Method and apparatus for broadband optical photothermal infrared spectroscopy

Abstract

To improve sampling time while collecting microscopic data on the composition of a sample over a wide field of view.SOLUTION: The system includes an infrared radiation source 200 configured to illuminate a broad area of a sample with a beam of infrared radiation, a probe radiation source 222 configured to illuminate the broad area of the sample with a beam of probe radiation, a collector 212 configured to collect as collected probe radiation at least a portion of the probe radiation from each of a plurality of spatially resolved locations on the sample, and at least one camera 250 configured to detect as collected probe radiation at least a portion of the probe radiation from each of the plurality of spatially resolved locations on the sample and generate a signal indicative of infrared absorption corresponding to each of the plurality of spatially resolved locations.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] This disclosure relates to the investigation and analysis of materials by optical means, i.e., using infrared, visible, or ultraviolet light.

[0002] Priority Various aspects of the embodiments and techniques described in this application are described in U.S. Provisional Application No. 62 / 679,588, filed June 1, 2018, the disclosure of which is incorporated herein by reference. [Background technology]

[0003] The present disclosure relates to infrared spectroscopy and imaging with spatial resolution down to the submicron scale using optical photothermal detection techniques. Several optical photothermal techniques are described, for example, in U.S. Patent Nos. 9,091,594 and 9,841,324. These references often refer to the techniques by different names and acronyms. For purposes of this application, these techniques will be collectively referred to herein as optical photothermal infrared (OPTIR).

[0004] Several research groups have worked on this general area of ​​OPTIR, including researchers from the Naval Research Laboratory, Purdue University, University of Notre Dame, Boston University, and Massachusetts Institute of Technology. Instruments developed in these laboratories use visible light to probe the photothermal response of samples in response to the absorption of infrared radiation. Possibly relevant background publications and patents include: (1) R. Furstenberg, C. Kendziora, ND Bassim, RA McGill, and VK Nguyen, US9091594 B2 (2015); (2) C. Li, D. Zhang, MN Slipchenko and J.-X. Cheng, Anal. Chem., 89, 9, 4863-4867 (2017); (3) D. Zhang, C. Li, C. Zhang, MN Slipchenko, G. Eakins, and J.-X. Cheng, Science Advances, 2, 9, e1600521 (2016). (4) Z. Li, K. Aleshire, M. Kuno, and GV Hartland, The Journal of Physical Chemistry B, 121, 37, 8838-8846 (2017); (5) Z. Li, M. Kuno, and G. Hartland, "Super-resolution imaging with mid-IR photothermal microscopy on the single particle level", in SPIE Nanoscience+ Engineering (International Society for Optics and Photonics, 2015), p. 954912-954912-954918; (6) Z. Li, M. Kuno, and G.Hartland, "Super-resolution Mid-infrared Imaging using Photothermal Microscopy," in Conference on Lasers and Electro-Optics (Optical Society of America, San Jose, California, 2016), p. ATu3J.7; (7) A. Mërtiri, A. Totachawattana, H. Liu, MK Hong, T. Gardner, M.Y. Sander, and S. Erramilli, "Label-free mid-IR photothermal imaging of bird brain with quantum cascade laser," in CLEO: Applications and Technology (Optical Society of America, 2014), p. AF1B.4; (8) M.Y. Sander, "Mid-infrared photothermal imaging," in Laser Science (Optical Society of America, 2015), p. LM1I.2; (9) U.S. Patent No. 9,091,594 B2, assigned to the Secretary of the Navy, entitled "Chemical mapping using thermal microscopy at the micro and nano scales,”.

[0005] There are also devices using off-axis illumination and camera sensors to detect variations in light scattering, as discussed, for example, in RM Sullenberger, SM Redmond, D. Crompton, AM Stolyarov, and WD Herzog, Opt. Lett., 42, 2, 203-206 (2017), as well as photothermal modulation of laser speckles, as discussed, for example, in AM Stolyarov, RM Sullenberger, DR Crompton, TH Jeys, BG Saar, and WD Herzog, Opt. Lett., 40, 24, 5786-5789 (2015). However, these approaches are not suitable for microscopy applications at submicron dimensions due to focal length / numerical aperture limitations in the sample imaging optics.

[0006] A key limitation of conventional photothermal imaging and spectroscopy techniques is the extremely small photothermal effect due to IR absorption. For example, the overall intensity modulation in the collected probe beam due to the absorption of IR radiation by the sample can be 3 to 6 orders of magnitude smaller than the average intensity of all collected probe beams. This can make it difficult to detect small absorptions of IR radiation from either weakly absorbing samples, samples with weak photothermal responses, or microscopically small amounts of sample material. Increasing the measurement time to improve accuracy is often impractical. While measurement accuracy increases proportionally to the square root of the sampling time, increasing the accuracy of OPTIR detectors by increasing the sampling time is practically limited by the need to increase the sampling time by a factor of 100, for example, to improve accuracy by one order of magnitude. Summary of the Invention

[0007] According to embodiments described herein, an apparatus for microscopic analysis of a sample is provided by simultaneously characterizing infrared absorption properties at multiple spatially resolved locations, the multiple spatially resolved locations corresponding to large areas of the sample. The apparatus includes an infrared radiation source configured to illuminate the large area of ​​the sample with a beam of infrared radiation, a probe radiation source configured to illuminate the large area of ​​the sample with a beam of probe radiation, a collector configured to collect at least a portion of the probe radiation from each of the multiple spatially resolved locations on the sample as collected probe radiation, and at least one camera configured to detect at least a portion of the probe radiation from each of the multiple spatially resolved locations on the sample as collected probe radiation and generate signals indicative of infrared absorption corresponding to each of the multiple spatially resolved locations.

[0008] The present application relates to a novel instrument and method for improving measurement throughput through measurements of IR absorption at the microscopic scale of submicron dimensions simultaneously over a large area of ​​a sample. The above summary is not intended to describe each illustrated embodiment or every implementation of the subject matter herein. The following figures and detailed description more particularly exemplify various embodiments. [Brief explanation of the drawings]

[0009] The subject matter herein can be more fully understood in consideration of the following detailed description of various embodiments in conjunction with the accompanying figures. [Figure 1] Figure 1 shows a schematic diagram of the photothermal principle employed in optical photothermal infrared (OPTIR) spectroscopy and imaging. [Figure 2] FIG. 2 is a schematic diagram of an embodiment of a wide-field OPTIR system. [Figure 3] Figure 3 shows an example of time-resolved measurements of the photothermal response to IR absorption. [Figure 4] Figure 4 shows an example of gating the IR absorption signal from the probe beam. [Figure 5] 5A-5C show probe beam illumination according to three embodiments. [Figure 6] FIG. 6 illustrates various illumination schemes according to one embodiment. [Figure 7] FIG. 7 illustrates various illumination schemes according to one embodiment. [Figure 8] FIG. 8 illustrates various illumination schemes according to one embodiment. [Figure 9] FIG. 9 shows an embodiment in which multiple discrete sampling locations over a large area are measured simultaneously. [Figure 10] FIG. 10 shows an output illustrating various substances detected by a sensor according to one embodiment described herein. DETAILED DESCRIPTION OF THE INVENTION

[0010] While various embodiments are susceptible to various modifications and alternative forms, details thereof have been shown by way of example in the drawings and will be described in detail. It should be understood, however, that the intention is not to limit the claimed invention to the particular embodiments described herein. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the subject matter as defined by the claims.

[0011] This specification describes methods and apparatus for performing optical photothermal infrared (OPTIR) imaging and spectroscopy with improved sensitivity, improved signal-to-noise ratio, and reduced background signal. The use of wide-area techniques allows for simultaneous parallel measurements to improve scan speed and accuracy.

[0012] Below are some definitions of phrases used throughout the application:

[0013] "Illumination" means directing radiation onto the surface of an object, e.g., a sample. Illumination can include any arrangement of radiation sources, pulse generators, modulators, reflective components, focusing elements, and any other beam steering or beam conditioning components.

[0014] In the context of light interacting with a sample, the term "interacts" means that the light illuminating the sample is at least one of scattered, refracted, absorbed, aberrated, diverted, diffracted, transmitted, and reflected as it passes through or exits the sample.

[0015] "Infrared source" and "infrared radiation source" refer to one or more light sources that generate or emit radiation in the infrared wavelength range, generally between 2 and 25 microns. The radiation source may be one of a number of radiation sources, including thermal or globar sources, supercontinuum laser sources, frequency combs, difference frequency generators, sum frequency generators, harmonic generators, optical parametric oscillators (OPOs), optical parametric generators (OPGs), quantum cascade lasers (QCLs), nanosecond, picosecond, femtosecond, and attosecond laser systems, CO2 lasers, microscopic heaters, and other light sources that generate a radiation beam. In a preferred embodiment, the light source emits infrared radiation, but alternatively or as well, it can emit radiation in other wavelength ranges, for example, from ultraviolet to THz. The light source may be, for example, a laser with a spectral width of 10 cm. -1 Less than or equal to 1 cm -1 It may be narrow band or less than 10 cm -1 Larger than 100cm -1 Greater than or equal to 500cm -1 It may be a larger wideband.

[0016] A "probe beam" is a beam of light that is focused onto a sample to detect photothermal or other optical distortions resulting from the interaction of the sample with IR radiation, for example, detecting absorption of IR radiation by the sample.

[0017] "Probe radiation source" refers to a radiation source that generates a probe beam. Probe radiation sources generally generate a probe beam with one or more emission wavelengths in the visible to ultraviolet wavelength range, but may also or instead generate radiation at shorter or longer wavelengths, e.g., in the near-infrared, extreme ultraviolet, or X-ray ranges. Probe radiation sources can include, for example, gas lasers, laser diodes, diode-pumped solid-state lasers, superluminescent diodes (SLDs), UV and / or visible laser beams generated, e.g., by sum or difference frequency generation, or other lasers or other coherent radiation. They can also include incoherent light sources, e.g., incandescent light sources, light-emitting diodes, or other light sources. In one example, they can also include any other UV or visible light that can be focused to a spot on the scale of less than 2.5 micrometers, preferably less than 1 micrometer.

[0018] "Spectrum" refers to the measurement of one or more properties of a sample as a function of wavelength, or equivalently (and more generally) as a function of wavenumber.

[0019] "Optical property" refers to an optical property of a sample, including, but not limited to, refractive index, absorption coefficient, reflectance, absorptance, scattering, real and imaginary components of the refractive index, real and imaginary components of a derivative function of the sample, and / or any property that can be mathematically derived from one or more of these optical properties.

[0020] "Optical response" refers to the result of the interaction of radiation with a sample. The optical response is related to one or more of the optical properties defined above. The optical response can be absorption of radiation, temperature increase, thermal expansion, photoinduced forces, light reflection or scattering, or other response of a material upon interaction with illuminating radiation.

[0021] A "signal indicative of" refers to a signal mathematically related to a property of interest. The signal may be an analog signal, a digital signal, and / or one or more numbers stored in a computer or other digital electronic device. The signal may be a voltage, a current, or any other signal that can be easily converted and recorded. The signal may be mathematically identical to the property being specifically measured, such as an absolute phase signal or an absorption coefficient. It may also be a signal mathematically related to one or more properties of interest, such as by linear or other scaling, offsetting, inversion, or complex mathematical manipulation.

[0022] A "scanner" is one or more scanning mechanisms used to generate relative motion between a probe and a sample, allowing the probe to interact with multiple locations on the sample and measure its properties. The scanning mechanism can move either the probe, the sample, or a combination of these. Scanning mechanisms are typically piezoelectric devices, but electromagnetic, electrostatic, electrostrictive, and other drive mechanisms that induce desired motion in response to a given control signal or command can also be used. Scanners include, but are not limited to, piezoelectric tubes, piezoelectric stacks, piezoelectrically driven flexure stages, voice coils, and other mechanisms that provide high-precision translation.

[0023] "Controller" refers to a system that facilitates data collection and system control. The controller may be a single, integrated electronic enclosure or may include multiple distributed elements. Control elements can provide control for probe, illumination, and sample positioning and scanning. They can also collect data regarding probe modulation, profile motion, or other responses, and control source power, polarization, modulation, steering, focus, and other functions. Control elements can include computer-programmed or digital logic methods and can be implemented using a combination of various computing devices (computers, personal electronic devices), analog and / or digital discrete circuit components (e.g., transistors, resistors, capacitors, inductors, diodes), programmable logic, microprocessors, microcontrollers, application-specific integrated circuits, field-programmable gate arrays, or other circuit elements. Memory configured to store a computer program may be implemented with discrete circuit components to perform one or more of the processes described herein. We also recognize and anticipate that advances in computing, including, but not limited to, quantum devices (qubits), photonic circuits, and molecular electronics, may be useful. We also recognize and anticipate useful advances in software, including genetic algorithms, neural networks, GPU TensorFlow and other artificial intelligence and deep machine learning techniques.

[0024] A "lock-in amplifier" is a device or algorithm that demodulates the response of a system at one or more reference frequencies. Lock-in amplifiers may be electronic assemblies comprising analog electronics, digital electronics, or a combination of the two. They may also be computational algorithms implemented in digital electronic devices such as microprocessors, microcontrollers, field-programmable gate arrays (FPGAs), digital signal processors, and personal computers. Lock-in amplifiers can generate signals indicative of various metrics of a resonant system, including amplitude components, phase components, or any combination of the above, in the in-phase (X) and quadrature-phase (Y) components. Lock-in amplifiers in this context can also generate such measurements at both the reference frequency and harmonics of the reference frequency, and / or at sideband frequencies of the reference frequency.

[0025] "Photothermal distortion" refers to a change in the properties of a sample due to the absorption of optical energy, e.g., IR radiation. Photothermal distortion may also refer to a change in refractive index, reflectivity, thermal expansion, surface distortion, or other effects that can be detected with a probe beam.

[0026] "Camera" refers to an array-based photodetector containing multiple light-sensitive pixels. A camera may include one or more technologies, including, but not limited to, CCD, EM-CCD, CMOS, s-CMOS, or other photosensitive array technologies. A camera may support frame rates ranging from a few frames per second to hundreds of frames per second, or even thousands of frames per second or higher.

[0027] "Figure of merit" refers to any metric or indicator of the relative quality of a signal or measurement. A figure of merit can be, for example, measurement sensitivity, signal strength, noise level, signal-to-noise ratio, background level, signal-to-background ratio, any combination of these, or any other metric that can rank the relative quality of signals or measurements.

[0028] Optical Photothermal Infrared (OPTIR) Imaging and Spectroscopy FIG. 1 illustrates a conventional OPTIR imaging system. Other similar systems are described, for example, in co-pending PCT patent application PCT / US17 / 63807, the disclosure of which is incorporated by reference in its entirety. A region 100 of a sample 102 is illuminated with a beam of infrared radiation 104, which induces a temperature increase, i.e., a photothermal response, in an IR-absorbing region of the sample. To measure the photothermal response resulting from IR absorption, at least a portion of the IR-absorbing region is also illuminated with a beam of probe radiation 106. In the illustrated configuration, this probe beam 106 is then reflected along an illumination path and transmitted through the sample (108). In addition to a diagram of the beam waist showing the focused probe radiation 106, FIG. 1 also includes arrows indicating both the reflected and transmitted portions. Temperature changes in the IR-illuminated sample or the environment (e.g., air or fluid above or surrounding the sample) due to IR absorption by the sample cause changes in the phase, polarization, intensity, and angle of the light (110) reflected / scattered from the sample, and changes in the phase / polarization / intensity / angle of the light (112) transmitted through the IR-absorbing region. By measuring the variations in the collected probe light, a signal can be generated that indicates the absorption of IR light by the absorbing region of the sample. In the configuration shown, the probe light is collected in a reflection configuration, but measurements are possible in a transmission and scattering configuration as well.

[0029] In many conventional systems, the probe beam can comprise a beam of visible or ultraviolet light, i.e., a beam with a wavelength significantly shorter than that of IR light. The reason for using UV or visible light is that the shorter wavelength of UV / visible light allows it to be focused to a much smaller spot than IR radiation. It can therefore be used to measure IR absorption with a spatial resolution that is more than ten times better than conventional IR microscopes. In far-field microscopes, optical diffraction limits the achievable spatial resolution to a length scale on the order of the wavelength of the light used. Specifically, the minimum detectable separation δ between two objects using the Rayleigh criterion is given by: Equation 1 δ = 0.61 λ / (n NA)

[0030] where λ is the wavelength of the infrared light used, n is the refractive index of the surrounding medium, and NA is the effective numerical aperture of the microscope objective lens used. High-power IR objective lenses typically have a maximum NA in the range of 0.7-0.81, which sets the best achievable spatial resolution under the Rayleigh criterion at approximately 75%-87% of the wavelength used. For example, at λ = 10 μm, the spatial resolution δ is approximately 8 μm. The spatial resolution achievable by OPTIR techniques under Equation 1 is set by the wavelength of the visible probe beam, not the wavelength of the IR beam. The spatial resolution improvement achievable with OPTIR techniques is determined by the ratio of the IR wavelength to the probe wavelength. For example, λ IR λ = 10 μm probe = 0.532 μm, the spatial resolution of the OPTIR technique can be improved by a factor of (10 / 0.532) ~ 19 times over conventional IR microscopy with the same objective. probe Using the same NA as mentioned above, =0.532 μm, the OPTIR technique produces a spatial resolution of about 0.4 μm. Even higher spatial resolution can be achieved using objectives with higher NA, e.g., objectives optimized for visible light microscopy, and probe beams with shorter wavelengths, e.g., in the blue / UV range.

[0031] In some embodiments, short-duration (on the order of hundreds of nanoseconds) periodic pulses of IR illumination are used. The use of short IR pulses in combination with synchronous detection techniques can provide high sensitivity and spatial resolution. A signal indicative of IR absorption can be measured as a function of the wavelength of the IR radiation to generate an IR absorption spectrum. The IR absorption signal can be measured as a function of relative sample position to generate a spatially resolved map of the sample's chemical composition. A spatially resolved map can be generated by measuring IR absorption at a selected wavelength (or equivalent wavenumber) as a function of sample position, and / or the entire IR absorption spectrum can be measured at multiple locations on the sample. In so-called hyperspectral imaging, the data cube has lateral dimensions x and y along the sample surface, and the third dimension comprises an IR absorption spectrum used to identify molecules by their excitation mode (e.g., stretching, bending, twisting, etc.).

[0032] However, a key limitation of conventional photothermal imaging and spectroscopy techniques is measurement throughput. Photothermal spectra are typically acquired at rates of 0.1–100 seconds per spectrum. While this is acceptable for point spectra or small arrays of spectra, this measurement time becomes unacceptable for hyperspectral arrays containing a large number of spectra, e.g., thousands or millions of spectra. Described below are embodiments for achieving high-throughput OPTRI measurements by enabling simultaneous parallel measurements at multiple points on a sample.

[0033] FIG. 2 shows one embodiment of an OPTIR instrument configured for wide-field measurements. In this embodiment, an infrared (IR) light source 200 generates a beam of IR light 202. The beam of IR light 202 is optionally directed by one or more mirrors 204 and 206 before passing through a beam combiner 208 (typically a dichroic beam splitter, although a polarizing beam splitter can also be used). In the configuration shown, the beam combiner 208 is substantially transparent to the IR radiation and reflective to the probe radiation, so that the IR beam passes through a dichroic 210. The beam combiner can be alternately reflective to IR and transparent to the probe light, and the configuration can be adjusted accordingly. The dichroic 210 reflects the IR light to a focusing element 212, which focuses a beam of IR radiation 214 to a spot 216 on an area of ​​a sample 218. The sample 218 may be mounted on a translation stage or scanner 220 to change the position of the sample 218 relative to the IR beam 214. A probe beam source 222 generates a beam of ultraviolet or visible light 224. The probe beam 224 is directed to optional mirrors 228 and 230. After the optional mirrors, the probe beam 232 is sent to the beam combiner 208.

[0034] In the illustrated configuration, probe beam 232 is reflected to the right by beam combiner 208, reflected by dichroic 210, and then reflected by focusing lens 212. Focusing lens 212 focuses probe beam 236 to focal spot 217 on sample 218. Focusing spot 217 generally at least partially overlaps focal spot 216 of IR beam 214. In an alternative configuration, the IR and probe beams can be delivered to the sample by separate focusing lenses. As discussed below, in this embodiment, at least the probe beam, and optionally the IR beam, are positioned to illuminate a large area of ​​the sample. That is, unlike conventional OPTIR measurements, the probe beam is not focused to a single diffraction-limited spot but instead illuminates a large area or at least multiple locations simultaneously, enabling parallel measurements of IR absorption at multiple locations on the sample simultaneously. In one example, both the IR and probe beams illuminate an area of ​​the sample greater than 100 microns in diameter.

[0035] At least one of mirrors 204, 206, 228, and 230 is preferably electronically controllable to adjust and optimize the overlap of the focused IR and probe light beams 202 and 224 to produce overlapping IR and probe beams 214 and 234 at sample 218. The overlap need not be symmetrical or centered one over the other.

[0036] The probe light reflected or scattered from the sample can be collected by a collection lens or "collector." The collector may include a focusing lens 212. That is, the same lens can be used to illuminate and collect light from the sample. To achieve high spatial resolution, it is desirable for the collector to have a high numerical aperture. For example, the collector may include an objective lens with a numerical aperture of at least 0.4, or more preferably greater than 0.6. In one example, the collector is a Cassegrain-type reflective objective lens with 40x magnification and an NA of 0.78. In another example, the collector is a Cassegrain-type objective lens with 20x magnification and an NA of 0.70. The arrangement of FIG. 2 has a significant advantage in that it allows collection of not only specularly reflected light, but all scattered light within the collector's numerical aperture cone. This means that the embodiment shown in FIG. 2 has the ability to capture the most intense portion of the probe light returning from the sample and, therefore, can generate a strong signal indicative of IR absorption by the sample with submicron spatial resolution. Using the numerical apertures listed above, the resolution given by Equation 1 is 0.42 μm for a 0.78 NA objective and 0.46 μm for a 0.70 NA objective. High NA objectives generally have short working distances, often less than a few millimeters. This short working distance makes the back-reflection configuration shown in Figure 2 advantageous because the proximity of the objective to the sample dramatically limits access to other off-axis collection paths.

[0037] In another embodiment, an IR refractive objective can be used instead of a reflective objective. For example, Pike Technologies manufactures 20x, 0.60 NA and 40x, 0.85 NA refractive objectives. Compensation lenses can be used in either the probe or IR beam path to compensate for focal length / dispersion differences between the IR and probe wavelengths.

[0038] Alternatively, the probe light that reflects, passes through, scatters, or otherwise interacts with the sample can be collected by another collecting lens (not shown). For samples that transmit the probe light, a separate collecting objective can be placed below the sample to collect the transmitted probe light. This is advantageous for highly transparent samples, as it allows most of the probe light to be recollected after interacting with the IR-absorbing region of the sample. In this case, the collecting objective can be a high-NA visible objective. This also benefits spatial resolution, as visible objectives with numerical apertures greater than 0.9NA are available. A 0.9NA objective collector used in the transmission configuration can provide a spatial resolution of 0.36 μm at a probe wavelength of 532 nm.

[0039] Returning to the reflective configuration shown in Figure 2, the collected probe light is then reflected back towards filter mirror 210. Filter mirror 210 may be partially reflective, e.g., a beam splitter, and may direct a portion of the reflected light towards camera 250 or a similar array detector. Camera 250 may be used to measure changes in the collected probe light due to IR absorption by the sample at multiple locations on the sample in parallel.

[0040] Controller 252 is used to perform several functions. First, controller 252 synchronizes IR source 200, probe beam source 222, and camera 250 with trigger signal 254. Specifically, the trigger signal ensures that (1) the camera frame is exposed in sync with the IR laser pulse and (2) the probe beam does not unnecessarily expose the sensor in camera 250 in a manner that would lead to camera saturation. The trigger scheme is described in more detail below.

[0041] Controller 252, or a separate controller (not shown), is used to collect data from camera 250 and measure the sample's photothermal response due to IR absorption, thereby enabling measurement of a signal indicative of IR absorption by the sample. By measuring this IR absorption signal as a function of wavelength or equivalently wavenumber, an IR absorption spectrum 256 can be measured, providing chemical characterization and material identification of the sample's chemical constituents. The IR absorption of the sample can be measured at multiple locations on the sample by using the parallel measurement capabilities of camera 250 or by repeating measurements at multiple locations on sample scanner 220.

[0042] There are several challenges associated with performing wide-field IR measurements, particularly with using a camera / array detector instead of a single point detector. Using OPTIR techniques to enable simultaneous IR absorption measurements at multiple locations on a sample with submicron spatial resolution involves dynamic range, sample illumination, spatial resolution, and other factors. Each of these is discussed in turn below.

[0043] Sensitivity and Dynamic Range Achieving sufficient sensitivity using a camera-based detector versus a single-point detector can be challenging because the photothermal response can be very small, i.e., smaller than can be detected within the dynamic range of a typical camera. For example, changes in refractive index and thermal expansion can be as small as about 10 for polymeric materials. -4 / K, and will be smaller for many other materials. The changes in the detected probe beam intensity are generally of the same order of magnitude as these small changes in the index / expansion coefficient; i.e., the collected probe beam intensity also decreases by about 10 -4 / K. It is often desirable to minimize the temperature rise of the sample to limit the possibility of thermally induced changes to the sample. Therefore, a good goal is to keep the temperature rise below 10 K. This results in a maximum fractional change in refractive index or thermal expansion of 10 K x 10 -4 / K=10 -3In IR spectroscopy, it is also desirable to have a wide dynamic range to accommodate the difference in absorption between the strongest and weaker absorption bands. For example, a weak IR band may be 10 to 100 times smaller than a strong band. To resolve a band that is 100 times weaker than the SNR of 10, a 10 -3 / 10 / 100=10 -6 Unfortunately, this requires the ability to detect fractional changes of 10 -6 This means a dynamic range of 10,000-60,000, far exceeding that of most, if not all, commercially available cameras. Cameras have a finite well depth, which sets the maximum number of photoelectrons that can accumulate before the exposure is stopped and the camera's pixels are read out. The dynamic range of a camera sensor is set by the ratio of the well depth to the pixel read noise. High-end scientific CMOS cameras have a dynamic range of about 10,000-60,000, several orders of magnitude lower than the dynamic range required to perform high-speed IR spectroscopy with enough dynamic range to resolve minor bands.

[0044] By stacking multiple frames, a sufficiently high dynamic range can be achieved. In this case, the signal increases proportionally to the number of stacked frames, while the noise is partially canceled out and only increases as the square root of the number of frames. Therefore, the dynamic range increases as the square root of the number of stacked frames. For example, if a camera sensor with a dynamic range of 50,000 has a dynamic range of 10, 6 To achieve this, you need to accumulate (10 6 / 50,000) 2 = 400 frames would be required. Multiple camera frames can be accumulated by the controller 252 and / or by any additional frame summing electronics not shown, including, for example, an FPGA, embedded computer, or other electronics capable of capturing and summing frames.

[0045] throughput We now turn to the measurement throughput achievable under the embodiments described herein. For wide-field photothermal measurements, the relevant figure of merit for a camera is the total time t required to achieve a desired dynamic range DR, given the camera's intrinsic dynamic range DR¬0. This measurement time t is given by: t = (DR / DR0) 2 / f where f is the frame rate of the camera. Different cameras have dramatically different frame rates, from tens of frames per second to thousands of frames per second or more. A higher frame rate is not necessarily better, if it comes at the expense of a significant reduction in dynamic range. Scientific CMOS cameras offer a good balance between frame rate and dynamic range. For example, using a camera such as the Tucsen Dyhana 95, the dynamic range is about 62,100, and the frame rate for 256x256 pixels is 192 frames per second. Using the above formula, 10 frames per second is obtained in 1.35 seconds. 6 A dynamic range of 10 ... 6 This represents a spectral rate 100-1000 times faster than the 1-10 seconds / spectrum rate of acquiring OPTIR spectra using a single point detector. Thus, the wide-field approach allows for dramatically higher measurement speeds, even with a lower inherent dynamic range. The embodiments described herein achieve a 10-1000 times faster measurement rate for a spectrum. 4 , 10 5 and 106 It is possible to measure IR absorption at multiple locations on a sample simultaneously with spectral throughputs of over 20 spectra per second, over 50 spectra per second, and over 90 spectra per second, while achieving a high dynamic range of .

[0046] In some implementations, it may be desirable to accumulate camera frames both when the IR light source is pulsed on and when it is off. The accumulated frames with the IR light on and with the IR light off can be used to generate a differential measurement that indicates changes in the collected probe light due to IR absorption by the sample. In some configurations, the camera system may have onboard memory for accumulating multiple frames, which can reduce the frequency required for data transfer to the host. It is also possible to accumulate two or more separate images in the camera. For example, one accumulator for frames with the IR on and another for frames with the IR off. Some cameras, such as Princeton PI-MAX® brand cameras, also have the ability to acquire two different image buffers with a variable delay between the camera's exposure to the two buffers. In this configuration, an initial trigger signal can trigger the first acquisition, such as the probe beam's response to the IR pulse. The second image buffer is accumulated after a short delay, such as after thermal relaxation has occurred and the probe intensity has returned to its baseline value. By subtracting or comparing the two images, the spatially resolved photothermal response due to IR absorption by the sample can be measured.

[0047] Alternatively, for example, digital lock-in techniques or computations aimed at simulating lock-in can be utilized, i.e., multiplying the time-dependent data by one or more periodic basis functions (e.g., sine and cosine, gated pulses, wavelets, or other functions), and then filtering the resulting output to convert the camera data into a time-based data stream that is synchronously demodulated. In either of these cases, the goal is to measure dynamic changes in the collected camera measurements of probe intensity via IR absorption modulation of the collected probe light, thereby measuring a signal indicative of IR absorption in a region of the sample.

[0048] Sample illumination In one embodiment, the IR and probe beams illuminate an area approximately 100 μm in diameter. In another embodiment, an area as large as 1 mm in diameter may be illuminated by the IR and probe beams. Selecting an illumination area of ​​approximately 100 μm offers several advantages. First, the illumination area is only a few times larger than the diffraction-limited spot size of the infrared beams used in conventional OPTIR instruments. For example, the size of the focused IR spot may range from 10 to 30 μm, depending on the NA and quality of the focusing objective lens used. Therefore, expanding the IR beam to a diameter of approximately 100 μm only increases the beam area by 10 to 100 times. Therefore, maintaining the same IR optical power density used in conventional OPTIR measurements requires IR power 10 to 100 times greater than that currently used for single-point measurements. Typical OPTIR single-point measurements are performed with peak power (i.e., the power during the IR pulse) of approximately 5 to 20 mW.

[0049] Available tunable light sources in the mid-infrared can generate output beams on the scale of hundreds of milliwatts to several watts of optical power. For example, M Square Laser's Firefly-IR can produce mid-infrared radiation with an average output power exceeding 250 mW. For example, Ekspla's nanosecond optical parametric oscillator (OPO) can generate 450 μJ at 1 kHz or 450 mW of IR power. OPO lasers from Amplitude Systemes can accept input powers up to 50 W and generate mid-infrared output power with efficiencies exceeding 12%, generating, for example, 6 W of mid-infrared energy. Jiang et al. have demonstrated a high-power pulsed OPO with a power of up to 8.5 W at a 3.3 μm wavelength (DOI: 10.1364 / OE.23.002633). Peng et al. have demonstrated a tunable mid-infrared laser with an output power exceeding 27 W (DOI: 10.1134 / S1054660X1201015X). Hemming et al. demonstrate a high-power mid-infrared ZGP ring OPO with optical power exceeding 30 W (DOI:10.1364 / CLEO_SI.2013.CW1B.7).

[0050] Each of these mid-infrared sources then provides enough power to illuminate increasingly larger areas of the sample at the same average power density as used for microscope focusing. For example, for a microscope, assume that the IR beam is focused to a 10 μm spot with 1 mW of power to obtain sufficient sensitivity. We were able to use 30 W of IR power from Jiang et al.'s OPO to illuminate an area 122 times larger in diameter than the 10 μm focused spot (via the square root of the power ratio of 30 W to 1 mW). This therefore corresponds to the ability to illuminate a circular area 1220 μm in diameter. The net effect is that there are multiple IR sources with sufficient intensity to illuminate large areas on the 100 μm diameter scale, and several sources sufficient to support circular areas greater than 1 mm in diameter. Larger IR beam sizes can be generated by using a beam reducer to reduce the effective numerical aperture of the IR beam focus or by using an IR-compatible diffuser. Low-cost thermal sources, such as globals, are large in size and therefore necessarily illuminate a larger area of ​​the sample. These light sources can be made suitable for OPTIR measurements with high spatial resolution by coupling them to high-speed modulators, such as photoelastic modulators or high-speed choppers. For example, Hind Instruments manufactures suitable photoelastic modulators, and Scientec manufactures optical choppers capable of modulating beams at frequencies above 100 kHz.

[0051] On the viewing side, going from an area of ​​roughly 1 μm to an area of ​​100 μm requires an illumination area roughly 100,000 times larger. One might assume that this would require illumination with over 100,000 times the laser power. Traditional single-point OPTIR measurements are performed with input probe powers in the range of approximately 1–100 mW, so requiring over 100,000 times the power may seem prohibitive. However, there are several aspects that can mitigate this and reduce the required probe power. The Tucsen Dyhana 95 has a quantum efficiency of 95% and a full well capacity of 90,000 photoelectrons. At a wavelength of 532 nm, this leads to a saturation dose of 2 nJ at 256 × 256 pixels. Operating at the Tucsen Dyhana 95 camera's maximum frame rate of 192 frames per second means that that saturation dose is delivered every 1 / 192 seconds, or 5 milliseconds. An exposure of 2 nJ every 5 ms corresponds to an average probe power across the camera of 2 nJ / 5 ms = 0.4 μW. By comparison, the probe power levels incident on a single-point detector in conventional OPTIR measurements are on the order of 20–400 μW (depending on the sample's reflectivity and damage threshold). Therefore, the camera pixels saturate at power levels up to 50–1000 times lower than typically used in single-point detectors. This means that the average power per pixel requirement can be significantly reduced by using a camera-based detector.

[0052] Assuming a saturation dose of 2 nJ or an average power target of 0.4 μW, we can estimate the required input pulse energy / power. Assuming a 4% sample reflectivity, 60% optical throughput through the Cassegrain objective in both directions, two beam splitter losses of 50% each, and 80% optical throughput elsewhere, we estimate that approximately 400 times higher input power is required than will be detected by the detector. Therefore, a probe beam source capable of delivering approximately 800 nJ per pulse or an average power of 160 μW is required. This is actually a fairly modest amount of power, not 100,000 times more than that used by single-point detectors, as first impressions might suggest. Several visible probe sources exist that may be suitable. For example, gas lasers, diode lasers, superluminescent diodes, and LED sources can provide sufficient optical power for this application. Other incandescent light sources can also be used. However, a higher-power source offers another advantage: it supports shorter probe pulses, which can improve spatial resolution.

[0053] Spatial resolution and thermal diffusion A shorter probe beam pulse is advantageous for maintaining high spatial resolution. Thermal diffusion can, in principle, impair spatial resolution because heat generated in the IR-absorbing region diffuses to adjacent non-absorbing regions of the sample. Using a short probe beam pulse allows the photothermal response to be measured in a selected time window after the onset of the IR pulse. The thermal diffusion length L is given by: L = (α t) 1 / 2 Lα is the thermal diffusivity of the sample material, and t is the diffusion time. For example, 10 -7 m 2 Assuming a thermal diffusivity of 1 / sec (typical for polymers), the thermal diffusion length corresponding to a 200 μsec long probe pulse duration is about 4 μm, while a 2 μsec probe pulse results in a thermal diffusion length of about 0.4 μm.

[0054] Figure 3 shows the photothermal response 300 on a polymer film captured with a single-point detector in a time-resolved measurement. The measurement in Figure 3 shows the photothermal response 300 on a polymer film captured with a single-point detector in a time-resolved measurement. The photothermal response 300 was measured using a quantum cascade laser (QCL)-based IR light source at 1729 cm -1 An IR pulse with a pulse duration of 260 nsec was irradiated onto the polymer film while the intensity of the reflected probe light was measured with a single-point silicon detector. In the configuration used, the probe beam intensity dropped sharply during the duration of the IR pulse (point 304) compared to the intensity before the IR pulse (302). The resulting thermal relaxation is observed at 306 when the probe signal returns to baseline. Figure 3 is plotted to show only the AC changes in the collected probe light (i.e., the baseline is shifted to zero).

[0055] Figure 3 shows that the photothermal response of a sample can last for more than 100 μsec. To maximize the total detected signal, it may be desirable to integrate the photothermal response over the entire decay time (or a significant portion thereof). However, if the photothermal response is integrated over a long period, spatial resolution may be compromised due to thermal diffusion to adjacent non-IR-absorbing regions. In some implementations, to limit the loss of spatial resolution due to thermal diffusion, it is desirable to construct an IR absorption signal using the photothermal response corresponding to a short period after the IR laser pulse. This can be achieved in several ways. First, by rapidly recording the collected probe multiple times and then constructing the IR absorption signal using selected probe beam measurements only from gate durations spanning a selected period after the onset of the IR pulse. Alternatively, a similar result can be achieved using a short camera exposure selected to begin synchronously with the IR laser pulse. In this case, the camera exposure time and phase act as the gate. In one embodiment, the probe beam is also synchronously pulsed, and the effective camera exposure time can be controlled by the duration of the probe beam pulse. In any of these cases, the gate time (eg, probe beam pulse duration or camera exposure time) can be adjusted to optimize between spatial resolution and sensitivity.

[0056] Figure 4 shows an example of gating that can be used to construct an IR absorption signal from a probe beam. Trace 400 shows an example of a pulse train of trigger pulses from an IR source, such as the synchronization or trigger input to a pulsed laser source. Trace 402 shows the collected probe light intensity as a function of time. As shown in Figure 3, trace 402 shows a rapid decrease in the collected probe light for the duration of the IR pulse, followed by a more gradual thermal decay back to the baseline. Trace 404 shows an example of a gating function that can be used to select a portion 406 of trace 402 for use in calculating a signal indicative of IR absorption by the sample. Note that in the example shown, the gating pulses in trace 404 are arranged to occur twice as frequently as the IR pulses and photothermal response in 400 and 402, if desired, to allow for differential measurements. Specifically, the probe beam intensity can be sampled alternately at two different times. (a) in response to IR absorption, e.g., portion 408 of trace 406; and (b) during a baseline period before or after the IR pulse, e.g., portion 410 of trace 406. Subtracting or comparing the two IR-on (408) and IR-off (410) signals produces a differential signal indicating the change in the photothermal signal in response to IR absorption and, therefore, the amount of IR absorption. The gate pulse shown in trace 404 can be used in several ways. For example, it can be used in time-resolved measurements to select a region of interest in trace 402 and select portions 408 and 410 of trace 406. Alternatively, gate pulse 404 can be used to set the start and end times of a camera exposure. The gate pulse can also or instead control the emission time of the probe beam pulse. For example, the gate pulse can be used to modulate the diode current in an LED-based probe light source or a diode-pumped solid-state laser or other probe light source with an electronically controllable output. The gate pulse can alternately control a shutter or modulator located in front of the camera or probe beam light source.Electro-optic modulators, acousto-optic modulators, photoelastic modulators, high-speed choppers, and other related devices are examples of devices that can be used to periodically modulate the intensity of a CW laser source to provide a probe light pulse that is synchronized with the IR light pulse to illuminate the sample.

[0057] The length of the gate time determines which portion of the thermal decay is sampled. To optimize spatial resolution, it may be desirable to use a short gate time. For example, to accommodate thermal diffusion lengths of less than 0.5 μm, it may be desirable to use probe beam pulses of less than 2 μs, as described above. The required maximum probe beam power is set by dividing the desired probe pulse energy by the pulse duration. Thus, if one wishes to deliver 2 nJ of probe energy in 2 μs (to fill the camera pixel well within the 2 μs thermal diffusion time), a maximum power of 2 nJ / 2 μs = 1 mW is required for the camera. Applying the 400x optical loss between the probe source and the camera mentioned above suggests that the probe beam source should provide at least 400 mW of optical power for 2 μs to achieve optimal spatial resolution and fully utilize the dynamic range of the camera sensor.

[0058] Suitable high-brightness LED light sources are also available. An LED light source for the probe beam can be advantageous because an incoherent light source can minimize speckle and interference artifacts. For example, Thorlabs manufactures monochromatic, high-power LED illuminators for microscopes. Their SOLIS brand of products, for example, produces up to 3–7 W of light in many wavelength options and has the ability to be pulsed and modulated at frequencies up to 250 kHz using available LED power supplies / controllers. Based on laboratory measurements, 7 W of optical power from an LED microscope illuminator impinging on an optically transparent polymer film through a 40× 0.78 NA Cassegrain objective lens returns enough light to the camera to saturate the central area of ​​a scientific CMOS camera sensor within 70 microseconds. Therefore, a 7W LED light source is sufficient to provide a light pulse as short as 70 μs while using the maximum dynamic range of the camera sensor. (These measurements were performed on a testbed system with many optical surfaces and associated losses. An optimal design with a minimum number of optical surfaces and appropriate low-loss coatings can achieve optical throughput that is approximately twice as good.) For highly reflective samples or samples measured in transmission, the amount of probe light collected can be much greater. For example, a transparent polymer may reflect only about 4% of the incident probe light, but a highly reflective sample or a sample on a highly reflective substrate may reflect more than 90% of the incident probe light, resulting in 22 times more light being detected by the camera. This allows for probe light pulses as short as 70 μs / 22 = 3.2 μs. Similar results can be obtained in transmission through samples that are largely transparent at the wavelength of the probe light. Note that maximizing the dynamic range of the camera pixels is not necessarily necessary. If achieving the highest spatial resolution is the primary goal, short probe beam pulses can be used. This allows the collected light to be well below the well depth of the camera pixels, and many more camera frames can be co-added to achieve the desired dynamic range.

[0059] High-power laser sources are also available that are intense enough to illuminate large areas of a sample. Visible, UV, and near-infrared pump lasers are also available with very high powers. For example, a 532 nm green laser system with output powers up to 6 W is available from Optronics. Other low-noise scientific lasers with powers in excess of 20 W are also available. Coherent manufactures a 1064 nm laser with optical powers up to 55 W.

[0060] Probe beam illumination can be configured in several different ways. Figures 5A, 5B, and 5C show three configurations. Figure 5A shows a subset of one embodiment of the present invention. In this subset, sample 500 has region 502, the topmost region illuminated by IR beam 504 and probe beam 506, which significantly overlap in an area beyond the spatial resolution of the imaging system. Region 502 allows for simultaneous detection of IR absorption from multiple points within the illuminated region. This diagram shows the illumination as a Gaussian distribution with a waist at the surface. However, a more uniform illumination (also known as a top-hat intensity profile) is preferred, and this can be generated in a number of ways, including a combination of Gaussian and donut laser modes.

[0061] Figure 5B shows an alternative embodiment in which the IR beam 524 is captured from the side. The low NA required to provide a spot approximately 1 mm in diameter makes IR side illumination very practical. Stretching in one axis can produce an ellipse on the surface that is two or three times its width. Side IR beam delivery allows for better objectives that work only with visible light (refractive, not reflective). For example, focusing element 212 in Figure 2 can be a visible / refractive objective. High NA objectives for 532 nm light are abundant and affordable. A Mitutoyo 100x Plan-Apochromat has an NA of 0.7 and a working distance of 6 mm, making it accessible enough for side IR beam delivery. Refractive objectives can also provide better imaging and optical throughput than IR-compatible Cassegrain-style objectives.

[0062] FIG. 5C shows an alternative embodiment in which two beams are counter-propagating. In this case, an IR beam 534 is shown illuminating the sample from below, for example, through an IR-transparent substrate. Meanwhile, a probe beam 506 illuminates the sample from above. In this case, it may be convenient for a camera positioned to collect the probe light transmitted through the sample. It is also possible to swap the configuration of the IR beam and the probe beam in both FIGS. 5B and 5C. For example, the probe beam may illuminate the sample from the angle of FIG. 5B, and the IR beam may be perpendicular to the sample. In FIG. 5C, the IR beam can be from above and the probe beam from below.

[0063] Wide-field illumination by a probe beam can be achieved in many possible configurations. As an example, conventional Köhler illumination can be used to project an image of a probe illumination source onto the back focal plane of a focusing objective, such as focusing element 212 in FIG. 2 . A simplified schematic diagram of such an arrangement is shown in FIG. 6 . In FIG. 6 , a probe beam source 600, such as a visible light LED emitter, generates a beam of probe light 601, at least a portion of which is collected by a first focusing lens 602, such as an aspheric lens. The focusing lens, optionally in combination with other lenses, focuses an image of the probe source 600 onto an image plane near aperture stop 604. This image of the probe source is then combined by one or more lenses, such as lenses 606 and 610, to generate an image of the probe source near the back focal plane of objective lens 616. The focusing lens and other lenses can be arranged to generate an image of the probe source at the back focal plane of objective lens 616. The back focal plane is appropriately matched to the input pupil of the objective lens. An optional field stop 608 can adjust the area of ​​the sample illuminated by the probe beam. For more uniform illumination by the probe beam, it is desirable to make the image of the probe beam somewhat larger than the input pupil—for example, 20–50% larger, depending on the need for uniform illumination relative to light throughput requirements. The IR source 622 can be collinearly combined with the probe beam using a dichroic mirror 614. Optional beam steering mirrors, such as 204, 206, 228, and 230 in Figure 2, are not shown in Figure 6, but can be used to control and optimize the overlap between the IR and probe beams. In this way, the IR beam 624 and the probe beam 618 can be focused onto the sample to illuminate a large area of ​​the sample 620. The probe light scattered or reflected from the sample can be collected by a camera 650 and analyzed as described above to generate a signal indicative of IR absorption at multiple locations on the sample.

[0064] Alternatively, a critical illumination method can be used, for example, by forming an image of the probe beam source (e.g., an LED emitter) directly on the sample. Figure 7 shows a simplified schematic diagram of this arrangement. Figure 7 is substantially similar to Figure 6, and the same numerical references are used, with the relevant descriptions applying as appropriate. However, Figure 7 uses a simplified illumination scheme. In this scheme, the probe beam source 600 is directly imaged onto the sample 620 using a combination of a condenser lens 602 and an objective lens 616. The image of the probe beam source can be magnified or demagnified as needed based on the relative size of the probe beam source emitter to the desired size of the illuminator. For example, for a 1 mm LED emitter, a 10x ratio between the focal length of the condenser lens and the objective lens focal length would project a 100 μm diameter image of the LED emitter onto the sample surface. In another embodiment, the LED emitter can be positioned directly above the sample, for example, attached to the bottom of the focusing element 212.

[0065] FIG. 8 illustrates an alternative embodiment for providing IR and visible illumination to a sample. FIG. 8 is similar to FIG. 6 , employing some of the same reference numerals, with the relevant descriptions applying where appropriate. In FIG. 8 , a probe beam source 800 generates a beam of probe light 801. This beam of probe light 801 is incident on one or more optional lenses 802, which can be used to collect, collimate, magnify / demagnify, and / or focus the light from the probe beam source 800. The probe beam 801 then is incident on a small mirror 804, which directs a probe light beam 806 toward the sample 618. In the case of a Cassegrain objective lens used for focusing element 616, the objective lens has a central obstruction. Therefore, mirror 804 can be attached or positioned at the bottom of objective lens 616 without interfering with the path of IR light beam 624, which is also directed toward the sample.

[0066] FIG. 9 shows an example of an alternative illumination scheme using a lenslet array to provide an array of individual illumination spots. In this embodiment, a sample 900 is illuminated from below by an IR beam 904. A probe beam 906 is directed toward the sample 900 from above. However, before the probe beam 906 strikes the sample, it strikes a lenslet array 920, which includes multiple small lenses 922. Each lens 922 produces a discrete focal point 928 that illuminates a portion of the sample 900. Large areas of the sample can be measured by repeating measurements at multiple locations on the sample, for example, by raster scanning the sample on a sample scanner as shown in FIG. 2. The scanner needs to have a range that roughly corresponds to the spacing between the lenslet focal spots so that it can generate a continuous image of the sample's IR absorption properties.

[0067] FIG. 10 shows an example of wide-field OPTIR data measured under the embodiment shown in FIG. 2. The sample was a cross-section of a multilayer polymer film. The sample was illuminated with a probe light source comprising a 7 W, 445 nm wavelength LED light source in CW operation, and focused onto the sample with a 40× 0.78 NA Cassegrain objective. The sample was measured at 1293 cm -1 The sample was simultaneously illuminated with pulses of IR light from a QCL-based light source operating at 1000 kHz. The probe light reflected and scattered from the sample was collected with the same Cassegrain objective and directed onto a 1200 x 1200 pixel scientific CMOS camera. Five hundred frames were co-added with the IR pulse on and the IR pulse off. Chemical image 1000 of the sample is the difference between the image with the IR laser on and the image with the IR laser off. Chemical image 1000 shows a 400 x 400 pixel image at a scale of 3.04 pixels per micron, thus corresponding to an image size of 131 x 131 microns. Therefore, under the illustrated embodiment, a camera-based sensor can be used to measure signals indicative of IR absorption at multiple locations on the sample, covering a measurement area of ​​more than 100 x 100 microns.

[0068] The embodiments described herein are exemplary. Modifications, rearrangements, alternative processes, alternative elements, etc. may be made to these embodiments and still fall within the teachings described herein. One or more of the steps, processes, or methods described herein may be performed by one or more suitably programmed processes or digital devices.

[0069] Depending on the embodiment, certain acts, events, or functions of any of the method steps described herein may be performed in a different order and may be added, merged, or omitted entirely (e.g., not all of the acts or events described may be required for the implementation of an algorithm). Furthermore, in certain embodiments, acts or events may be performed simultaneously rather than sequentially.

[0070] The various illustrative logic blocks, optical components, control components, and method steps described in connection with the embodiments disclosed herein can be implemented as electronic hardware, computer software, or a combination of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, and steps have been described above generally in terms of their functionality. Whether such functionality is implemented as hardware or software depends on the particular application and design constraints imposed on the overall system. The described functionality can be implemented in various ways for each particular application, and such implementation decisions should not be interpreted as causing a departure from the scope of the present disclosure.

[0071] The various illustrative logic blocks and modules described in connection with the embodiments disclosed herein may be implemented or performed by a machine such as a processor configured with specific instructions, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A processor may be a microprocessor, but alternatively, a processor may be a controller, microcontroller, or state machine, combinations thereof, etc. A processor may also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, multiple microprocessors, one or more microprocessors in combination with a DSP core, or any other such configuration.

[0072] Elements of a method, process, or algorithm described in connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by a processor, or in a combination of the two. The software module may reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, a hard disk, a removable disk, a CD-ROM, or any other form of computer-readable storage medium known in the art. An exemplary storage medium may be coupled to the processor such that the processor can read information from, and write information to, the storage medium. Alternatively, the storage medium may be integrated into the processor. The processor and the storage medium may reside in an ASIC. The software module may include computer-executable instructions that cause a hardware processor to execute the computer-executable instructions.

[0073] Conditional language used herein, particularly "can," "might," "may," "may," "for example," and the like, is intended to generally convey that certain embodiments include certain features, elements, or conditions, while other embodiments do not, unless otherwise specified or understood within the context of use. Thus, such conditional language generally does not imply that the features, elements, or conditions are in any way necessary to one or more embodiments, or that one or more embodiments necessarily include logic, with or without author input or prompting, for determining whether those features, elements, or conditions are included in or performed in any particular embodiment. Terms such as "comprises," "includes," "has," and "involves" are synonymous and are used inclusively in an open-ended fashion and do not exclude additional components, functions, acts, operations, etc. Similarly, the term "or" is used in its inclusive (not exclusive) sense; for example, when used to connect a list of components, the term "or" means one, some, or all of the components in the list.

[0074] Disjunctive language, such as the phrase "at least one of X, Y, or Z," should be understood in context as generally used to indicate that an item, term, etc. can be either X, Y, or Z, or any combination thereof (e.g., X, Y, and / or Z), unless otherwise specified. Thus, such disjunctive language is not, and should not be, intended to generally require that a particular embodiment require that at least one of X, at least one of Y, or at least one of Z, respectively, be present.

[0075] Terms such as "about" or "approximately" are synonymous and are used to indicate that the value modified by the term has a range of understanding associated therewith, where the range may be ±20%, ±15%, ±10%, ±5%, or ±1%. The term "substantially" is used to indicate that a result (e.g., a measurement) is close to a target value, where close may mean, for example, that the result is within 80% of the value, within 90% of the value, within 95% of the value, or within 99% of the value.

[0076] Unless otherwise specified, articles such as "a" and "an" should generally be construed to include one or more described items. Thus, a phrase such as "an apparatus configured to" is intended to include one or more described apparatuses. Such one or more described apparatuses may also be collectively configured to perform the specified description. For example, "a processor configured to perform descriptions A, B, and C" may include a first processor configured to perform description A in conjunction with a second processor configured to perform descriptions B and C.

[0077] While the foregoing detailed description illustrates, describes, and points out novel features as applied to the exemplary embodiments, it will be understood that various omissions, substitutions, and changes in the form and details of the described devices or methods may be made without departing from the spirit of the present disclosure. It will be recognized that some features can be used or practiced separately from other features, and therefore particular embodiments described herein may be embodied in a form that does not provide all of the features and advantages described herein. All changes that come within the meaning and range of equivalency of the claims are intended to be embraced within their scope.

[0078] Various embodiments of systems, devices, and methods have been described herein. These embodiments are provided by way of example only and are not intended to limit the scope of the claimed invention. It should be understood that various features of the described embodiments can be combined in various ways to produce numerous additional embodiments. Furthermore, while various materials, dimensions, shapes, configurations, and arrangements, etc., have been described for use in the disclosed embodiments, others than those disclosed may be utilized without departing from the scope of the claimed invention.

[0079] Those skilled in the relevant art will recognize that the subject matter herein may include fewer features than those shown in the individual embodiments above. The embodiments described herein are not intended to be an exhaustive listing of ways in which various features of the subject matter herein can be combined. Thus, the embodiments are not mutually exclusive combinations of features. Rather, various embodiments may include combinations of different individual features selected from different individual embodiments, as would be understood by one skilled in the art. Furthermore, components described with respect to one embodiment can be implemented in other embodiments, even if not described in such embodiments, unless otherwise specified.

[0080] Although a dependent claim may refer to a specific combination with one or more other claims in the claim, other embodiments may also include combinations of the dependent claim with the subject matter of each other dependent claim, or combinations of one or more features with other dependent or independent claims, and such combinations are suggested herein unless it is stated that a particular combination is not intended.

[0081] Any incorporation by reference of the above documents is limited so that no subject matter contrary to the express disclosure of this specification is incorporated. Any incorporation by reference of the above documents is further limited so that no claims contained in the documents are incorporated herein by reference. Furthermore, any incorporation by reference of the above documents is further limited so that any definitions provided in the documents are not incorporated herein by reference, unless expressly included herein.

[0082] It is expressly intended that the provisions of 35 U.S.C. § 112(f) will not be invoked for purposes of construing a claim unless the specific terms "means for" or "step for" appear in the claim.

Claims

1. 1. An apparatus for microscopic analysis of a sample by simultaneously characterizing infrared absorption properties of a plurality of spatially resolved locations corresponding to a large area of ​​the sample, comprising: an infrared radiation source adapted to illuminate a large area of ​​the sample with a beam of infrared radiation; a probe radiation source adapted to illuminate a large area of ​​the sample with a beam of probe radiation; a collector adapted to collect at least a portion of the probe radiation emanating from each of a plurality of spatially resolved locations on the sample as collected probe radiation; at least one camera adapted to detect at least a portion of the collected probe radiation to generate signals indicative of infrared absorption corresponding to each of a plurality of spatially resolved locations; An apparatus characterized in that the signal indicative of infrared absorption has a spatial resolution of less than 1 micrometer.

2. 10. The apparatus of claim 1, wherein the infrared radiation source is adjustable to produce an infrared beam of variable wavelength, and wherein signals indicative of infrared absorption of the sample are detected at a plurality of infrared wavelengths.

3. 3. The apparatus of claim 2, wherein the signal indicative of infrared absorption at a plurality of infrared wavelengths comprises an infrared absorption spectrum.

4. 10. The apparatus of claim 1, further comprising an image co-adder, wherein the image co-adder sums a plurality of camera frames to generate at least 10 ４ 2. An apparatus for constructing a co-addition image having a dynamic range of .

5. 10. The apparatus of claim 1, wherein the co-added image is at least 10 ５ The apparatus has a dynamic range of .

6. 10. The apparatus of claim 1, wherein the co-added image is at least 10 ６ The apparatus has a dynamic range of .

7. 10. The apparatus of claim 1, wherein the locations of the sample that are measured simultaneously have an area of ​​at least 100 [mu]m in diameter.

8. 10. The apparatus of claim 1, wherein the signal indicative of IR absorption at multiple locations on the sample achieves a spatial resolution of less than 0.5 μm.

9. 10. The apparatus of claim 1, further comprising a gating function for limiting the duration over which the camera detects probe radiation collected from multiple locations on the sample.

10. 10. The apparatus of claim 9, wherein the gating function limits at least one of (a) the exposure time of the camera and (b) the length of the pulse duration of the probe beam.

11. 10. The apparatus of claim 1, wherein signals indicative of infrared absorption at a plurality of locations on the sample are generated by comparing a first set of camera frames collected with an IR source illuminating the sample and a second set of camera frames collected with an IR source not illuminating the sample.

12. 10. The apparatus of claim 1, wherein the collector comprises an objective lens having a numerical aperture (NA) of at least 0.

4.

13. 10. The apparatus of claim 1, wherein the collector comprises an objective lens having a numerical aperture (NA) of at least 0.

6.

14. 2. The apparatus of claim 1, wherein the probe radiation source is an incoherent light source.

15. 10. The apparatus of claim 1, wherein the probe radiation source comprises at least one light emitting diode.

16. 1. A method of operating a system for simultaneously characterizing infrared absorption properties of multiple locations on a sample, comprising: illuminating the sample at a plurality of locations with an infrared radiation source to generate infrared illumination spots on the sample; illuminating a plurality of locations on the sample with a probe radiation source; collecting probe radiation from a plurality of locations on the sample; detecting the collected probe radiation with at least one camera; analyzing the probe radiation detected by the camera to generate signals indicative of infrared absorption at a plurality of locations on the sample; The method is characterized in that the signal indicative of infrared absorption at a plurality of locations on the sample achieves a spatial resolution of less than 1 μm.

17. 17. The method of claim 16, further comprising adjusting an infrared radiation source to produce an infrared beam having a variable wavelength, wherein signals indicative of infrared absorption of the sample are detected at a plurality of infrared wavelengths.

18. 17. The method of claim 16, wherein the signal indicative of infrared absorption comprises an infrared absorption spectrum.

19. 20. The method of claim 18, wherein the infrared absorption spectra are measured at a rate of greater than 20 spectra per second.

20. 20. The method of claim 18, wherein the infrared absorption spectra are measured at a rate of greater than 50 spectra per second.

21. 20. The method of claim 18, wherein the infrared absorption spectra are measured at a rate of greater than 90 spectra per second.

22. 17. The method of claim 16, wherein the system further comprises an image co-adder, and wherein the steps of using the co-adder and combining the multiple camera frames to generate at least 10 ４ and constructing a co-addition image having a dynamic range of

23. 23. The method of claim 22, wherein the co-added image is at least 10 ５ A method characterized in that the method has a dynamic range of

24. 23. The method of claim 22, wherein the co-added image is at least 10 ６ A method characterized in that the method has a dynamic range of

25. 17. The method of claim 16, further comprising the step of simultaneously measuring an area of ​​at least 100 μm in diameter.

26. 17. The method of claim 16, wherein the signals indicative of IR absorption at multiple locations on the sample achieve a spatial resolution of less than 0.5 μm.

27. 17. The method of claim 16, further comprising applying a gating function to limit the duration over which the camera detects the probe radiation collected from multiple locations on the sample.

28. 28. The method of claim 27, wherein the gating function limits at least one of (a) the exposure time of the camera and (b) the length of the pulse duration of the probe beam.

29. 29. The method of claim 28, wherein the signal indicative of IR absorption at a plurality of locations on the sample achieves a spatial resolution of less than 0.5 μm.

30. 17. The method of claim 16, wherein signals indicative of infrared absorption at a plurality of locations on the sample are generated by comparing a first set of camera frames collected with an IR source illuminating the sample and a second set of camera frames collected with an IR source not illuminating the sample.

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