g), all three beams are misaligned with respect to each other. These misalignments can be any reasonable value depending on the requirements of the imaging session. Figures 44h and 44i highlight situations where the central beam axes form an angle between themselves and the sample; this angle can generally range from 5 to 90 degrees relative to the sample surface. Figure 44h shows the case where two beams remain coaligned and the angle of the third beam is varied. Figure 44i shows the case where each beam holds an individual angle relative to the other beams. Finally, Figures 44j and 44k show two different cases for scanning the sample (4410).In Figure 44k, the sample is placed directly in the path of the beam, or in Figure 44j, there is some scattering medium or optical window (4411) placed in the beam path before the sample; an example of a common medium in the beam path before the sample is a glass slide or cover slip to contain the sample. This illustration is not limiting, and there are obvious extensions to the system with more than three beams.

[0113] Figure 45 shows another high-level example of PARS signal enhancement. Because the PARS signal has a strong dependence on backscattered light and thus the local scattering characteristics of the sample, this information can be leveraged to recreate a more accurate visualization of the optical absorption contrast by removing the residual scattering contrast inherent in standard PARS acquisition. In some applications, scattering components from the detection laser, signal-enhancement laser, or excitation laser may be collected separately. These signals may be subtracted or added to the PARS signal and analyzed separately based on their amplitude, phase, polarization, and frequency content to yield additional information about the sample. In the example shown, the local backscattering amplitude is extracted separately from the PARS signal to decouple the PARS signal amplitude from the local scattering efficiency. In this example, both PARS signals appear to have the same amplitude, but there is significantly less backscattering in example "b." To reduce the effects of local scattering, the PARS signal is normalized to its measured local scattering amplitude. As exemplified here, this normalization serves to amplify signals with low scattering amplitudes and reduce signals with strong scattering amplitudes. Optical properties of either detection, signal enhancement, or excitation may be collected, including polarization, frequency, phase content, fluorescence, etc. This information can be mixed with, subtracted from, added to, or separately enhanced from the PARS signal to achieve the desired signal modification results.

[0114] In at least some PARS embodiments, perhaps only absorption contrast (from the excitation light source) is measured. In SD-PARS embodiments, both scattering contrast (detection light source only) and absorption contrast (due to excitation and detection light sources) are measured. This measurement can be used directly to generate visualizations, i.e., giving each image a different color (PARS absorption and scattering) and overlaying the results. Alternatively, wavelength-specific absorption and scattering can be exploited to reveal or suppress information in the sample.

[0115] In SD-PARS, the detection wavelength can be intentionally selected to suppress photoacoustic or PARS signals originating from certain regions. For example, if a desired target is located immediately adjacent to a large blood vessel (which could otherwise overwhelm the signal from the desired target), the detection wavelength can be selected to suppress the signal from the blood vessel by collecting the absorbed energy level prior to detection. In SD-PARS, the detection wavelength can also be selected to highlight scattering contrast resulting from regions of interest in the sample. For example, when imaging tissue, highly scattering wavelengths can be selected to highlight morphological structures.

[0116] In SD-PARS, specific signal extraction algorithms may be applied that rely on the time-domain behavior of both PARS absorption and PARS scattering. SD-PARS surveys may be selected to emphasize the sample's inherent scattering, absorption, fluorescence, polarization content, frequency content, and phase content. This additional information encoded in SD-PARS surveys can be applied to improve signal fidelity, enhance image contrast, and recover information about the sample's shape, size, and dimensions. SD-PARS survey characteristics can be processed in a variety of ways depending on the desired features and applications. For example, processing techniques may include machine learning methods, extensive feature extraction, feature extraction based on multidimensional decomposition and frequency content, and signal processing methods. In contrast to standard PARS, processing techniques can be enhanced by leveraging information resulting from the survey light source interaction rather than the PARS excitation event. This information can also be applied to enhance multiplex imaging by suppressing signal from structures by collecting absorption energy levels prior to detection. This information can also be applied to highlight specific scattering features in the sample.

[0117] One such implementation of SD-PARS focuses on unmixing the absorption contrast due to the detection light source and is processed as follows.

[0118] 1) The PARS absorption signal is collected in the "standard" way by bandpass filtering the raw photodiode output before capturing the signal with a high frequency digitizer. 2) Characteristic PARS amplitudes are extracted from the digitized time-domain signal by any one of the following methods: maximum projection, frequency analysis, feature decomposition, etc.

[0119] 3) This completes one embodiment of the PARS process. Following this point, the SD-PARS process may include additional processing compared to the "standard" PARS process. This processing can be divided into three general sections:

[0120] Section 1: SD-PARS absorption signal processing The extracted PARS data from 2) above can be analyzed to highlight signals specific to absorption contrast from the detection light source. Low-amplitude PARS signals, resulting from high absorption from the detection light source or low absorption from the excitation light source, are extracted using a histogram-based processing technique. First, the histogram is nonlinearly scaled. Here, a gamma shift is used to "stretch" the lower region of the histogram. This shifts the image contrast, emphasizing low-amplitude PARS signals and suppressing high-amplitude PARS signals. The result is then windowed based on a statistical measure of the low-amplitude PARS signals, isolating the signals of interest. In this case, the segmented image shows tissue features with low excitation absorption or high detection absorption.

[0121] Section 2: SD-PARS scattering signal processing The light scattering contrast of the detection is collected to explicitly separate the contrast due to absorption of the detection light source. The scattering signal is collected separately from the PARS absorption signal. The scattering signal is collected by capturing the raw, unfiltered photodiode output (unlike traditional PARS signals). The backscatter intensity is determined for each location as a weighted average of the time-domain output of the photodiode.

[0122] Section 3: SD-PARS Unmixing Unmixing is then performed using the detected scattering intensity at each location and the corresponding PARS absorption signal. The PARS absorption signal is decomposed into a linearly weighted sum of the detected absorption contrast and the excitation absorption contrast. This decomposition follows the ratio:

[0123] PARS ABS ∝Det Sc (Det abs +Ext abs ) A combined PARS absorption and PARS scattering visualization can be generated by assigning different color ranges to each image (PARS absorption and PARS scattering). For example, low absorption and scattering signals can be assigned white values, while high-value such signals can be assigned unique colors to mimic the appearance of a multi-stained tissue specimen. In this example, the contrast resulting from a given location is the combined scattering and absorption contrast captured at that location. The different contrasts can be combined using several methods, such as a linear blending algorithm or a nonlinear color blending algorithm. The resulting SD-PARS visualization then provides both absorption and scattering visualization. In other examples, any signal characteristic can be used to define the coloring. Amplitude-based thresholding is one possible implementation, but is not limited to it. Coloring can focus on any characteristic, such as magnitude, phase, polarization, or frequency content. Furthermore, coloring can be performed in a variety of ways, such as linear or nonlinear blending, or AI-based techniques.

[0124] Figure 46 shows an example of an SD-PARS combined scattering and absorption contrast implementation. The SD-PARS system can be utilized to simultaneously collect both scattering and absorption signals. Here, the wavelength of SD-PARS detection is selected to specifically target the optical properties of the sample. This targeted contrast can be used to directly generate an enhanced visualization that highlights both the SD-PARS scattering and absorption contrast. When generating the combined visualization, the SD-PARS scattering signal can be analyzed based on its amplitude, phase, polarization, frequency content, etc. to extract information. The extracted SD-PARS scattering contrast can then be subtracted, added, or blended with the PARS absorption signal using various methods, such as linear or nonlinear color blending or artificial intelligence-based techniques. In the illustrated example, the absorption contrast of a cell nucleus is captured using PARS (Figure 46(a)). Simultaneously, the SD-PARS optical scattering contrast is captured separately from the absorption signal. In this example, infrared light is used to capture the scattering contrast of tissue, including nuclear structures (b). The two visualizations (a) absorption and (b) scattering are then blended to form the enhanced representation. Here, next, the tissue structure and accompanying nuclear contrast are visible (c). In this case, the two images are combined using a basic linear mixing technique. However, this combination can be performed using any number of techniques, including nonlinear mixing, or AI-based methods. Additionally, this combination can target any optical property of the sample, including polarization, frequency, phase content, fluorescence, etc. This information can be mixed with, subtracted from, added to, or separately augmented from the PARS signal to achieve the desired signal modification results.

[0125] Figure 47 shows examples of different contrast potentials for SD-PARS. Here, the SD-PARS detection wavelengths are chosen to target different optical properties of the sample. In this example, scattering images of thin sections of preserved human breast tissue are captured. (a) uses a near-infrared 1310 nm SD-PARS detection source, and (b) uses a visible 405 nm SD-PARS detection source. Each detection source highlights unique structures in the tissue sample. This targeted contrast forms the basis of the SD-PARS mechanism and enhanced visualization.

[0126] Figure 48 shows one embodiment of autofluorescence-sensitive PARS. A multi-wavelength fiber excitation laser (4812) is used to generate a PARS signal. An excitation beam (4817) passes through a multi-wavelength unit (4840) and a lens system (4842) to adjust its focus on the sample (4818). The optical subsystem used to adjust the focus can be constructed using components known to those skilled in the art, including, but not limited to, beam expanders, adjustable beam expanders, adjustable collimators, adjustable reflective expanders, telescope systems, etc. The optical subsystem used to adjust the focus can be constructed using components known to those skilled in the art, including, but not limited to, beam expanders, adjustable beam expanders, adjustable collimators, adjustable reflective expanders, telescope systems, etc. The acoustic signature is interrogated using either a short-coherence length or long-coherence length probe beam (4816) from a detection laser (4814), which is confocalized and co-aligned with an excitation spot (4818) on the sample. The interrogation / probe beam (4816) passes through a lens system (4843), a polarizing beam splitter (4844), and a quarter-wave plate (4856) to direct the reflected light (4820) from the sample (4818) to a photodiode (4846). However, this architecture is not limited to including the polarizing beam splitter (4844) and the quarter-wave plate (4856). The aforementioned components may be replaced with equivalent fiber-based components, such as nonreciprocal elements such as circulators, couplers, WDMs, and / or double-clad fibers. Such an element receives light from a first path, but can then redirect said light to a second path. The interrogation beam (4816) is combined with the excitation beam using another beam combiner (4830). The combined beam (4821) is scanned by a scanning unit (4819). This beam passes through an objective lens (4855) and is focused onto the sample (4818). The reflected beam (4820) returns following the same path.The reflected beam is filtered by a beam combiner / splitter (4831) to separate the detection beam (4816) from any autofluorescence light returning from the sample. The autofluorescence light (4890) passes through a lens system (4845) and is focused onto an autofluorescence-sensitive photodetector (4891). The separated detection beam (4820) is sent through a beam splitter (4831) to a signal collection / analysis path, where the returning detection light is redirected by a polarizing beam splitter (4844). The detection path consists of a photodiode (4846), an amplifier (4848), a high-speed data acquisition card (4850), and a computer (4852). The autofluorescence-sensitive photodetector can be any such device, including a camera, photodiode, photodiode array, etc. The autofluorescence detection path may include a Morse beam splitter and a photodetector to further separate and detect light of specific wavelengths.

[0127] Figure 49 shows an example of visualization that can potentially be achieved with autofluorescence-sensitive total absorption PARS (TA-PARS). There are a limited number of interactions that can occur when a sample absorbs light. The absorbed energy is converted to temperature and pressure or to light of another wavelength. Temperature and pressure signals are captured by the PARS detection beam, while light emission can be detected by the autofluorescence-sensitive PARS. In this way, the total absorption of light by the tissue (whether in the form of generated pressure, generated temperature, or fluorescence) can be captured by the PARS system. With this architecture, any portion of the light returning from the sample can be collected, excluding the detection beam, and analyzed based on wavelength. By isolating specific wavelengths of light emission from the sample, specific molecules of interest can be visualized. For example, autofluorescence-sensitive PARS can be applied to tissue imaging. Here, PARS excitation was selected to capture the nuclear absorption contrast. In this case, UV excitation was used to generate pressure and temperature signals due to nuclei in the tissue. At the same time, the autofluorescence contrast generated by PARS excitation is captured. In this case, non-nuclear regions of the tissue are highly fluorescent. In this way, nuclear and non-nuclear structures of the tissue can be visualized simultaneously, and furthermore, the resulting visualization may require only a single excitation wavelength to be captured.

[0128] For example, autofluorescence-sensitive PARS can be implemented in our PARS absorption spectrometer to accurately measure the total absorption of light by a sample. Furthermore, autofluorescence-sensitive PARS can be used to measure the proportion of absorbed energy that is converted to heat and pressure, or light, respectively. This may enable the most sensitive quantum efficiency measurements to date.

[0129] The TA-PARS signal may also be collected with a single detector, as highlighted in Figure 50. Given that significant components of the TA-PARS signal may appear distinct from one another, a single detector may adequately characterize these components. For example, the initial signal level (scattering) may represent the unperturbed intensity reflectance of the detection beam from the sample at the interrogation location, encoding the scattering intensity. Next, following excitation with an excitation pulse (100 ns in the figure), PARS excitation signals related to heat, temperature, and fluorescence can be observed as unique superposition signals (labeled PA and AF in the figure). If these excitation signals are significantly distinct from one another, they can be resolved from the composite signal to extract their magnitudes along with their characteristic lifetimes. This wealth of information may be useful for improving the resulting contrast, providing additional multiplexing capabilities, and providing characteristic molecular signatures of the constituent chromophores. Additionally, such an approach offers practical advantages in that only a single detector and detection path may be required, dramatically reducing the complexity and cost of physical hardware.

[0130] It will be apparent that other examples may be designed using different fiber-based or free-space components to achieve similar results. Other alternatives may include light sources of various coherence lengths, the use of balanced photodetectors, interrogation beam modulation, the incorporation of optical amplifiers in the return signal path, etc.

[0131] During in vivo imaging experiments, no drugs or ultrasound coupling media are required. However, the target can be prepared with any liquid, such as water or oil, before the non-contact imaging session. Similarly, in some cases, an intermediate window, such as a cover slip or glass window, can be placed between the imaging system and the sample.

[0132] All light sources, including but not limited to PARS excitation, PARS detection, PARS signal enhancement, and OCT light sources, can be implemented as continuous beams, modulated continuous beams, or short-pulse lasers whose pulse widths can range from a few attoseconds to several milliseconds. These light sources can be set to any wavelength suitable for exploiting the sample's optical (or other electromagnetic) properties, such as scattering and absorption. Wavelengths can also be selected to intentionally enhance or suppress detection or excitation photons from different absorbers. Wavelengths can range from nanometers to micrometer (micron) scales. Continuous wave beam power can be set to any appropriate power range, such as attowatts to watts. Pulsed light sources can use pulse energies appropriate for the particular sample being tested, such as in the range of a few attojoules to several joules. Various coherence lengths may be implemented to exploit interference effects. These coherence lengths can range from a few nanometers to several kilometers. Similarly, pulsed light sources can use any repetition rate deemed appropriate for the sample being tested, such as from continuous wave to the gigahertz range. The light source can be tunable, monochromatic or polychromatic.

[0133] SD-PARS can use detection wavelengths that are purposefully selected to suppress PARS signals originating from certain regions. For example, if a desired target is located immediately adjacent to a large blood vessel (which could otherwise overwhelm the signal from the desired target), the detection wavelength may be selected to suppress the signal from the blood vessel by collecting the absorbed energy level prior to detection.

[0134] The TA-PARS, TE-PARS, TS-PARS, SR-PARS, SE-PARS, SD-PARS, PARS-OCT, or EPARS-OCT subsystems can use any interferometer design, including common-path interferometers (using specially designed interferometer objectives), Michelson interferometers, Fizeau interferometers, Ramsey interferometers, Fabry-Perot interferometers, Mach-Zehnder interferometers, and optical quadrature detection. Interferometers can be free-space, fiber-based, or some combination. The basic principle is that phase and amplitude variations of a probing receiver beam can be detected using an interferometer and at AC, RF, or ultrasonic frequencies using a variety of detectors.

[0135] The TA-PARS, TE-PARS, TS-PARS, SR-PARS, SE-PARS, or SD-PARS subsystems can use and implement a non-interferometric detection design to detect amplitude modulation in the signal. The non-interferometric detection system can be free-space, fiber-based, or some combination of these.

[0136] The TA-PARS, TE-PARS, TS-PARS, SD-PARS, SR-PARS, SE-PARS, PARS-OCT or EPARS-OCT subsystems can use a variety of optical fibers, such as photonic crystal fibers, image guide fibers, and double-clad fibers.

[0137] The PARS subsystem can be implemented as conventional photoacoustic remote sensing (PARS), non-interferometric photoacoustic remote sensing (NI-PARS), camera-based photoacoustic remote sensing (C-PARS), coherence-gated photoacoustic remote sensing (CG-PARS), single-source photoacoustic remote sensing (SS-PARS), or an extension of any of these.

[0138] The OCT subsystem can be implemented as spectral-domain optical coherence tomography (SD-OCT), swept-source optical coherence tomography (SS-OCT), time-domain optical coherence tomography (TD-OCT), full-field optical coherence tomography (FF-OCT), line-field optical coherence tomography (LF-OCT), polarization-sensitive optical coherence tomography (PS-OCT), Gabor-domain optical coherence tomography (GD-OCT), etc.

[0139] In PARS-OCT and EPARS-OCT, the PARS and OCT subsystems operate separately as a single imaging system and can acquire images independently as stand-alone imaging devices.

[0140] In one example, all beams can be scanned together. In this way, PARS excitation can be detected over the same and largest area as it is generated. OCT detection can also be performed in the same location as PARS to aid in alignment. Other configurations can also be used, including keeping one or more of the beams fixed while the other beams are scanned, or vice versa.

[0141] Optical scanning can be performed by galvanometer mirrors, MEMS mirrors, polygon scanners, stepper / DC motors, etc. Mechanical scanning of the sample can be performed by a stepper stage, a DC motor stage, a linear drive stage, a piezo drive stage, a piezo stage, etc.

[0142] Both optical and mechanical scanning techniques can be utilized to generate one-, two-, or three-dimensional scans around a sample. Adaptive optics such as TAG lenses and deformable mirrors can be used to perform axial scans within a sample.

[0143] Both optical and mechanical scanning can be combined to form a hybrid scanner. This hybrid scanner employs one or two optical axes to capture large areas or strips in a short time. The mirrors can potentially be controlled using custom control hardware to create customized scan patterns that improve scanning efficiency in terms of speed and quality. For example, one optical axis can be used to scan at high speed while simultaneously moving the sample using one mechanical axis. This creates a ramp-like scan pattern that can then be interpolated. In another example, custom control hardware can be used to step the mechanical stage only when the fast axis movement is finished, resulting in a Cartesian coordinate-like grid that may eliminate the need for any interpolation.

[0144] PARS can achieve 3D imaging by optical or mechanical scanning of the beam, or by mechanical scanning of the sample or imaging head, or by a combination of mechanical and optical scanning of the beam, optics, and sample, which can enable high-speed en-face or 3D imaging of structures and features.

[0145] One or more pinholes may be employed to remove out-of-focus light when scanning the beam optically or mechanically, or when scanning the sample or imaging head mechanically, or when scanning the beam, optics, and sample mechanically and optically. These pinholes can improve the signal-to-noise ratio of the resulting image.

[0146] The beam combiner can be implemented using dichroic mirrors, prisms, beam splitters, polarizing beam splitters, WDMs, etc. The beam paths can be focused onto the sample using different optical paths. Single or multiple PARS excitation, detection, signal enhancement, etc. and OCT paths can each use separate focusing elements toward the sample, or all can share a single path or any combination. The beam paths may return from the sample using unique optical paths that are different from the paths used to focus onto the sample. These unique optical paths can interact with the sample at normal incidence or at an angle, with the central beam axis forming an angle ranging from 5 to 90 degrees with the sample surface.

[0147] The beam configurations shown in Figures 19e and 19f allow for additional spatial rejection of unwanted randomly scattered photons and allow for the detection of only photons modulated by the excitation or signal-enhancing laser. Because the PARS imaging region is defined by the overlap of the excitation, detection, and, in the case of TE-PARS, thermally enhanced, and rearward detection / reflection beam paths, when these paths are all aligned together, the interrogation region on the sample can be defined by a lateral radial distribution that is generally shorter than the axial distribution. As shown in Figures 19e and 19f, by angling the beams relative to one another, overlap can be defined between two or more radial distribution combinations. In this way, the lateral resolution of one of the beams can improve the axial performance achieved by another beam. To maximize this effect, it may be most advantageous to distribute each beam evenly in azimuth and at approximately 45 degrees relative to the sample surface. In some embodiments, the elevation angle can be varied between the beam paths.

[0148] In some applications, such as ophthalmic imaging, the imaging head may not implement any primary focusing elements, such as an objective lens, to tightly focus the light onto the sample. Instead, the beam may be collimated or loosely focused (creating a spot size much larger than the optical diffraction limit) while being directed toward the sample. For example, ophthalmic imaging devices direct a collimated beam toward the eye, allowing the eye's lens to focus the beam onto the retina.

[0149] The imaging head can focus the beam to a depth of at least 50 nm into the sample. The imaging head can focus the beam to a depth of up to 10 mm into the sample. The increased depth compared to previous PARS systems arises from the novel use of a deeper penetrating detection wavelength, as described above.

[0150] The light may be amplified by an optical amplifier before interacting with the sample or before detection. Light can be collected by photodiodes, avalanche photodiodes, phototubes, photomultiplier tubes, CMOS cameras, CCD cameras (including EM-CCD, intensified CCD, and backside thin-film cooled CCD), spectrometers, etc.

[0151] The detected signal can be amplified by an RF amplifier, a lock-in amplifier, a transimpedance amplifier, or other amplifier configuration. The modality can be used to obtain A-scan, B-scan or C-scan images for in vivo, ex vivo or phantom studies.

[0152] TA-PARS, TE-PARS, TS-PARS, SD-PARS, SR-PARS, SE-PARS, PARS-OCT, or EPARS-OCT can take the form of any embodiment common to microscopy and biological imaging techniques, some of which may include, but are not limited to, devices embodied as tabletop microscopes, inverted microscopes, handheld microscopes, surgical microscopes, endoscopes, or ophthalmic devices, etc. These can be constructed based on principles known in the art.

[0153] TA-PARS, TE-PARS, TS-PARS, SD-PARS, SR-PARS, SE-PARS, PARS-OCT, or EPARS-OCT can be optimized to utilize a multi-focus design that improves the depth of focus for 2D and 3D imaging. The chromatic aberrations of the collimating objective lens pair can be used to refocus the light from the fiber onto the object, so that each wavelength is focused at a slightly different depth. These chromatic aberrations can be used to encode depth information into the recovered PARS signal, which can then be recovered using wavelength-specific analysis techniques. These wavelengths can also be used simultaneously to improve the depth of field and signal-to-noise ratio (SNR) of the PARS image. Depth scanning by wavelength tuning can also be performed during imaging.

[0154] In PARS, lateral or axial discrimination of the sample can be performed by spatially encoding the detection area, e.g., by using several pinholes, or by the spectral content of a broadband beam.

[0155] TA-PARS, TE-PARS, TS-PARS, SR-PARS, SE-PARS, SD-PARS, PARS-OCT, or EPARS-OCT systems may be combined with other imaging modalities, such as stimulated Raman microscopy, fluorescence microscopy, two-photon and confocal fluorescence microscopy, coherent anti-Raman-Stokes microscopy, Raman microscopy, other photoacoustic systems, and ultrasound systems. This combination can also enable simultaneous imaging of microcirculation, blood oxygenation parameters, and other molecular-specific targets—potentially important tasks that are difficult to perform using fluorescence-based microscopy alone. Multi-wavelength visible laser sources may also be implemented to generate photoacoustic signals for functional or structural imaging.

[0156] A polarization analyzer may be used to resolve the detected light into its respective polarization states, with the detected light at each polarization state providing information about the sample. A phase analyzer may be used to decompose the detected light into its phase components, which provides information about the sample.

[0157] The TA-PARS, PARS, TE-PARS, TS-PARS, SR-PARS, SE-PARS, or SD-PARS systems can detect the resulting signal as the detection beam returns from the sample. These perturbations can include, but are not limited to, changes in intensity, polarization, frequency, phase, absorption, nonlinear scattering, and nonlinear absorption, and can be caused by a variety of factors such as pressure, thermal effects, etc.

[0158] Analog-based signal extraction may be implemented along the electrical signal path. Some examples of such analog devices may include, but are not limited to, lock-in amplifiers, peak detection circuits, etc.

[0159] The PARS subsystem can detect temporal information encoded in the back-reflected detection beam. This information can be used to identify chromophores, enhance contrast, improve signal extraction, etc. This temporal information may be extracted using analog and digital processing techniques. These techniques include, but are not limited to, the use of lock-in amplifiers, Fourier transforms, wavelet transforms, intelligent algorithmic extraction, etc. In one example, lock-in detection can be utilized to extract PARS signals that resemble known expected signals for extracting specific chromophores such as DNA, cytochromes, red blood cells, etc.

[0160] The OCT subsystem may detect the generated PARS, thermal, and pressure signals as perturbations in the back-reflected detection beam. These perturbations can include changes in intensity, polarization, phase, frequency, absorption, nonlinear scattering, and nonlinear absorption. The OCT subsystem can detect these perturbations by tracking the changes during successive OCT scans. The OCT subsystem can also detect vibrations or surface vibrations generated by the PARS system.

[0161] The OCT and PARS subsystems may be used to detect sample absorption properties by spectroscopic techniques. This subsystem can be used to detect either PARS-induced absorption, OCT-induced absorption, or both.

[0162] The imaging head of the system may include closed-loop or open-loop adaptive optics components, including but not limited to wavefront sensors, deformable mirrors, TAG lenses, etc. for wavefront and aberration correction. Aberrations may include defocus, astigmatism, coma, distortion, third-order effects, etc.

[0163] The signal-enhancing beam may also be used to suppress signals from undesired chromophores by intentionally inducing saturation effects such as photobleaching. Various types of optical components may be utilized to take advantage of their respective advantages. For example, an axicon may be used as the primary objective to generate Bessel beams with a greater depth of focus compared to that obtained with standard Gaussian beam optics. Such optical components may also be used elsewhere in the beam path as deemed appropriate. Cathodic optical components may also replace the respective refractive elements, such as using a reflective objective instead of a standard compound objective.

[0164] The optical path may include nonlinear optical elements for various related purposes such as wavelength generation and wavelength shifting. The beam foci may overlap at the sample, but may also be slightly offset from one another laterally and axially, if appropriate.

[0165] A TA-PARS, PARS, TE-PARS, TS-PARS, SR-PARS, SE-PARS or SD-PARS system may be used as a spectrometer for sample analysis.

[0166] Other advantages inherent in the structure will be apparent to those skilled in the art. The embodiments described herein are exemplary and do not limit the scope of the claims, which should be interpreted in light of the specification as a whole.

[0167] Purpose It should be understood that the systems described herein may be used in a variety of ways, such as for purposes described in the prior art, and may be used differently to take advantage of the aspects described above. A non-exhaustive list of uses is discussed below.

[0168] This system may be used to image angiogenesis in a variety of preclinical tumor models. The system may be used to unmix targets by utilizing different wavelengths, different pulse widths, different coherence lengths, repetition rates, exposure times, etc. based on the absorption, scattering or frequency content of the target.

[0169] The system may be used for imaging with resolution up to and beyond the diffraction limit. The system may be used to image anything that absorbs light, including exogenous and endogenous targets and biomarkers.

[0170] Some potential surgical applications of the system include functional and structural imaging during brain surgery, use to assess internal bleeding and cauterization verification, imaging perfusion sufficiency of organs and organ transplants, imaging vascularization around pancreatic islet transplants, imaging skin grafts, imaging tissue scaffolds and biomaterials to assess angiogenesis and immune rejection, imaging to assist in microsurgery, and guidance to avoid cutting critical blood vessels and nerves.

[0171] The system may also have several gastroenterological applications, such as imaging the vascular bed and depth of invasion in Barrett's esophagus and colorectal cancer. Depth of invasion, at least in some embodiments, is key to prognosis and metabolic potential. This depth can be used for virtual biopsies, monitoring Crohn's disease and IBS, and carotid artery examinations. For gastroenterological applications, the PARS system may be combined with or piggybacked off a clinical endoscope. The miniaturized PARS system can be designed as a standalone endoscope or fit within the accessory channel of a clinical endoscope.

[0172] The system may also be used for clinical imaging of microcirculation, macrocirculation, and pigment cells, and may find use in: (1) the eye, possibly augmenting or replacing fluorescein angiography; (2) imaging of skin lesions such as melanoma, basal cell carcinoma, hemangioma, psoriasis, eczema, dermatitis, etc.; imaging for Mohs surgery; imaging to verify tumor margin resection; (3) peripheral vascular disease; (4) diabetic ulcers and pressure ulcers; (5) burn imaging; (6) plastic surgery and microsurgery; (7) imaging of circulating tumor cells, particularly melanoma cells; (8) imaging of lymph node angiogenesis; (9) imaging of response to photodynamic therapy, including those with vascular destruction mechanisms; (10) imaging of response to chemotherapy, including anti-angiogenic drugs; (11) imaging of response to radiation therapy;

[0173] The system may also be used for several histopathology imaging applications, such as frozen pathology, creating H&E-like images from tissue samples, virtual biopsy, etc. The system may be used for a variety of tissue captures, such as formalin-fixed paraffin-embedded tissue blocks, formalin-fixed paraffin-embedded tissue slides, frozen pathology sections, freshly resected specimens, etc. Within these samples, visualization of macromolecules such as DNA, RNA, cytochromes, lipids, and proteins may be performed.

[0174] The system may be useful for estimating oxygen saturation using multi-wavelength PARS excitation in applications including (1) estimating venous oxygen saturation when pulse oximetry is unavailable, including estimating cerebral venous oxygen saturation and central venous oxygen saturation. This system may potentially be an alternative to catheterization, which can be dangerous, especially in small children and infants.

[0175] Oxygen flux and oxygen consumption can also be estimated using PARS imaging to estimate oxygen saturation and blood flow in the vessels flowing into and out of a region of tissue.

[0176] The system can be useful for separating prominent histological chromophores such as cell nuclei and surrounding cytoplasm by exploiting their respective absorption spectra. The system may be used to unmix a target with its absorption content, scattering, phase, polarization or frequency content by utilizing different wavelengths, different pulse widths, different coherence lengths, repetition rates, fluences, exposure times, etc.

[0177] Other example applications include imaging of contrast agents in clinical or preclinical applications, identification of sentinel lymph nodes, non-invasive or minimally invasive identification of tumors in lymph nodes, imaging of genetically encoded reporters such as tyrosinase, color proteins, fluorescent proteins, etc. for preclinical or clinical molecular imaging applications, imaging of actively or passively targeted light-absorbing nanoparticles for molecular imaging, and imaging of thrombi and possibly staging of thrombus age.

[0178] Other example applications include clinical and preclinical ophthalmology applications, such as measuring oxygen saturation and retinal metabolic rate in diseases such as age-related macular degeneration, diabetic retinopathy, and glaucoma, imaging of the peripheral vasculature and stem cells, imaging of corneal nerves and neovasculature, assessing changes in Schlemm's canal in patients with glaucoma, imaging of choroidal neovasculature, imaging of anterior and posterior segment blood flow, and blood flow status.

[0179] The system may be used to measure and estimate metabolism within a biological sample by leveraging the capabilities of both PARS and OCT. In this example, OCT may be used to estimate volumetric blood flow within a region of interest, and the PARS system may be used to measure oxygen saturation within the blood vessels of interest. In this case, combining these measurements provides an estimate of metabolism within that region.

[0180] The system may be used for head and neck and skin cancer types, functional brain activity, examining the vasculature of stroke patients to help locate blood clots, monitoring changes in gut bacterial composition, neuronal and brain function / development as a result of changes in atherosclerotic plaques, monitoring oxygen sufficiency after flap reconstruction, satisfaction after plastic or cosmetic surgery, and imaging of cosmetic injectables.

[0181] The system may be used for topological tracking of surface deformations. For example, OCT may be used to track the position of the sample surface. Corrections can then be applied to the tightly focused PARS device using mechanisms such as adaptive optics to maintain alignment with the surface as the scan continues.

[0182] The system can be implemented in a variety of different form factors that are suitable for applications such as tabletop microscopes, inverted microscopes, handheld microscopes, surgical microscopes, ophthalmic microscopes, and endoscopes.

[0183] Embodiment 1. A photoacoustic remote sensing and optical coherence tomography system for functional, structural, and multiplexed visualization of subsurface structures in a sample, comprising: one or more light sources configured to generate pressure and thermal signals in the sample at the excitation location; one or more light sources configured to generate an interrogation beam or collection of interrogation beams incident on the sample at an excitation location, with a portion of the interrogation beam or collection of interrogation beams returning from the sample indicative of the generated pressure and thermal signals; one or more light sources configured to generate an interrogation beam or a collection of interrogation beams incident on the sample at an excitation location, wherein a portion of the interrogation beam or collection of interrogation beams exhibits light scattering returning from the sample; a detector or collection of detectors configured to detect a return portion of the interrogation beam or collection of interrogation beams; an optical system configured to focus the beam onto the sample; A system including a processor configured to calculate an image of the sample based on a detected portion of the interrogation beam return from the sample.

[0184] A system including a nonlinear optical element configured to generate or modify beam characteristics. A system that uses the same light source for one or more of the PARS excitation / surveys and OCT surveys.

[0185] The system includes different embodiments such as tabletop, handheld, surgical microscope, ophthalmic microscope, endoscope, etc. The system wherein the light source may be any continuous, pulsed or modulated source of electromagnetic radiation having a wavelength ranging from about 50 nm to 100 μm.

[0186] A system including a nonlinear optical element configured to generate or modify beam characteristics. A system that uses the same light source for one or more of the PARS excitation / probe / signal enhancement beams.

[0187] The system includes different embodiments such as tabletop, handheld, surgical microscope, ophthalmic microscope, endoscope, etc. In some applications, the imaging head may not include any focusing elements.

[0188] A system with the first, second and third focal points located at depths ranging from 50 nm to 10 mm below the sample surface. A system in which all beams are focused onto and collected from the sample using the same focusing optics.

[0189] A system in which the beam is focused onto and collected from the sample using separate focusing optics. A system in which the focusing optics are normal to the surface.

[0190] A system in which the central axis of the focusing optic forms an angle between 0 and 85 degrees with the surface normal. A system in which the beam combiner is implemented using free-space optics.

[0191] A system in which the beam combiner is implemented using a fiber-based device. A system in which the imaging head performs optical scanning using a galvanometer mirror, MEMS mirror, polygon scanner, stepper / DC motor, etc.

[0192] A system in which a mechanical scanner, such as a stepper stage, DC motor stage, linear drive stage, piezo drive stage, or piezo stage, is used to scan the sample around the imaging head, or the imaging head around the sample, or both simultaneously.

[0193] A system in which the detector is an interferometer. A system in which the detector is a non-interferometric detector. A system in which the portion of the beam returning from the sample encodes the generated pressure and thermal signals as changes in [intensity, polarization, frequency, phase, fluorescence, nonlinear scattering, nonlinear absorption].

[0194] A system in which part of the beam returning from the sample is amplified by an optical amplifier. A system in which adaptive optics are used to adjust beam properties such as aberrations, focus, and to compensate for surface roughness.

[0195] The system is configured to generate structures on a sample through a [glass window, air, water, vacuum, or other material]. The system is configured with OCT detection to detect PARS modulation in the sample. In this case, OCT detection can function as short-coherence PARS interference detection, which can facilitate omitting PARS detection all together or enable depth-sensitive optical absorption recovery from within the sample. The system detects the PARS initial pressure signal at the origin, providing unique information about the optical absorption of the sample.

[0196] A system in which OCT detection is configured to detect vibrations and fluctuations generated by a PARS signal, which detects vibrations caused by PARS pressure propagation on and below the surface of the sample, providing unique information about the optical absorption of the sample.

[0197] The system wherein the OCT detection is configured to detect the topology of the sample. The system is configured such that the OCT detection detects the surface roughness of the sample. A dual-modality photoacoustic remote sensing combined with optical coherence tomography (PARS-OCT) system for visualizing details of a sample, the system comprising one or more light sources configured to generate (1) one or more excitation beams configured to generate signals in the sample at one or more first locations below the surface of the sample, (2) one or more interrogation beams incident on the sample at one or more second locations, (3) a sample beam, and (4) a reference beam, wherein a portion of the one or more interrogation beams returning from the sample represents the generated signals, and the system further comprises one or more light sources configured to generate one or more excitation beams configured to generate signals in the sample at one or more first locations below the surface of the sample, (2) one or more interrogation beams incident on the sample at one or more second locations, (3) a sample beam, and (4) a reference beam, wherein a portion of the one or more interrogation beams returning from the sample represents the generated signals, the sample arm is configured to direct a sample beam from one or more light sources to a third location, the reference arm is configured to direct a reference beam from one or more light sources into the path, the portion of the sample beam returning from the sample arm represents scattering collected by the sample arm, the portion of the reference beam returning from the reference arm represents scattering collected by the reference arm, and the interferometer is configured to detect the portion of the return from the one or more sample arms and the one or more reference arms.

[0198] A PARS-OCT system in which the signals generated by the one or more excitation beams include ultrasound signals, thermal signals, photoacoustic signals, and / or pressure signals, and a portion of the return of the one or more interrogation beams represents the generated ultrasound signals, thermal signals, photoacoustic signals, and / or pressure signals.

[0199] The PARS-OCT system further comprising one or more beam combiners configured to combine the at least one excitation beam, the at least one interrogation beam, and / or the sample beam prior to delivery toward the sample.

[0200] A PARS-OCT system, wherein one or more beam combiners are configured to direct a return portion of at least one interrogation beam to one or more first photodetectors and are also configured to direct a return portion of the sample beam to an interferometer.

[0201] A PARS-OCT system further including a brightfield microscope light source, wherein one or more beam combiners are configured to combine light from the brightfield microscope light source with at least one excitation beam, at least one interrogation beam, and a sample beam before delivering it to the sample.

[0202] A PARS-OCT system configured to provide absorption and scattering contrast of a sample. A PARS-OCT system further including a scope, the scope including a collimator and imaging optics, and wherein one or more excitation beams, one or more interrogation beams, and / or a sample beam are passed through the scope before being delivered towards the sample.

[0203] A PARS-OCT system, wherein the one or more light sources include a first light source configured to generate one or more of an excitation beam, a sample beam, and a reference beam. A PARS-OCT system, wherein the one or more light sources include a second light source configured to generate one or more interrogation beams.

[0204] A PARS-OCT system, wherein the one or more light sources include a first light source configured to generate one or more of an interrogation beam, a sample beam, and a reference beam. A PARS-OCT system further including one or more optical systems configured to focus or direct (1) one or more excitation beams to one or more first focal points and (2) one or more interrogation beams to one or more second focal points, wherein one or more of the first and second focal points are below the surface of the sample.

[0205] A PARS system, wherein one or more light sources are configured to generate one or more signal-enhanced beams incident on the sample at one or more first locations, the one or more first photodetectors are configured to detect a return portion of the one or more signal-enhanced beams, the return portion of the one or more signal-enhanced beams returning from the sample representing a generated PARS signal.

[0206] A PARS-OCT system in which one or more excitation beams contain exactly one wavelength and one or more signal enhancement beams contain multiple wavelengths. The PARS-OCT system further comprising a controller configured to determine a temperature of the specimen based on the intensity of feedback from the one or more photodetectors.

[0207] A PARS-OCT system further including a processing unit configured to obtain images with resolution greater than the optical diffraction limit by exploiting nonlinear optical absorption contrast effects in the sample, including light intensity induced optical absorption decay or photobleaching and nonlinear thermal dependence of material properties including thermal expansion coefficients, the processing unit configured to use several scans of the sample as input such that nonlinear PARS signal generation occurs between acquisitions to allow application of Vandermonde matrix-based processing to separate Nth-order power relationships.

[0208] 10. A PARS-OCT system further including one or more optical systems configured to disperse the one or more interrogation beams based on a wavelength or spatial positioning of the one or more interrogation beams, wherein the one or more optical systems are configured to recombine the one or more interrogation beams based on a wavelength or spatial positioning of the one or more interrogation beams.

[0209] The PARS-OCT system further includes one or more pinholes or apertures configured to map desired light onto the one or more first photodetectors when optically or mechanically scanning the beam or when mechanically scanning the sample or image head.

[0210] The interferometer is configured to detect PARS modulations, or vibrations and fluctuations produced by one or more excitation beams, in the sample, allowing OCT to give optical absorption contrast, a PARS-OCT system.

[0211] A dual-modality photoacoustic remote sensing combined with optical coherence tomography (PARS-OCT) system for visualizing details of a sample, providing tissue absorption and scattering contrast, comprising a PARS subsystem configured to generate (1) one or more excitation beams configured to generate ultrasound, thermal, photoacoustic, and / or pressure signals within the sample at one or more first locations; (2) one or more interrogation beams incident on the sample at one or more interrogation locations; and one or more optical systems configured to focus or direct the one or more excitation beams to one or more first foci and the one or more interrogation beams to one or more second foci, wherein the one or more first foci and the second foci are below the surface of the sample. or a pressure signal, and one or more photodetectors configured to detect the return portions of the one or more interrogation beams; and an OCT subsystem including one or more light sources and one or more interferometers each having a sample arm and a reference arm, wherein the sample arm directs a sample portion of the one or more light sources to a third focal point and the reference arm directs a reference portion of the one or more light sources into a path of known length, wherein the portion of light returning from the sample arm represents scattering collected by the sample arm, and the portion of the reference beam returning from the reference arm represents scattering collected by the reference arm, and the one or more interferometers are configured to detect the return portions from the sample arm and the reference arm; and wherein (1) the PARS subsystem and the OCT subsystem share at least one light source, or (2) the PARS subsystem and the OCT subsystem have only separate light sources.

[0212] Applications include: imaging of histological samples, imaging of cell nuclei, imaging of proteins, imaging of DNA, imaging of RNA, imaging of lipids, imaging of blood oxygen saturation, imaging of tumor hypoxia, wound healing, diagnostic or surgical imaging of burns, imaging of the microcirculation, imaging of blood oxygenation parameters, estimation of blood flow in blood vessels entering or leaving an area of ​​tissue, imaging of molecular-specific targets, imaging of angiogenesis in preclinical tumor models, clinical imaging of the micro- and macrocirculation and pigment cells, ocular imaging, augmenting or replacing fluorescein angiography, imaging of skin lesions, imaging of melanoma, imaging of basal cell carcinoma, imaging of hemangiomas, imaging of psoriasis, imaging of eczema, imaging of dermatitis, Mohs surgery imaging, imaging to verify tumor margin resection, imaging of peripheral vascular disease, imaging of diabetic ulcers and / or pressure ulcers, burn imaging, plastic surgery, microsurgery, imaging of circulating tumor cells, imaging of melanoma cells, imaging of lymph node angiogenesis, photodynamic therapy imaging of response to chemotherapy, imaging of response to photodynamic therapy with vascular disruption mechanisms, imaging of response to chemotherapy, imaging of frozen pathology specimens, imaging of paraffin-embedded tissue, imaging of H&E-like images, imaging of oxygen metabolism changes, imaging of response to anti-angiogenic drugs, imaging of response to radiation therapy, estimation of oxygen saturation using multi-wavelength PARS excitation, estimation of venous oxygen saturation when pulse oximetry is not available, estimation of cerebral venous oxygen saturation and / or central venous oxygen saturation, estimation of oxygen flux and / or oxygen consumption, imaging of vascular beds and depth of invasion in Barrett's esophagus and / or colorectal cancer, functional and structural imaging during brain surgery, use for assessing internal bleeding and / or ablation verification, imaging of perfusion sufficiency of organs and / or organ transplants, imaging of angiogenesis around pancreatic islet transplants, imaging of skin transplants, imaging of tissue scaffolds and / or biomaterials to assess angiogenesis and / or immune rejection, imaging to assist microsurgery,guidance to avoid cutting blood vessels and / or nerves, imaging of contrast agents in clinical or preclinical applications, identification of sentinel lymph nodes, non-invasive or minimally invasive identification of tumors in lymph nodes, imaging of genetically encoded reporters including tyrosinase, color proteins, and / or fluorescent proteins for preclinical or clinical molecular imaging applications, imaging of actively or passively targeted light-absorbing nanoparticles for molecular imaging, imaging of thrombi, staging of thrombi age, remote or non-invasive intratumoral assessment of glucose concentration by detecting endogenous glucose absorption peaks, assessment of organoid growth, monitoring of developing embryos, assessment of biofilm composition, assessment of dental caries, assessment of non-living structures, assessment of painting formulations to non-invasively confirm authenticity, assessment of archaeological artifacts, manufacturing quality control, manufacturing quality assurance, replacement of catheter procedures, gastroenterological applications, single excitation pulse imaging across the entire field of view, imaging of tissues, imaging of cells, imaging of scattered light from an object surface, imaging of absorption-induced changes in scattered light, or non-contact imaging of light absorption. The technical concepts that can be understood from the above-described embodiment will be described below as supplementary notes. [Appendix 1] 1. A method for visualizing details in a sample, comprising: directing an excitation beam to an excitation location below the surface of the sample to generate a signal in the sample; directing an interrogation beam towards the excitation location of the sample; directing a signal-enhancing beam to the sample to increase the temperature of a portion of the sample compared to a temperature of the portion of the sample in the absence of the signal-enhancing beam, wherein the portion of the sample is within a focus of the interrogation beam; detecting a portion of the interrogation beam returning from the sample, the portion representing the generated signal. [Appendix 2] 2. The method of claim 1, wherein the excitation beam has a pulse width in the nanosecond or picosecond range, and the signal-enhancing beam has a longer pulse width or is a continuous beam. [Appendix 3] 2. The method of claim 1, further comprising calculating an image of the sample based on a portion of the interrogation beam returning from the sample. [Appendix 4] 2. The method of claim 1, further comprising detecting autofluorescent light returning from a portion of the sample. [Appendix 5] 2. The method of claim 1, wherein directing the signal-enhancing beam to the sample increases the value of the signal generated in the sample compared to the value of the signal generated in the sample in the absence of the signal-enhancing beam. [Appendix 6] 2. The method of claim 1, wherein directing the signal-enhancing beam to the sample alters an optical property of a portion of the sample compared to a value of the portion of the sample in the absence of the signal-enhancing beam. [Appendix 7] 7. The method of claim 6, wherein the optical property that is altered comprises a local refractive index or Gruneisen parameter of a portion of the sample. [Appendix 8] detecting a portion of the interrogation beam returning from the sample, the portion representing the generated signal, further comprises extracting an absorption signal from the returning portion of the interrogation beam; determining a scattered intensity from a return portion of the interrogation beam; 2. The method of claim 1, further comprising generating a composite image from the extracted absorption signal and the determined scattering intensity. [Appendix 9] 2. The method of claim 1, wherein the signal generated by the excitation beam includes an ultrasound signal, a thermal signal, a photoacoustic signal, and / or a pressure signal, and a portion of the return of the interrogation beam represents the generated ultrasound signal, thermal signal, photoacoustic signal, and / or pressure signal. [Appendix 10] 2. The method of claim 1, further comprising analyzing a portion of the interrogation beam returning from the sample for amplitude / intensity, frequency content, content related to polarization changes, fluorescence, second harmonic generation, and / or phase variations. [Appendix 11] 10. The method of claim 1, further comprising exploiting absorption-induced modulation effects including one or more of modulation of material reflectivity, scattering, polarization, phase accumulation, nonlinear absorption, and / or nonlinear scattering. [Appendix 12] The method comprises: Imaging of histological samples, Imaging of cell nuclei, Protein imaging, DNA imaging, RNA imaging, lipid imaging, Blood oxygen saturation imaging, Imaging tumor hypoxia, Imaging for wound healing, burn diagnosis, or surgery, Imaging of the microcirculation, Imaging of blood oxygenation parameters, Estimation of blood flow in blood vessels flowing into and out of tissue regions; Imaging of molecular-specific targets, Imaging angiogenesis in preclinical tumor models Clinical imaging of microcirculation and macrocirculation and pigment cells, Ocular imaging, Augmenting or replacing fluorescein angiography, Imaging of skin lesions, melanoma imaging, Imaging of basal cell carcinoma, Imaging of hemangiomas, Imaging of psoriasis, Eczema imaging, Dermatitis imaging, Mohs surgery imaging, Imaging to verify tumor margin resection; Imaging of peripheral vascular disease, Imaging of diabetic ulcers and / or pressure ulcers, Burn imaging, plastic surgery, Microsurgery, Imaging of circulating tumor cells, Imaging of melanoma cells, Imaging of lymph node angiogenesis, Imaging the response to photodynamic therapy, Imaging the response to photodynamic therapy with vascular disruption mechanisms Imaging response to chemotherapy Imaging of frozen pathology samples, Imaging of paraffin-embedded tissues, Imaging of H&E-like images, Imaging of changes in oxygen metabolism, Imaging the response to antiangiogenic drugs Imaging response to radiation therapy, Oxygen saturation estimation using multi-wavelength PARS excitation, Estimation of venous oxygen saturation when pulse oximetry is unavailable; Estimation of cerebral venous oxygen saturation and / or central venous oxygen saturation, Estimation of oxygen flux and / or oxygen consumption, Imaging of the vascular bed and depth of invasion in Barrett's esophagus and / or colorectal cancer functional and structural imaging during brain surgery, functional and structural imaging during brain surgery, Assessment of internal bleeding and / or cauterization verification; Imaging perfusion sufficiency of organs and / or organ transplants; Imaging of angiogenesis around pancreatic islet transplants Imaging of skin grafts, Imaging of tissue scaffolds and / or biomaterials to assess angiogenesis and / or immune rejection; Imaging to assist microsurgery, Guidance on how to avoid cutting blood vessels and / or nerves; Imaging contrast agents in clinical or preclinical applications, Identification of sentinel lymph nodes, Non-invasive or minimally invasive identification of tumors in lymph nodes; Imaging of genetically encoded reporters, including tyrosinase, color proteins, and / or fluorescent proteins, for preclinical or clinical molecular imaging applications; Imaging of actively or passively targeted light-absorbing nanoparticles for molecular imaging; thrombus imaging, Thrombosis age staging, Remote or non-invasive intratumoral assessment of glucose concentrations by detecting endogenous glucose absorption peaks; Assessment of organoid growth, monitoring developing embryos, Assessment of biofilm composition, evaluation of dental caries, Assessment of abiotic structures, Evaluation of painting formulations to non-invasively confirm authenticity; evaluation of archaeological remains; manufacturing quality control, Manufacturing quality assurance, catheterization replacement, gastroenterological applications, Single excitation pulse imaging across the entire field of view tissue imaging, Cell imaging, Imaging of scattered light from object surfaces, Imaging absorption-induced changes in scattered light, or 2. The method of claim 1, wherein the method is used in one or more applications of non-contact imaging of optical absorption. [Appendix 13] 1. A method for visualizing details in a sample, comprising: directing an excitation beam to an excitation location below the surface of the sample to generate a signal in the sample; directing an interrogation beam towards the excitation location of the sample; extracting an absorption signal from a return portion of the interrogation beam; determining a scattered intensity from a return portion of the interrogation beam; generating a composite image from the extracted absorption signal and the determined scattering intensity. [Appendix 14] 14. The method of claim 13, wherein extracting the absorption signal comprises extracting the absorption signal from a portion of a filtered return of the interrogation beam. [Appendix 15] 14. The method of claim 13, wherein determining the scattered intensity is determined from a portion of the unfiltered return of one or more interrogation beams. [Appendix 16] 14. The method of claim 13, further comprising unmixing constituent chromophores from within the sample by using one or more of the wavelength, pulse width, power, energy, coherence length, repetition rate, and exposure time of the excitation and / or detection beams. [Appendix 17] 14. The method of claim 13, further comprising using the collected time-domain behavioral features to improve signal fidelity, enhance image contrast, recover information about the shape, size, and dimensions of the sample, or perform multiplexing / functional imaging. [Appendix 18] 14. The method of claim 13, further comprising using lock-in amplification, machine learning methods, extensive feature extraction, multidimensional decomposition, and / or frequency content based feature extraction and signal processing methods. [Appendix 19] 14. The method of claim 13, further comprising unmixing the composition of the target based on absorption, temperature, polarization, frequency, phase, nonlinear absorption, fluorescence, nonlinear scattering, and scattering content of the target. [Appendix 20] 14. The method of claim 13, further comprising unmixing the size, shape, features, and dimensions of the target based on absorption, temperature, polarization, frequency, phase, nonlinear absorption, nonlinear scattering, and scattering content of the target. [Appendix 21] 14. The method of claim 13, further comprising unmixing the target with its absorption content, scattering content, fluorescence, polarization content, frequency content, and phase content by utilizing different wavelengths, different pulse widths, different coherence lengths, repetition rates, laser exposure times, and / or laser fluences. [Appendix 22] 1. A photoacoustic remote sensing (PARS-OCT) system for visualizing details of a specimen, comprising: one or more first laser light sources configured to generate (1) one or more excitation beams configured to generate a signal in the sample at one or more first locations, and (2) one or more interrogation beams incident on the sample at one or more second locations; a laser light source separate from the one or more first laser light sources, wherein a second light source is configured to generate one or more signal-enhanced beams incident on the sample at one or more third locations; the one or more first locations, the one or more second locations, and the one or more third locations are below a surface of the sample; a portion of the one or more interrogation beams returning from the sample represents a generated signal; The system further one or more first photodetectors configured to detect portions of the one or more interrogation beams returning from the sample; and a processor configured to calculate an image of the sample based on a portion of one or more interrogation beams returning from the sample, wherein the portion of the one or more interrogation beams returning from the sample is modified and / or enhanced by the one or more signal-enhancing beams compared to a signal generated without the one or more signal-enhancing beams. [Appendix 23] 1. A system for visualizing details in a specimen, providing tissue absorption and scattering contrast, said system comprising: one or more light sources configured to generate (1) one or more excitation beams configured to generate ultrasound, thermal, photoacoustic, and / or pressure signals in the sample at one or more first locations; and (2) one or more interrogation beams incident on the sample at one or more interrogation locations, the one or more interrogation beams having a different wavelength than the one or more excitation beams; one or more optical systems configured to focus or direct the one or more excitation beams to the one or more first focal points and the one or more interrogation beams to one or more second focal points, the one or more first and second focal points being below a surface of the sample; a portion of the one or more interrogation beams returning from the sample representing a generated ultrasonic signal, a thermal signal, a photoacoustic signal, and / or a pressure signal; one or more photodetectors configured to detect a return portion of said one or more interrogation beams; one or more processors, extracting an absorption signal from a return portion or portions of said one or more interrogation beams; determining a scattered intensity from a portion or portions of the return of said one or more interrogation beams; and the one or more processors configured to generate a composite image from the extracted absorption signals and the determined scattering intensities. [Appendix 24] 1. A photoacoustic remote sensing system for visualizing details of a sample, comprising: one or more lasers configured to generate (1) an excitation beam configured to generate a signal in the sample at an excitation location; and (2) an interrogation beam incident on the sample at the excitation location, a portion of the interrogation beam returning from the sample representing the generated signal; an optical system configured to focus the excitation beam and / or the interrogation beam below the surface of the sample; a filter configured to separate a return portion of the interrogation beam from autofluorescence light returning from the sample; a first detector for detecting a return portion of the interrogation beam; and a second detector that detects the autofluorescent light returning from the sample. [Appendix 25] 1. A method for visualizing details in a sample, comprising: directing an excitation beam to an excitation location below the surface of the sample to generate a signal in the sample; directing an interrogation beam towards the excitation location of the sample; detecting a portion of the interrogation beam returning from the sample, the portion representing the generated signal; and detecting autofluorescent light returning from said sample. [Appendix 26] 1. A system for visualizing details of a specimen, comprising: one or more light sources configured to generate (1) one or more excitation beams configured to generate a signal in the sample at one or more first locations below the surface of the sample, and (2) one or more interrogation beams incident on the sample at one or more second locations; a second light source, separate from the one or more light sources, configured to generate one or more signal-enhanced beams incident on the sample at the one or more first locations, the one or more signal-enhanced beams configured to increase a temperature of the sample within a focus of the one or more interrogation beams, any of the one or more signal-enhanced beams having a lower intensity than each of the one or more excitation beams; a portion of the one or more interrogation beams returning from the sample represents a generated signal; The system further comprises: one or more first photodetectors configured to detect portions of the one or more interrogation beams returning from the sample; the system further includes a processor configured to calculate an image of the sample based on portions of the one or more interrogation beams returning from the sample, wherein intensity modulation of the portions of the one or more interrogation beams returning from the sample is increased by the one or more signal-enhancing beams due to an increase in temperature of the sample within the focal points of the one or more interrogation beams altering the local refractive index of the sample compared to intensity modulation of the portions of the one or more interrogation beams returning from the sample generated without the one or more signal-enhancing beams; 10. A system wherein signals generated by the one or more excitation beams include ultrasound signals, thermal signals, photoacoustic signals, fluorescence signals, and / or pressure signals, and wherein a portion of the return of the one or more interrogation beams represents the generated ultrasound signals, thermal signals, photoacoustic signals, and / or pressure signals. [Appendix 27] 1. A system for providing tissue absorption and scattering contrast for visualizing details of a specimen, comprising: one or more light sources configured to generate (1) one or more excitation beams configured to generate ultrasound, fluorescence, thermal, photoacoustic, and / or pressure signals in at least a first target within the sample at one or more first locations; and (2) one or more interrogation beams incident on the sample at one or more interrogation locations, the one or more interrogation beams having a different wavelength than the one or more excitation beams, the wavelength of the one or more interrogation beams being configured to suppress any ultrasound, thermal, photoacoustic, and / or pressure signals generated by a second target within the sample due to the one or more excitation beams irradiating the sample by collecting an absorbed energy level of the second target, the first target and the second target having different chromophores; one or more optical systems configured to focus or direct the one or more excitation beams at the one or more first foci and the one or more interrogation beams at one or more second foci, the one or more first foci and second foci being below a surface of the sample, wherein a portion of the one or more interrogation beams returning from the sample represents a generated ultrasound signal, a thermal signal, a photoacoustic signal, a fluorescence signal, and / or a pressure signal; a portion of the one or more excitation beams returning from the sample representing a generated ultrasound signal, a thermal signal, a photoacoustic signal, a fluorescence signal, and / or a pressure signal; The system further comprises: one or more photodetectors configured to detect a return portion of said one or more interrogation beams; 1. A processor, comprising: unmixing the first target and the second target within the sample with respect to one another and assigning different colors to the first target and the second target based on an optical property of the first target and an optical property of the second target; and the processor configured to cause the different colors to be simultaneously shown on a single image, wherein the first target and the second target each comprise endogenous and / or exogenous chromophores, and the processor configured to unmix the first target and the second target after scanning the sample with the one or more light sources.

Claims

1. 1. A method for visualizing details in a sample, comprising: directing an excitation beam to an excitation location below the surface of the sample to generate a signal in the sample; directing an interrogation beam towards the excitation location of the sample; directing a signal-enhancing beam to the sample to increase the temperature of a portion of the sample compared to a temperature of the portion of the sample in the absence of the signal-enhancing beam, wherein the portion of the sample is within a focus of the interrogation beam; detecting a portion of the interrogation beam returning from the sample, the portion representing the generated signal.

2. 10. The method of claim 1, wherein the excitation beam has a pulse width in the nanosecond or picosecond range and the signal enhancement beam has a longer pulse width or is a continuous beam.

3. The method of claim 1 , further comprising calculating an image of the sample based on a portion of the interrogation beam returning from the sample.

4. The method of claim 1 further comprising detecting autofluorescent light returning from the portion of the sample.

5. 2. The method of claim 1, wherein directing the signal-enhancing beam to the sample increases the value of the signal generated in the sample compared to the value of the signal generated in the sample in the absence of the signal-enhancing beam.

6. 10. The method of claim 1, wherein directing the signal-enhancing beam to the sample alters an optical property of a portion of the sample compared to a value of the portion of the sample in the absence of the signal-enhancing beam.

7. The method of claim 6 , wherein the optical properties that are altered include the local refractive index or Gruneisen parameters of a portion of the sample.

8. detecting a portion of the interrogation beam returning from the sample, the portion representing the generated signal, further comprises extracting an absorption signal from the returning portion of the interrogation beam; determining a scattered intensity from a return portion of the interrogation beam; and generating a composite image from the extracted absorption signal and the determined scattering intensity.

9. 2. The method of claim 1, wherein the signal generated by the excitation beam comprises an ultrasound signal, a thermal signal, a photoacoustic signal, and / or a pressure signal, and a portion of the interrogation beam return represents the generated ultrasound signal, a thermal signal, a photoacoustic signal, and / or a pressure signal.

10. 10. The method of claim 1, further comprising analyzing a portion of the interrogation beam returning from the sample for amplitude / intensity, frequency content, content related to polarization changes, fluorescence, second harmonic generation, and / or phase variations.

11. The method of claim 1 , further comprising exploiting absorption-induced modulation effects including one or more of modulation of material reflectivity, scattering, polarization, phase accumulation, nonlinear absorption, and / or nonlinear scattering.

12. The method comprises: Imaging of histological samples, Imaging of cell nuclei, Protein imaging, DNA imaging, RNA imaging, lipid imaging, Blood oxygen saturation imaging, Imaging tumor hypoxia, Imaging for wound healing, burn diagnosis, or surgery, Imaging of the microcirculation, Imaging of blood oxygenation parameters, Estimation of blood flow in blood vessels flowing into and out of tissue regions; Imaging of molecular-specific targets, Imaging angiogenesis in preclinical tumor models Clinical imaging of microcirculation and macrocirculation and pigment cells, Ocular imaging, Augmenting or replacing fluorescein angiography, Imaging of skin lesions, melanoma imaging, Imaging of basal cell carcinoma, Imaging of hemangiomas, Imaging of psoriasis, Eczema imaging, Dermatitis imaging, Mohs surgery imaging, Imaging to verify tumor margin resection; Imaging of peripheral vascular disease, Imaging of diabetic ulcers and / or pressure ulcers; Burn imaging, plastic surgery, Microsurgery, Imaging of circulating tumor cells, Imaging of melanoma cells, Imaging of lymph node angiogenesis, Imaging the response to photodynamic therapy, Imaging the response to photodynamic therapy with vascular disruption mechanisms Imaging response to chemotherapy Imaging of frozen pathology samples, Imaging of paraffin-embedded tissues, H&E-like imaging, Imaging of changes in oxygen metabolism, Imaging the response to antiangiogenic drugs Imaging response to radiation therapy, Oxygen saturation estimation using multi-wavelength PARS excitation; Estimation of venous oxygen saturation when pulse oximetry is unavailable; estimation of cerebral venous oxygen saturation and / or central venous oxygen saturation; Estimation of oxygen flux and / or oxygen consumption; Imaging of the vascular bed and depth of invasion in Barrett's esophagus and / or colorectal cancer; functional and structural imaging during brain surgery, functional and structural imaging during brain surgery, Assessment of internal bleeding and / or cauterization verification; Imaging perfusion sufficiency of organs and / or organ transplants; Imaging of angiogenesis around pancreatic islet transplants Imaging of skin grafts, Imaging of tissue scaffolds and / or biomaterials to assess angiogenesis and / or immune rejection; Imaging to assist microsurgery, Guidance to avoid cutting blood vessels and / or nerves; Imaging contrast agents in clinical or preclinical applications, Identification of sentinel lymph nodes, Non-invasive or minimally invasive identification of tumors in lymph nodes; Imaging of genetically encoded reporters, including tyrosinase, color proteins, and / or fluorescent proteins, for preclinical or clinical molecular imaging applications; Imaging of actively or passively targeted light-absorbing nanoparticles for molecular imaging; thrombus imaging, Thrombosis age staging, Remote or non-invasive intratumoral assessment of glucose concentrations by detecting endogenous glucose absorption peaks; Assessment of organoid growth, monitoring developing embryos, Assessment of biofilm composition, evaluation of dental caries, Assessment of abiotic structures, Evaluation of painting formulations to non-invasively confirm authenticity; evaluation of archaeological remains; manufacturing quality control, Manufacturing quality assurance, catheterization replacement, gastroenterological applications, Single excitation pulse imaging across the entire field of view, tissue imaging, Cell imaging, Imaging of scattered light from object surfaces, Imaging absorption-induced changes in scattered light, or Non-contact imaging of optical absorption The method of claim 1 for use in one or more of the following applications:

13. 1. A photoacoustic remote sensing (PARS-OCT) system for visualizing details of a sample, comprising: one or more first laser light sources configured to generate (1) one or more excitation beams configured to generate a signal in the sample at one or more first locations, and (2) one or more interrogation beams incident on the sample at one or more second locations; a laser light source separate from the one or more first laser light sources, wherein the second light source is configured to generate one or more signal-enhanced beams incident on the sample at one or more third locations; the one or more first locations, the one or more second locations, and the one or more third locations are below a surface of the sample; a portion of the one or more interrogation beams returning from the sample represents a generated signal; The system further one or more first photodetectors configured to detect portions of the one or more interrogation beams returning from the sample; and a processor configured to calculate an image of the sample based on a portion of one or more interrogation beams returning from the sample, wherein the portion of the one or more interrogation beams returning from the sample is modified and / or enhanced by the one or more signal-enhancing beams compared to a signal generated without the one or more signal-enhancing beams.

14. 1. A system for visualizing details of a specimen, comprising: one or more light sources configured to generate (1) one or more excitation beams configured to generate a signal in the sample at one or more first locations below the surface of the sample, and (2) one or more interrogation beams incident on the sample at one or more second locations; a second light source, separate from the one or more light sources, configured to generate one or more signal-enhanced beams incident on the sample at the one or more first locations, the one or more signal-enhanced beams configured to increase a temperature of the sample in a focus of the one or more interrogation beams, any of the one or more signal-enhanced beams having a lower intensity than each of the one or more excitation beams; a portion of the one or more interrogation beams returning from the sample represents a generated signal; The system further comprises: one or more first photodetectors configured to detect portions of the one or more interrogation beams returning from the sample; the system further includes a processor configured to calculate an image of the sample based on portions of the one or more interrogation beams returning from the sample, wherein intensity modulation of the portions of the one or more interrogation beams returning from the sample is increased by the one or more signal-enhancing beams due to an increase in temperature of the sample within the focal points of the one or more interrogation beams altering the local refractive index of the sample compared to intensity modulation of the portions of the one or more interrogation beams returning from the sample generated without the one or more signal-enhancing beams; wherein the signals generated by the one or more excitation beams include ultrasound signals, thermal signals, photoacoustic signals, fluorescence signals, and / or pressure signals, and wherein a portion of the return of the one or more interrogation beams represents the generated ultrasound signals, thermal signals, photoacoustic signals, and / or pressure signals.

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