Patent Grant
11592652
Certain implementations pertain generally to photoacoustic imaging, and more specifically, to photoacoustic spectroscopic and imaging systems and methods with enhanced-resolution.
Photoacoustic imaging is based on the photoacoustic effect where pulsed or modulated radiation is delivered to a sample and some of the delivered energy is absorbed and converted into heat, leading to transient thermoelastic expansion generating ultrasonic emissions. The ultrasonic emissions can be detected by an ultrasonic transducer device and analyzed to produce photoacoustic images. A photoacoustic image depends on the optical absorption properties of the sample being imaged. As a consequence, it offers greater molecular specificity than conventional ultrasound imaging with the ability to detect hemoglobin, lipids, water and other light-absorbing chromophores, but with greater penetration depth than pure optical imaging modalities that rely on ballistic photons. These attributes lend photoacoustic imaging to a wide variety of applications in clinical medicine, preclinical research and basic biology for studying cancer, cardiovascular disease, abnormalities of the microcirculation and other conditions.
Certain aspects pertain to an apparatus for measuring infrared absorption of a sample during operation. In some aspects, the apparatus includes a first radiation source configured to emit pulses of infrared radiation and a second radiation source configured to emit pulses with shorter wavelength than the pulses of infrared radiation emitted from the first radiation source. The apparatus also includes one or more optical elements configured to deliver of pulses of infrared radiation (e.g., mid-infrared radiation) to a region of the sample and to the deliver pulses from the second radiation source to a sub-region within the region. In addition, the apparatus also includes an ultrasonic transducer acoustically coupled to the sample to detect photoacoustic signals induced by at least one of the radiation sources and one or more processors and memory configured to analyze one or more properties of the detected photoacoustic signals to create a signal indicative of infrared absorption of the sub-region of the sample. In one aspect, the one or more processors and memory are further configured to create an infrared image of the sample using the one or more analyzed properties of the detected photoacoustic signals. In one aspect, the second radiation source is configured to emit ultraviolet radiation pulses. In one aspect, the second radiation source is configured to emit pulses with a wavelength between about 100 nm and about 2000 nm. In one aspect, the one or more processors and memory are configured to analyze the infrared absorption of the sub-region of the sample by calculating a difference in amplitude between a first photoacoustic signal induced by a radiation pulse from the second radiation source and a second photoacoustic signal induced by another radiation pulse from the second radiation source.
In some aspects, an apparatus for measuring infrared absorption of a sample during operation includes a first radiation source configured to emit mid-infrared radiation pulses. In one aspect, the apparatus is further configured to deliver: (i) a first ultraviolet radiation pulse to the sub-region, the first ultraviolet radiation pulse inducing a first photoacoustic signal; (ii) a mid-infrared radiation pulse to the region of the sample; and (iii) a second ultraviolet radiation pulse to the sub-region of the sample the second ultraviolet radiation pulse inducing a second photoacoustic signal. In this aspect, the one or more processors and memory may be optionally further configured to calculate a difference between the first and second photoacoustic signals, the difference being indicative of an infrared absorption property of the sub-region of the sample.
In one aspect, an apparatus also includes a pulser configured to generate trigger pulses, at least one of which is configured to trigger the second radiation source to emit a first ultraviolet radiation pulse at a first time before or after the first radiation source is triggered to emit a first infrared radiation pulse, and wherein at least one of the trigger pulses is configured to trigger the second radiation source to emit a second ultraviolet radiation pulse at a second time after the first radiation source is triggered to emit the first infrared radiation pulse. Optionally, the one or more processors and memory are configured to analyze the infrared absorption of the sub-region of the sample by calculating a difference in amplitudes of a first photoacoustic signal induced by the first ultraviolet radiation pulse and a second photoacoustic signal induced by the second ultraviolet radiation pulse.
In one aspect of an apparatus for measuring infrared absorption of a sample during operation, the apparatus also includes a tunable infrared radiation source that may optionally include at least one of an optical parametric oscillator and a quantum cascade laser.
In one aspect of an apparatus for measuring infrared absorption of a sample during operation, the apparatus also includes a broadband infrared radiation source or a fixed wavelength radiation source optically coupled to the one or more optical elements and configured to emit the infrared radiation pulses.
In one aspect of an apparatus for measuring infrared absorption of a sample during operation, the one or more processors and memory are configured to measure the signal indicative of infrared absorption of the sub-region with a spatial resolution of less than 1,000 nm. In another aspect, the one or more processors and memory are configured to measure the signal indicative of infrared absorption of the sub-region with a spatial resolution of less than 500 nm.
In one aspect of the apparatus for measuring infrared absorption of a sample during operation, the apparatus further includes a photodiode configured to measure an intensity of pulses emitted from the second radiation source. In this aspect, the one or more processors and memory may be optionally configured to normalize the photoacoustic signals by compensating for variations in pulse energy from pulses emitted from the second radiation source using measurements from the photodiode.
In one aspect of an apparatus for measuring infrared absorption of a sample during operation, the apparatus further includes a scanning mechanism configured to move at least one of the sample or one or more of the optical elements such that the infrared radiation pulse is scanned to a plurality of regions in a field-of-view of the sample and the first and second ultraviolet radiation pulses are scanned to one or more sub-regions within each region of the plurality of regions. Optionally, the one or more processors and the memory are further configured to use the signal indicative of infrared absorption of the sub-region in each of the plurality of regions to determine an infrared image of the sample.
In one aspect of the apparatus for measuring infrared absorption of a sample during operation, the signal indicative of infrared radiation is measured at a plurality of wavelengths of the first radiation source to construct a spectrum of infrared absorption of the sub-region.
In one aspect of an apparatus for measuring infrared absorption of a sample during operation, the apparatus further comprises a microlens array to generate an array of infrared radiation pulses and an array of additional radiation pulses, wherein the one or more optical elements further are configured to deliver the array of infrared radiation pulses and the array of additional pulses to a plurality of regions of the sample.
In one aspect of an apparatus for measuring infrared absorption of a sample during operation, the first radiation source is configured to emit pulses with wavelengths between about 3,000 and about 8,000 nanometers, between about 5,800 and about 6,200 nanometers, and the second radiation source is configured to emit pulses with wavelengths between about 200 and about 300 nanometers.
Certain aspects pertain to a method for measuring infrared absorption of a sample. In some aspect, the method includes: (i) initiating delivery of a first radiation pulse of shorter wavelength than infrared radiation to a sub-region of a region of a sample; (ii) initiating delivery of an infrared radiation pulse to the region of the sample; (iii) initiating delivery of a second radiation pulse of shorter wavelength than infrared radiation to the sub-region of the sample, wherein the second radiation pulse is initiated at a first delay time after (ii); (iv) receiving, from an ultrasonic transducer acoustically coupled to the sample, photoacoustic signals induced by the first radiation pulse and the second radiation pulse; and (v) analyzing one or more properties of the detected photoacoustic signals to determine a signal indicative of infrared absorption of the sub-region of the sample.
In one aspect, the first radiation pulse is initiated before (ii) or at a second delay time after (iii). In one aspect, operation (v) comprises determining the signal indicative of infrared absorption of the sub-region of the sample by calculating a difference in amplitudes of photoacoustic signals induced by the first and second radiation pulses.
In one aspect of a method for measuring infrared absorption of a sample, the method further comprises scanning relative positions of the infrared radiation pulse, the first radiation pulse, and the second radiation pulse to a plurality of regions of the sample.
In one aspect of a method for measuring infrared absorption of a sample, the first radiation pulse and the second radiation pulse have wavelength between about 100 nm and about 2000 nm.
In one aspect of a method for measuring infrared absorption of a sample, the first radiation pulse and the second radiation pulse are ultraviolet radiation pulses and the infrared radiation pulse is a mid-infrared radiation pulse.
In one aspect of a method for measuring infrared absorption of a sample, the first delay time is less than about 1,000 nanoseconds or less than about 500 nanoseconds. In another aspect, the first delay time is between about 100 nanoseconds and about 500 nanoseconds.
In one aspect of a method for measuring infrared absorption of a sample, the method further comprises initiating delivery of one or more additional radiation pulses of shorter wavelength than infrared radiation to additional sub-regions of the illuminated region, wherein the additional radiation pulses are initiated within a the first delay time after (ii). Optionally, the first delay time is less than or equal to a thermal confinement period of the sample and/or between about 100 nanoseconds and about 500 nanoseconds after (ii).
In one aspect of a method for measuring infrared absorption of a sample, (ii) further comprises tuning a tunable infrared radiation source to generate infrared radiation pulses at a plurality of infrared wavelengths.
In one aspect of a method for measuring infrared absorption of a sample, (i) comprises initiating delivery of a first ultraviolet radiation pulse to the sub-region, the first ultraviolet radiation pulse inducing a first photoacoustic signal; and (iii) comprises initiating delivery of a second ultraviolet radiation pulse to the sub-region during the first delay time after (ii), the second ultraviolet radiation pulse inducing a second photoacoustic signal. Optionally the method further includes measuring, using a photosensor, an amplitude of the first ultraviolet radiation pulse; measuring, using the photosensor, an amplitude of the second ultraviolet radiation pulse; and normalizing the amplitudes of the first and second photoacoustic signals based on the measured amplitudes of the first and second ultraviolet radiation pulses.
In some aspects, a method for measuring infrared absorption of a sample further comprises creating an infrared image of the sample using the one or more analyzed properties of the detected photoacoustic signals. In one aspect, the infrared image has a spatial resolution of less than 1,000 nm. In another aspect, the infrared image has a spatial resolution of less than 500 nm. In another aspect, the infrared image has a spatial resolution finer than one-tenth of a wavelength of the infrared radiation pulse.
In one aspect, a method for measuring infrared absorption of a sample further comprises, before (v), normalizing the photoacoustic signals by compensating for variations in the first and second radiation pulses using measured amplitudes of the first and second radiation pulses.
In one aspect, a method for measuring infrared absorption of a sample further comprises scanning the infrared radiation pulse, the first radiation pulse, and the second radiation pulse to a plurality of regions of the sample.
In one aspect of a method for measuring infrared absorption of a sample, the signal indicative of infrared radiation is measured at a plurality of wavelengths of the first radiation source to construct a spectrum of infrared absorption of the sub-region.
These and other features are described in more detail below with reference to the associated drawings.
These and other features are described in more detail below with reference to the associated drawings.
Different aspects are described below with reference to the accompanying drawings. The features illustrated in the drawings may not be to scale. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the presented embodiments. The disclosed embodiments may be practiced without one or more of these specific details. In other instances, well-known operations have not been described in detail to avoid unnecessarily obscuring the disclosed embodiments. While the disclosed embodiments will be described in conjunction with the specific embodiments, it will be understood that it is not intended to limit the disclosed embodiments.
Mid-infrared (MIR) microscopy has been exploited for applications ranging from material characterization to label-free histologic analysis. Examples of material characterization with MIR microscopy are described by Wetzel, D. L. & LeVine, S. M., “Imaging molecular chemistry with infrared microscopy,” Science 285, 1224-1225 (1999); Koenig, J. L., “Microspectroscopic Imaging of Polymers,” American Chemical Society, (1998); and Prati, S., Joseph, E., Sciutto, G. & Mazzeo, R., “New advances in the application of FTIR microscopy and spectroscopy for the characterization of artistic materials,” Acc. Chem. Res. 43, 792-801 (2010), which are hereby incorporated by reference in their entireties. Examples of label-free histologic analysis with MIR microscopy are described by Diem, M., Romeo, M., Boydston-White, S., Miljkovic, M. & Matthaus, C., “A decade of vibrational micro-spectroscopy of human cells and tissue,” (1994-2004). Analyst 129, 880-885 (2004); Fernandez, D. C., Bhargava, R., Hewitt, S. M. & Levin, I. W., “Infrared spectroscopic imaging for histopathologic recognition,” Nat. Biotechnol. 23, 469-474 (2005); Baker, M. J. et al., “Using Fourier transform IR spectroscopy to analyze biological materials,” Nat. Protoc. 9, 1771-1791 (2014); and Diem, M. et al., “Molecular pathology via IR and Raman spectral imaging.” J. Biophoton. 6, 855-886 (2013), which are hereby incorporated by reference in their entireties.
In the last two decades, the sensitivity and speed of MIR microscopy have been significantly improved. Examples of improvements in the sensitivity and speed of MIR microscopy are described by Griffiths, P., “Fourier transform infrared spectrometry,” Science 21, 297-302 (1983); Lewis, E. N. et al., “Fourier transform spectroscopic imaging using an infrared focal-Plane array detector,” Anal. Chem. 67, 3377-3381 (1995); Miller, L. M., Smith, G. D. & Carr, G. L., “Synchrotron-based biological microspectroscopy: From the mid-infrared through the far-infrared regimes,” Journal of Biological Physics 29, 219-230 (2003); Nasse, M. J. et al., “High-resolution Fourier-transform infrared chemical imaging with multiple synchrotron beams,” Nat. Methods 8, 413-416 (2011); Kole, M. R., Reddy, R. K., Schulmerich, M. V., Gelber, M. K. & Bhargava, R., “Discrete frequency infrared microspectroscopy and imaging with a tunable quantum cascade laser,” Anal. Chem. 84, 10366-10372 (2012); and Haas, J. & Mizaikoff, B., “Advances in mid-infrared spectroscopy for chemical analysis,” Annu. Rev. Anal. Chem. 9, 45-68 (2016), which are hereby incorporated by reference in their entireties.
Despite the aforementioned improvements in MIR microscopy, there are still various limitations to MIR microscopy in biomedical and other applications. As a first example, conventional transmission MIR microscopy can image only dried or thin samples (See Wetzel, D. L. & LeVine, S. M., “Imaging molecular chemistry with infrared microscopy,” Science 285, 1224-1225 (1999)), which requires complicated and time-consuming sample preparation (See Baker, M. J. et al., “Using Fourier transform IR spectroscopy to analyze biological materials,” Nat. Protoc. 9, 1771-1791 (2014)). In addition, the lateral resolution of MIR microscopy is diffraction limited to approximately the long MIR wavelength at a typically used numerical aperture (˜0.5). Furthermore, for fresh biological samples, the broadband and strong MIR absorption of water creates a huge background, compromising imaging contrast and interfering with molecular analysis.
As described below, many techniques have improved MIR microscopy by addressing one or two of the above limitations, yet there are still various limitations to MIR microscopy in biomedical and other applications.
Attenuated total reflection-Fourier transform infrared (ATR-FTIR) microspectroscopic imaging can measure fresh samples in reflection mode with improved spatial resolution, but its penetration depth—due to the use of evanescent waves—is limited to only 1-2 μm. Examples of ATF-FTIR imaging are described by Sommer, A. J., Marcott, C., Story, G. M. & Tisinger, L. G., “Attenuated total internal reflection infrared mapping microspectroscopy using an imaging microscope,” Appl. Spectrosc. 55, 252-256 (2001) and Chan, K. L. A. & Kazarian, S. G., “New opportunities in micro- and macro-attenuated total reflection infrared spectroscopic imaging: spatial resolution and sampling versatility,” Appl. Spectrosc. 57, 381-389 (2003), which are hereby incorporated by reference in their entireties.
Atomic force microscopy (AFM) has achieved nanoscale resolution by detecting thermal expansion, light scattering, or force induced by MIR laser absorption; however, the surface contact or near-field detection poses challenges in scanning fresh biological samples. Examples of AFM involving detection of thermal expansion are described by Dazzi, A., Prazeres, R., Glotin, F. & Ortega, J. M., “Local infrared microspectroscopy with subwavelength spatial resolution with an atomic force microscope tip used as a photothermal sensor,” Opt. Lett. 30, 2388-2390 (2005); Lu, F., Jin, M. & Belkin, M. A., “Tip-enhanced infrared nanospectroscopy via molecular expansion force detection,” Nat. Photon. 8, 307-312 (2014); and Dazzi, A. & Prater, C. B., “AFM-IR: technology and applications in nanoscale infrared spectroscopy and chemical imaging,” Chem. Rev. 117, 5146-5173 (2017), which are hereby incorporated by reference in their entireties. Examples of AFM involving detection of light scattering are described by Knoll, B. & Keilmann, F., “Near-field probing of vibrational absorption for chemical microscopy,” Nature 399, 134-137 (1999), which is hereby incorporated by reference in its entirety. Examples of AFM involving detection of force are described by Nowak, D. et al., “Nanoscale chemical imaging by photoinduced force microscopy,” Sci. Adv. 2, e1501571 (2016), which is hereby incorporated by reference in its entirety.
Photothermal MIR imaging, which employs a continuous-wave visible or near-IR laser beam to detect the MIR thermal lensing effect, greatly improves the resolution and somewhat reduces the water background, but the scattering-based detection method restricts its applications to only translucent samples. Examples of photothermal MIR imaging are described by Furstenberg, R., Kendziora, C. A., Papantonakis, M. R., Nguyen, V. & McGill, R. A., “Chemical imaging using infrared photothermal microspectroscopy.” In Proceedings of SPIE Defense, Security, and Sensing (eds Druy, M. A. & Crocombe, R. A.) 837411 (SPIE, 2012); Li, Z., Kuno, M. & Hartland, G., “Super-resolution imaging with mid-IR photothermal microscopy on the single particle level,” In Proceedings of SPIE Physical Chemistry of Interfaces and Nano-materials XIV (eds Hayes, S. C. & Bittner, E. R.) 954912 (International Society for Optics and Photonics, 2015); Zhang, D. et al., “Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution,” Sci. Adv. 2, e1600521 (2016); and Li, Z., Aleshire, K., Kuno, M. & Hartland, G. V., “Super-resolution far-field infrared imaging by photothermal heterodyne imaging,” J. Phys. Chem. B 121, 8838-8846 (2017), which are hereby incorporated by reference in their entireties.
Stimulated Raman scattering (SRS) imaging has demonstrated label-free chemical mapping of biological cells and tissues at high spatial resolution and contrast. Examples of SRS imaging are described by Lu, F.-K. et al., “Label-free DNA imaging in vivo with stimulated Raman scattering microscopy,” Proc. Natl Acad. Sci. USA 112, 11624-11629 (2015); Cheng, J.-X. & Xie, X. S., “Vibrational spectroscopic imaging of living systems: an emerging platform for biology and medicine,” Science 350, aaa8870 (2015); and Ji, M. et al., “Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy,” Sci. Transl. Med. 7, 309ra163 (2015), which are hereby incorporated by reference in their entireties.
Far-field super resolution has been achieved using stimulated emission depletion. Examples of which are described by Gong, L. & Wang, H., “Breaking the diffraction limit by saturation in stimulated-Raman-scattering microscopy: a theoretical study,” Phys. Rev. A 90, 13818 (2014) and Ruchira Silva, W., Graefa, C. T. & Frontiera, R. R., “Toward label-free super-resolution microscopy,” ACS Photon. 3, 79-86 (2016), which are hereby incorporated by reference in their entireties.
Photoacoustic infrared (PAIR) detection is capable of spectroscopy and sensing of totally opaque or highly light-scattering materials. Examples of PAIR detection are described by Rockley, M. G., “Fourier-transformed infrared photoacoustic spectroscopy of polystyrene film,” Chem. Phys. Lett. 68, 455-456 (1979); Patel, C. K. N. & Tam, A. C., “Pulsed optoacoustic spectroscopy of condensed matter,” Rev. Mod. Phys. 53, 517-550 (1981); Tam, A. C., “Applications of photoacoustic sensing techniques,” Rev. Mod. Phys. 58, 381-431 (1986); and Michaelian, K. H., Photoacoustic Infrared Spectroscopy (Wiley, 2003), which are hereby incorporated by reference in their entireties. PAIR detection has also been demonstrated for imaging thick and scattering fresh biological samples without thin slicing, examples of which are described by Sim, J. Y., Ahn, C.-G., Jeong, E.-J. & Kim, B. K., “In vivo microscopic photoacoustic spectroscopy for non-invasive glucose monitoring invulnerable to skin secretion products,” Sci. Rep. 8, 1059 (2018), which is hereby incorporated by reference in its entirety. However, PAIR detection does not address the drawbacks on spatial resolution and water background.
Various aspects disclosed herein relate to systems and methods of ultraviolet-localized mid-infrared photoacoustic microscopy and/or spectroscopy (ULM-PAM). In some aspects, these ULM-PAM techniques can be used to achieve high-resolution and water-background—free mid-infrared (MIR) imaging of fresh biological samples. In at least some of the disclosed aspects, a pulsed mid-infrared laser thermally excites a sample at a focal spot, and a pulsed ultraviolet (UV) laser photoacoustically detects the resulting transient temperature rise, thereby enabling measurement of the intensity of the MIR absorption by the sample (e.g., enabling measurement of an absorption coefficient of the sample at the wavelength of the MIR laser). This detection and measurement scheme is based on the fact that a temperature rise in a sample enhances photoacoustic signals, a phenomenon called the Grüneisen relaxation effect. Examples of the Grüneisen relaxation effect are described by Wang, L., Zhang, C. & Wang, L. V., “Grueneisen relaxation photoacoustic microscopy,” Phys. Rev. Lett. 113, 174301 (2014), Lai, P., Wang, L., Tay, J. W. & Wang, L. V., “Photoacoustically guided wavefront shaping for enhanced optical focusing in scattering media,” Nat. Photon. 9, 126-132 (2015), and U.S. published patent application US2016/0305914, published on Oct. 20, 2016, which are hereby incorporated by reference in their entireties.
While the ULM-PAM imaging methods disclosed herein reveal MIR absorption contrast, the lateral resolution is not determined by the MIR wavelength but is determined by the UV wavelength, which is one order of magnitude or more shorter than the MIR wavelength. In addition, UV laser pulses in the range of 200-300 nm and especially 200- 230 nm are highly absorbed by most biomolecules, such as lipids, proteins, and nucleic acids. Examples of UV absorption in biomolecules and water are described by Kunitz, M., “Crystalline desoxyribonuclease; isolation and general properties; spectrophotometric method for the measurement of desoxyribonuclease activity,” J. Gen. Physiol. 33, 349-362 (1950); Beaven, G. H. & Holiday, E. R., “Ultraviolet absorption spectra of proteins and amino acids,” Adv. Protein Chem 7, 319-386 (1952); Yao, D.-K., Maslov, K. I., Wang, L. V., Chen, R. & Zhou, Q., “Optimal ultraviolet wavelength for in vivo photoacoustic imaging of cell nuclei,” J. Biomed. Opt. 17, 056004 (2012); and Quickenden, T. I. & Irvin, J. A., “The ultraviolet absorption spectrum of liquid water,” J. Chem. Phys. 72, 4416-4428 (1980), which are hereby incorporated by reference in their entireties. Water, however, is highly transmissive in the UV wavelength range, thus by using UV light to detect IR absorption, the strong water background of MIR absorption is suppressed in the disclosed ULM-PAM aspects. Therefore, ULM-PAM enables high-resolution and photoacoustic MIR imaging of fresh thick and scattering biological samples with little or no water background. By combining the UV and MIR spectral regimes, the ULM-PAM imaging methods described herein provide high-resolution and water-background—free photoacoustic MIR imaging of fresh biological samples.
Furthermore, ultraviolet light can penetrate up to 100 μm or more, which is generally greater than MIR penetration in fresh specimens, and the photoacoustic signal can propagate in biological tissues with negligible scattering. Examples of the penetration depth of ultraviolet light and mid-infrared light are described in Wong, T. T. W. et al., “Fast label-free multilayered histology-like imaging of human breast cancer by photoacoustic microscopy,” Sci. Adv. 3, e1602168 (2017) and Zhang, D. et al., “Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution,” Sci. Adv. 2, e1600521 (2016), which are hereby incorporated by reference in their entireties.
The generation of photoacoustic signals by a sample depends on both the optical absorption coefficient (of the sample at the wavelength being observed) as well as the temperature of the sample prior to photo-stimulation (e.g., the pre-laser-pulse temperature). When an object absorbs a short laser pulse, thermal expansion causes it to emit a photoacoustic signal. The amplitude of the photoacoustic signal is proportional to the absorbed optical energy, with a coefficient called the Grüneisen parameter (I′), which depends on the expansion coefficient and the speed of sound, both of which are temperature-dependent and quasi-linearly proportional to the pre-pulse temperature. As a result, in the physiological temperature range (e.g., between approximately 10° C. and 50° C.), the Grüneisen parameter depends substantially linearly on the pre-pulse temperature (T). Near 20° C. and for water-rich soft biological tissues, the change in the Grüneisen parameter (e.g., ΔI′) can be determined using the equation ΔI′/I′≈0.04 ΔT (wherein Δ denotes a small change). Examples of temperature variations of the Grüneisen parameter are described in Danielli, A. et al., “Label-free photoacoustic nanoscopy,” J. Biomed. Opt. 19, 086006 (2014) and Xu, S., Scherer, G. W., Mahadevan, T. S. & Garofalini, S. H., “Thermal expansion of confined water,” Langmuir 25, 5076-5083 (2009), which are hereby incorporated by reference in their entireties. Therefore, a pre-pulse temperature rise of 1° C. can enhance the photoacoustic signal by ˜4%. This Grüneisen-based change in photoacoustic signal effect is relatively large effect and is highly advantageous as it permits infrared analysis of biological and other materials with minimal increases in the temperature in the specimen. This is a significant advantage compared photothermal techniques in which the relative signal change is of order 10−4/° C. To achieve a 4% change in a conventional photothermal measurement would require sufficient absorption of infrared radiation to raise the temperature of the specimen by 400° C. which would cause damage to most specimens under study. By comparison, the 1° C. change need to achieve a 4% change photoacoustic signal is readily tolerated by most samples.
This relationship has been used to photo-acoustically measure temperature in tissues as described by Larina, I. V. Latin, K. V. & Esenaliev, R. O., “Real-time optoacoustic monitoring of temperature in tissues,” J. Phys. D 38, 2633-2639 (2005); Shah, J. et al., “Photoacoustic imaging and temperature measurement for photothermal cancer therapy,” J. Biomed. Opt. 13, 034024 (2008); and Yao, J., Ke, H., Tai, S., Zhou, Y. & Wang, L. V., “Absolute photoacoustic thermometry in deep tissue,” Opt. Lett. 38, 5228-5231 (2013), which are hereby incorporated by reference in their entireties. When a pulsed laser induces a local transient temperature rise in a sample, the local Grüneisen parameter of the sample increases within the thermal confinement time (that is, the time before the local heat diffuses away), which is termed the Grüneisen relaxation effect. Examples of the Grüneisen relaxation effect are described in Wang, L., Zhang, C. & Wang, L. V., “Grueneisen relaxation photoacoustic microscopy,” Phys. Rev. Lett. 113, 174301 (2014) and Lai, P., Wang, L., Tay, J. W. & Wang, L. V., “Photoacoustically guided wavefront shaping for enhanced optical focusing in scattering media,” Nat. Photon. 9, 126-132 (2015).
ULM-PAM imaging and spectroscopic analysis may be realized as a two-step measuring scheme, as illustrated in
As shown in the top-down view of
In
At another point in time, such as time t2, a MIR heating pulse 120 is generated by a second radiation source and focused on the sample. Shortly thereafter at time t3, a UV probing pulse 130 is generated by the first radiation source and focused on the sample, which in turn emits a second photoacoustic signal 132 (PAUV2). The MIR heating pulse 120 induces a local temperature rise in the sample and the UV probing pulse 130 illuminates the sample before the local temperature rise has had a chance to dissipate. In one embodiment, the time interval (Δt) between the MIR heating pulse 120 and the UV probing pulse 130 is chosen to be a very short delay time after the MIR heating pulse, for example a sub-microsecond time delay that may be on the scale of picoseconds to hundreds of nanoseconds. The advantage of this approach are twofold: (1) the second UV pulse can be timed to be close in time to the maximum temperature rise of the sample; and (2) the delay can be chosen to be shorter than the thermal confinement time (or thermal diffusion time) of the sample. The latter ensures that the technique can achieve high spatial resolution as it ensures that the IR absorption profile of the sample is measured before the heat has substantially spread and diffused.
The local temperature rise (ΔT) induced by the MIR laser pulse is approximately proportional to the MIR absorption coefficient. Because the local temperature rise increases the local Grüneisen parameter, the second photoacoustic signal 132 (PAUV2) is stronger than the first photoacoustic signal 112 (PAUV1). In particular, the second photoacoustic signal 132 (PAUV2) has a larger amplitude 134 than the amplitude 114 of the first photoacoustic signal 112 (PAUV1). Note that amplitudes 114 and 134 can be measured in any number of ways, for example the peak to peak amplitude (as illustrated), the half amplitude, the curve may be rectified and the total integrated area measured, or an appropriate transform may be applied (e.g. Fourier transform or wavelet transform) and the amplitude of one of more of the transform components could be used. It is also possible to employ a lock-in amplifier and use a lock-in amplitude at the pulse repetition rate or any harmonic thereof. Any similar analysis that produces a signal indicative of the strength of the photoacoustic response may be suitable.
The fractional change in photoacoustic amplitude, % ΔPA (defined as ΔPA/PAUV1, where ΔPA=PAUV2−PAUV1), is proportional to ΔT, which is proportional to the absorption coefficient at the MIR wavelength (of the MIR heating pulse 120). However, the ultraviolet wavelength may be a magnitude shorter than the MIR wavelength and the spatial resolution is mainly determined by the focal diameter of the ultraviolet laser beam (See e.g., the UV focal spot 14 of
While
In some aspects, a single MIR heating pulse may be followed, within the thermal relaxation time, by multiple UV probing pulses. The multiple UV probing pulses may, in some aspects, be focused on the same UV focal spot (to obtain multiple measurements for averaging purposes, as an example). In other aspects, such as in the example shown in
In certain implementations, the UV source 410 can be a diode laser, a diode pumped solid state laser, an optical parametric oscillator, a nanosecond, picosecond, and/or femtosecond laser. The UV source 410 can be a fixed wavelength source, a tunable wavelength source, and/or a radiation source that emits a range of wavelengths simultaneously. The UV source 410 will have a shorter wavelength than the mid-infrared light source 412, such that the UV source 410 can be focused to a smaller spot size, thus illuminating a smaller sub-region of the region that the infrared radiation illuminates. The smaller focused spot size of the UV source 410 of radiation can result in an improvement in spatial resolution in the measurement of infrared absorption of the sample.
The ULM-PAM system 400 also includes a second radiation source 412, for example mid-infrared light source (e.g., a pulsed laser or other suitable mid-infrared source). In one aspect, the mid-infrared light source 412 is a pulsed optical parametric oscillator (OPO) tunable between 2,500 and 12,000 nanometers with a pulse duration of approximately 10.0 ns. Alternately, the mid IR light source can be a quantum cascade laser for example tunable in the range of 750-1900 cm−1 (or narrower or wider depending on the application need) with pulse durations in the range of 50 nsec to 1000 nsec or more. The first radiation source can also be a combination of multiple mid-IR sources, for example an OPO in combination with a QCL.
By tuning the wavelength mid-infrared light source 412, the ULM-PAM system 400 can measure a signal indicative of the absorption coefficient of a sample at multiple mid-infrared wavelengths. Thus, the ULM-PAM system 400 can obtain spectroscopic measurements (e.g. an infrared absorption spectrum) and images indicative of variations in the absorption coefficient of the sample as a function of wavelength/wavenumber and/or as a function of position within the sample.
In certain implementations, the mid-infrared light source 412 can be a tunable narrow band laser source, such as a quantum cascade laser, interband cascade laser, an optical parametric oscillator, or any other source of infrared radiation that can tuned over a plurality of wavelengths. MIR source 412 could also be a broadband laser source that simultaneously emits a range of wavelengths, for example a super-continuum source, a femtosecond laser, a frequency comb, or a thermal source (e.g. a “globar”). In these implementations, the first radiation source 412 may be a pulsed laser source that emits pulses, for example, with pulse durations in the microsecond, nanosecond or picosecond range. It may also be a continuous wave (CW) laser source that is chopped, modulated and/or gated.
In
In particular, the delay generator 415 may ensure that each probing ultraviolet pulse follows a mid-infrared pulse by less than the thermal confinement time (thermal diffusion time) of the sample 10. In the case of hydrated biological samples, typical thermal confinement times may be between approximately 100 nanoseconds and 500 nanoseconds, but can be longer for thick samples and/or those with low thermal conductivity. As a result, the delay generator 415 may be configured to delay probing ultrasonic pulses to impinge upon sample 10 between 0-500 ns after a corresponding mid-infrared pulse. In some embodiments, the delay generator 415 may begin a delay period upon generation of a mid-infrared pulse by mid-infrared light source 412 and, at the end of the delay period, the delay generator 415 may trigger ultraviolet light source 410 to emit an ultraviolet probing pulse. In various aspects, the delay added by delay generator 415 can be adjusted (e.g., by a user, by processor(s) 482, etc.), which may assist in adjusting for different types of samples 10 (having different thermal relaxation times) or lasers with different performance characteristics.
In
The ULM-PAM system 400 also includes an ultrasonic transducer 440 (e.g., an ultrasonic receiver) coupled to or otherwise in acoustic communication with sample 10 to detect photoacoustic signals from the illuminated regions of the sample 10. The ultrasonic transducer 440 may be acoustically coupled to sample 10 by an acoustic medium 12. The acoustic medium may be an acoustic gel, water, or other suitable medium capable of conveying ultrasound pulses from sample 10 to transducer 440. If desired, the acoustic medium 12 may be omitted. In some embodiments, the ultrasonic transducer 440 is a single element transducer. In other embodiments, the ultrasonic transducer 440 may be an array of transducers and, if desired, may be a steerable phased array with receive focusing capabilities. In some aspects, the ultrasonic transducer 440 may be an array of transducers that enable the collection of multiple photoacoustic signals in parallel, to enable faster imaging and spectroscopic analysis across an area of the sample 10.
The ULM-PAM system 400 may also include a scanning mechanism 430 coupled to one or more elements of the optical system 420. Optionally (denoted by a dotted line) the scanning mechanism 430 is coupled to the sample 10, in addition to or in the alternative to, the one or more elements of the optical system 420. The scanning mechanism 430 is coupled to one or more components of the ULM-PAM system 400 to be able to move the focal spots of the illumination beams (e.g., the MIR focal spot 13 and the UV focal spot 14 of
In
Returning to
In some implementations, the ULM-PAM system includes one or more communication interfaces (e.g., a universal serial bus (USB) interface). Communication interfaces can be used, for example, to connect various peripherals and input/output (I/O) devices such as a wired keyboard or mouse or to connect a dongle for use in wirelessly connecting various wireless-enabled peripherals. Such additional interfaces also can include serial interfaces such as, for example, an interface to connect to a ribbon cable. It should also be appreciated that the various system components can be electrically coupled to communicate with various components over one or more of a variety of suitable interfaces and cables such as, for example, USB interfaces and cables, ribbon cables, Ethernet cables, among other suitable interfaces and cables.
In
It would be understood that electrical communication between components of the various ULM-PAM systems described herein can be in wired or wireless form or a combination thereof. For simplicity, the sample 10 is illustrated as a block, it would be understood that the sample 10 can be in a variety of shapes and may have one or more objects of interest. Various blocks can also be combined within a single functional unit, for example the data acquisition unit 460 may be integrated with the computing device 480, for example as a data acquisition card in an expansion slot in a personal computer. The computing device 480 may also comprise a distributed system, for example including separate processors (CPU, digital signal processors, field programmable gate arrays, embedded controllers, single board computers, etc. that may be housed together and/or in separate enclosures.
During a data acquisition phase of an imaging process of the ULM-PAM system 400 according to one implementation, the processor(s) 482 executes instructions that send control signals to the ultraviolet laser 410 to deliver pulses of UV radiation to the sample 10, control signals to the mid-infrared light source 412 to deliver pulses of MIR radiation to the sample 10, control signals to the scanning mechanism 430 to scan the UV and MIR focal spots across (and/or through) the sample 10, control signals to the digitizer 462 to record photoacoustic signals received from the ultrasonic transducer device 440. The digitizer 462 records photoacoustic signals induced by the UV radiation pulses for each of the locations of the UV focal spots in sub-regions of the field-of-view of the sample 10 being imaged. During an image construction phase, the processor(s) 482 executes instructions to perform operations to construct a photoacoustic image, a spectroscopic image, or other information from the data in the photoacoustic signals.
The first radiation source 509 is capable of producing shorter wavelengths than the second (IR) radiation source. In one aspect the first radiation source 509 emits light in the ultraviolet range, but more broadly it can be of a broader range, for example within the wavelength range of 100-2000 nm (e.g. X-ray to near-IR).
One or both of the radiation sources 509 and 511 may be tunable to varying the wavelengths of the MIR or UV pulses. In such embodiments, computing device 580 may provide control signals to the radiation sources 509 and 511 to tune their wavelengths. Computing device 580 may be in one or more of the forms of computing device 480 as described associated with
In the example of
In the example of
The optical paths between sample 10 and the radiation sources 509 and 511 may be combined at a beam combiner 522, for example germanium dichroic mirror. Beam combiner 522 may be transmissive to IR and reflective to UV as in the configuration shown or the reverse (reflective to IR and transmissive to UV). The combined IR and UV beams are reflected by mirror 524 and then delivered to sample 10 through an objective 526. Objective 526 may for example be a reflective objective of a Schwarzschild (Cassegrain configuration). It may also be an off-axis parabolic mirror, and/or other mirror combination configured to focus the IR and UV pulses to focal spots on sample 10. Mirror arrangements can be optimal because they are generally wavelength independent, but it is possible to employ refractive approaches as well. It is also not necessary that the IR and UV beams be combined collinearly. It is possible to use two separate focusing elements to deliver the UV and IR beams separately, in which case the optics can be separately optimized for the respective wavelength ranges.
Pulses from both the UV and MIR lasers 509 and 511 are focused on the sample 10, which may be mounted on an IR transparent window (e.g. CaF2, ZnSe, ZnS, BaF2 or various other materials) attached to the bottom of a sample holder 510. Sample holder 510 optionally also serves as a reservoir (e.g., tank or liquid cell) to hold acoustic coupling fluid, for example water, buffer solution, acoustic gel or any other suitable coupling fluid. Configured in transmission mode, an ultrasonic transducer 540, which in one aspect has a 25 MHz center frequency, is configured to collect the photoacoustic signals generated by the UV and MIR lasers 509 and 511. Ultrasonic transducer 540 may optionally include a focusing element in which case it may be confocally aligned with the MIR and UV spots, i.e. the foci of the ultrasonic transducer 540 and the objective 526 are substantially aligned. Signals collected by transducer 540 may be optionally amplified by pre-amplifier 550 and then digitized by data acquisition system 560. Data acquisition system 560 may also include channels for collecting and digitizing signals from photodiodes 570 and 572 and may include trigger inputs and/or outputs to synchronize data acquisition with the radiation pulses from radiation sources 509 and/or 511.
In the example of
In some implementations, computer 580 may direct the components of system 500 to photoacoustically scan sample 10 along a x-axis line, translate the sample (or optics), and then scan sample 10 along a new x-axis line, repeating that process to form a two-dimensional (2D) image. If desired, the photoacoustic scanning may be repeated across a number of z-axis positions, to form a three-dimensional (3D) image. If desired, a 2D image may be formed that extends in the z-axis direction and one of the x-axis or y-axis directions.
In some embodiments, ULM-PAM imaging systems may be configured to operate in a transmission mode. An example of components of a ULM-PAM system 600 in a transmission mode configuration is shown in
In some other embodiments, ULM-PAM imaging systems may be configured to operate in a reflection mode. Examples of reflection mode configurations are shown in
At operation 910, the ULM-PAM system focuses one or more first ultraviolet pulses to one or more regions of a sample respectively. In one aspect, the ULM-PAM system focuses one first ultraviolet pulse to a region of the sample to induce a baseline photoacoustic signal (PAUV1) at operation 910. The ultrasonic transducer collects a baseline photoacoustic signal at a single position within the sample (e.g., a single UV focal spot). In another aspect, operation 910 involves delivering one or more first ultraviolet pulses to multiple regions respectively of the sample. In this aspect, the ultrasonic transducer detects one or more baseline photoacoustic signals (PAUV1) from different locations within the sample (e.g., a microlens array and ultrasound array as discussed in connection with
At operation 910, the ultraviolet light source generating the ultraviolet pulse(s) is either (i) triggered at a first time before the infrared light source is triggered at operation 912 to generate the infrared pulse(s) or (ii) triggered at a second delay time after the infrared light source is triggered in operation 912.
At operation 912, according to one aspect, the ULM-PAM system focuses a mid-infrared heating pulse to a focal spot at a region on the sample, resulting in localized heating of the sample at the focal spot of the MIR pulse in regions of the sample that absorb IR light at the selected wavelength of the MIR pulses. In another aspect, operation 912 involves delivering multiple MIR pulses to multiple regions of the sample simultaneously resulting in localized heating at the plurality of focal spots and/or simultaneously illuminating a large area of the sample with MIR light, e.g. though a large focused IR spot size. The amount of heating in the sample will vary depending on the absorption coefficient of the sample at the MIR wavelength (which is tunable). Thus it is possible to probe the samples IR absorption properties by measuring the relative amount of local sample heating due to IR absorption. The amount of heating is also dependent on the strength of the MIR pulse. In one embodiment, the strength of the MIR pulse used to heat the sample may be measured using photodiode 472 (or other device), to enable compensation for variations strength between different MIR pulses and variation in MIR pulse energy as a function of the MIR wavelength.
At operation 914, the ULM-PAM system focuses one or more second ultraviolet pulses on the sample. In one aspect, the ULM-PAM system focuses an ultraviolet pulse to a sub-region within the region heated by the infrared heating pulse in operation 912. The ultraviolet pulse induces a probing photoacoustic signal (PAUV2). The probing photoacoustic signal is from the sub-region of the sample heated in operation 912. In another aspect, the ULM-PAM system focuses multiple ultraviolet pulses to multiple sub-regions within the region heated by the infrared heating pulse in operation 912 and the ultraviolet pulses induce a plurality of probing photoacoustic signals (PAUV2) from the sub-regions. In yet another aspect, the ULM-PAM system focuses a plurality of first ultraviolet pulses to a plurality of sub-regions, each sub-region in a region heated by an infrared heating pulse in operation 912 and the second ultraviolet pulses induce a plurality of probing photoacoustic signals (PAUV2) from sub-regions in the plurality of regions.
The ULM-PAM system triggers an ultraviolet light source to generate the one or more second ultraviolet pulses in operation 914 within a predetermined period from the triggering of the infrared light source in operation 912. In most cases, the predetermined period is the thermal relaxation time (also referred to as thermal confinement time) of the sample (e.g., the duration before the heated region has substantially cooled or reached thermal equilibrium with the rest of the sample). In the case of hydrated biological samples, typical thermal confinement times may be between approximately 100 nanoseconds and 500 nanoseconds. Thus, when the sample is a hydrated biological sample, operation 914 may be performed within 100 nanoseconds, within 200 nanoseconds, within 250 nanoseconds, within 300 nanoseconds, within 400 nanoseconds, or within 500 nanoseconds of the triggering of the ultraviolet light source in operation 912.
Operation 914 also involves detecting a photoacoustic signal from a single position within the sample (e.g., a single UV focal spot) or collecting photoacoustic signals from a plurality of positions within the sample (e.g., a microlens array and ultrasound array as discussed in connection with
At operation 916, the ULM-PAM system determines if the desired field-of-view (of the sample) has been fully scanned. If the field-of-view is not fully scanned, the ULM-PAM system performs operation 917 by moving components of the system to move the focal spots of the light sources to a new region or new regions of the field-of-view. For example, operation 916 may involve processors(s) that send control commands to a scanning mechanism (e.g., scanning mechanism 430) to shift the UV and/or MIR focal spots to another region(s) of the field-of-view. Afterwards, the ULM-PAM method repeats operations 910, 912, and 914.
If the field-of-view is fully scanned, the ULM-PAM method performs operation 919 if spectroscopic measurements or images are desired. At operation 919, the ULM-PAM system tunes or adjusts the frequency (i.e. wavelength) of the infrared light source. As an example, operation 919 may involve processor(s) sending control commands to a tunable mid-infrared light source to re-tune to a new infrared frequency. Afterwards, the ULM-PAM system repeats operations 910, 912, and 914. With arrangements of this type, the ULM-PAM system can measure a signal indicative of the IR absorption coefficient at a plurality of infrared wavelengths to obtain spectroscopic information about the sample, e.g. an infrared absorption spectrum. Operation 919 may be performed repeatedly in combination with operations 910, 912, 914, and 916 as indicated by the arrows in which case a hyperspectral data cube will be created, i.e. a signal indicative of IR absorption is created at a multidimensional array of sample positions and IR wavelengths. It is also possible to perform these measurements at any subset of spatial or spectral dimensions. For example it is possible to obtain IR absorption measurements at a plurality of XY locations, but fixed MIR wavelength to produce a map of IR absorption for example at a single IR absorption band. It is also possible to remain at a single location on the sample and rapidly sweep the IR laser frequency (wavelength) to obtain an IR absorption spectrum at a single location. It is also possible to create IR absorption spectra at a pre-programmed array of points on the sample (e.g. a line array) and/or at discrete locations selected by a user.
After or concurrently with collecting the baseline and probing photoacoustic signals, the ULM-PAM system may perform normalization operation 922. At operation 922, the ULM-PAM system normalizes the collected photoacoustic signals. As an example, the ULM-PAM system may analyze the collected photodiode measurements indicative of the pulse energies of the IR and or UV pulses and scale the measured response by the strength of the IR/UV pulses. This may be performed in real-time, e.g., by simultaneously measuring the IR and/or UV pulse energy during the measurement of the ultrasonic responses, and/or it may be performed before or after. For example it is possible to separately measure the IR pulse energy as a function an IR frequency (wavelength) and use this as a normalization curve for the measured data. It is also possible to perform a reference measurement on a sample with either flat or known IR absorption properties and use the reference measurement to normalize the photoacoustic responses. This normalization step 922 can also follow rather than precede operation 924 below.
At operation 924, the ULM-PAM system analyzes the detected (and optionally normalized) amplitudes of the photoacoustic signals and identifies fractional changes resulting from the MIR pulse heating. As an example, the system may determine the change in amplitudes of the photoacoustic signals for each sub-region location (e.g., pixel) scanned within the sample and optionally for each MIR wavelength in the target spectrum, calculated as:
ΔPA=PAUV2−PAUV1 (Eqn. 1)
wherein PAUV1 is a baseline photoacoustic signal obtained before or after MIR heating and PAUV2 is a probing photoacoustic signal obtained synchronous with or following MIR heating but preferably within the thermal relaxation time of the sample (i.e. before the IR induced temperature increase has dissipated and/or the spatial resolution has been adversely affected due to spreading of heat).
As previously noted, the generation of photoacoustic signals by a sample depends on both the optical absorption coefficient (of the sample at the wavelength being observed, e.g., at the UV wavelength) as well as the temperature of the sample prior to photo-stimulation (e.g., the pre-laser-pulse temperature). Since each pair of baseline and probing photoacoustic pulses are collected using UV pulses at the same wavelength focused on the same portion of the sample, any differences in PAUV1 and PAUV2 results from the localized heating from the MIR pulse. The ULM-PAM system is able to use the differences in photoacoustic signals (each pair being associated with a particular focal position at sub-region and a particular MIR wavelength) to measure the amount of heating resulting from the MIR pulse. The temperature change resulting from each MIR pulse depends upon the strength of the MIR pulse as well as the absorption coefficient of the sample at the scan location. Thus, by measuring the amount of heating resulting from the MIR pulse, the ULM-PAM system is able to measure the absorption coefficient of the sample. Operation 924 may involve using feedback from photodiode 472 to compensate for variations in the strength of the MIR pulse such as by dividing every differential photoacoustic signal (ΔPA) by a signal representative of the strength of the MIR and/or UV pulses. In the description above the differencing operation 924 was described as operating between alternating pulse pairs. Note that it is also possible to obtain a plurality of baseline pulses (step 910) then a plurality of IR and second UV pulses (steps 912 and 914) and then perform the difference calculation (step 924) on the plurality of baseline and 2nd UV pulses.
At operation 926, the ULM-PAM system may construct an output from the differential photoacoustic signal(s) (ΔPA). As examples, the ULM-PAM system may construct a spectroscopic measurement (e.g., an absorption coefficient for a particular MIR wavelength at a single position in the sample) or a graph or image (e.g., a 1-D line graph, 2-D image, a 3-D image, or a 4-D image showing absorption coefficients of the sample as a function of at least one of MIR wavelength, x-position, y-position, and z-position within the sample).
To demonstrate the capabilities of ULM-PAM systems, a ULM-PAM system was used to obtain ULM-PAM measurements and imagery of various test samples. Various alternative systems were used to obtain alternative measurements of the test samples for the sake of comparison with the ULM-PAM results. As examples, a pure mid-infrared photoacoustic microscopy (MIR-PAM) system and an attenuated total reflection-Fourier transform infrared microscopy (ATR-FTIR) system were used to obtain comparison measurements.
The resolution of the ULM-PAM system was further demonstrated by imaging a carbon-nanotube (CNT) pattern deposited on an MgF2 substrate, which was developed as a broad-spectral resolution target for photoacoustic imaging.
As further demonstration investigating subcellular imaging, a ULM-PAM system was used to image the lipid and protein composition in freshly fixed 3T3 mouse fibroblast cells. The ULM-PAM UV wavelength was tuned to 224 nm, where almost all proteins, lipids, and nucleic acids have strong absorption. For lipid imaging, the MIR wavelength was set to the absorption peak at 3,420 nm (2924 cm−1), corresponding to the asymmetric stretching mode of the CH2 group—the dominant constituent of lipids. The ULM-PAM image of lipids in
In contrast, the MIR-PAM images of lipids (
Four line-profiles taken from
The ULM-PAM system was also used to image cells at different stages of their lifecycle.
Another major application of ULM-PAM systems is the label-free photoacoustic histology of thick tissue slices. Since the study of nerve fibers is important in brain science, and myelin is one of the main chemical components of nerves, a ULM-PAM system was used to study the structural distribution of myelin in the mouse brain ex vivo. A microtome sliced a coronal cerebrum section and a horizontal cerebellum section, both 300 μm thick, from a freshly fixed mouse brain. In the beginning, myelin images of these two sections (
To overcome the MIR-PAM limitations, a ULM-PAM system, configured with a UV wavelength of 224 nm and an MIR wavelength of 3,420 nm, was used to achieve higher-resolution and water-background—free MIR imaging of myelin. A MIR-PAM system was used to scan a small area of interest and the results are illustrated in the image of
The ULM-PAM image of myelin (
A similar set of images were obtained on another region of interest, this time within the cerebellum section of the brain (e.g., region 1202 in
Details of the systems used in obtaining the ULM-PAM, MIR-PAM, ATR-FTIR, and other sample images (of
The ULM-PAM system utilized an OPO mid-MIR laser (a NT242-SH, made by EKSPLA) and an OPO UV laser (a NT270, made by EKSPLA). The wavelength of the selected OPO mid-MIR laser was tunable from 2.5 to 12 μm (from 4,000 to 833 cm−1), with a pulse duration of about 10 ns, and an output pulse energy that varied from 10 to 100 μJ depending on the selected wavelength. For imaging lipids and proteins using ULM-PAM, the UV laser was tuned to 224 nm; for imaging nucleic acids using UV-PAM, it was set to 250 nm. Both the MIR and UV beams, with diameters of about 4 mm, were reflected by several UV-enhanced aluminum coated mirrors (PF10-03-F01, made by Thorlabs), and focused to a sample mounted on a CaF2 window through a 36× reflective objective (a 50102-02, made by Newport). Photoacoustic signals were detected in transmission mode by a focused ultrasonic transducer having a 25 MHz center frequency (a V324-SM, made by Olympus), which has an acoustic focal length of 12.7 mm and an element diameter of 6 mm. The acoustic coupling medium was de-ionized water. Photoacoustic signals were amplified by ˜50 dB using two low-noise amplifiers (a ZFL-500LN+, made by Mini-circuits), and then acquired by a DAQ card (a Razor 14, made by Gage) at 14 bits and 200 MS/s.
ULM-PAM images of the sample at selected wavelengths were obtained by raster scanning using two motorized stages (a PLS-85, made by PI miCos) coupled to the sample holder. To drive the scanning stages at high precision (e.g., a 50 nm step size) with minimal low-frequency vibration, two five-phase stepper motors (PKP546MN18B, made by Oriental Motor) and the associated drivers (CVD518-K, made by Oriental Motor) were used. Line scans along the x-axis were averaged to compensate for UV laser inter-pulse fluctuations. The laser tuning, data acquisition, and scanning systems were synchronized by a central computer via micro-controllers, using LabVIEW.
Note that all of the above system configuration information is provided as an illustrative example only. The ULM-PAM system can also be operated with many other alternative components, for example alternative MIR and UV laser sources described elsewhere herein, alternative data acquisition and computation arrangements, alternative scanning mechanisms, etc.
The scan step size varied for different samples. The point spread function of a 50-nm—diameter carbon nanobead was measured using a 50 nm step size. Imaging cells was done with a 200 nm step size. For imaging brain slices, to compensate for the slow laser repetition rates of the lasers, the UV beam was shrunk to reduce the effective NA to 0.16, which worsened the resolution to some extent, but accelerated the scanning with a larger step size (500 nm) and effectively extended the focal depth to accommodate uneven sample surfaces.
All ATR-FTIR spectra were measured on an ATR-FTIR spectrometer (a Nicolet 6700, made by Thermo) in the Molecular Materials Research Center at California Institute of Technology. The baseline was calibrated by measuring a blank sample.
Mouse embryonic fibroblast cells (NIH/3T3) were obtained from American Type Culture Collection (ATCC, Manassas, Va.) and maintained at 37° C. and 5% CO2 in Dulbecco's modified Eagle's medium (Invitrogen brand) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin (P/S, Invitrogen). The cells were seeded onto 1-mm—thick CaF2 substrates, grown for about two days, and then fixed in 3.7% formalin solution and washed with phosphate buffered saline.
The brain was extracted from Swiss Webster mice (Hsd: ND4, Harlan Laboratories), and fixed in 3.7% formalin solution at room temperature. Afterwards, the fixed brain was embedded in 4% agarose and then sectioned by a microtome (VT1200 S, Leica) into slices 300 μm thick. All experimental animal procedures were carried out in conformity with a laboratory animal protocol approved by the Animal Studies Committee of California Institute of Technology. After they are imaged by ULM-PAM, the thick slices underwent standard procedures for histological staining (including paraffinization, slicing into 10 μm in thickness, and staining), and were finally imaged in a digital pathology system (VENTANA iScan HT, Roche). It should be noted that some samples were fragmented and slightly distorted during the thin-slicing procedure, which however did not affect the comparison.
ULM-PAM provides a new high-resolution and water-background—free MIR microscopy modality capable of imaging fresh biological samples without staining. For cells, ULM-PAM provides sub-cellular MIR imaging of lipids and proteins with high contrast without a strong obscuring water background. For tissue slices, ULM-PAM produces label-free PA-histologic images without requiring thinly slicing and drying samples, unlike conventional MIR imaging. The ULM-PAM system can cover the full MIR spectral range, allowing exploration of a variety of molecules of interest. Among the existing far-field MIR imaging approaches, ULM-PAM is believed to have achieved the highest imaging resolution at 250 nm, which can be further improved by using a higher-NA objective lens and a shorter UV wavelength (as examples). In addition, ULM-PAM can potentially bridge the resolution gap from 100 nm to 400 nm in materials science studies between AFM-MIR and other MIR modalities, such as ATR-FTIR and photothermal MIR.
The ULM-PAM can be further improved in some aspects. As an example, although the signal-to-noise ratios of raw photoacoustic signals are high, their fractional changes can be noisy because they are derived from two successive but non-identical UV laser pulses. Thus, selection of stable UV and MIR lasers, with low pulse-to-pulse variations, is beneficial to a ULM-PAM system. Alternately, however, the ULM-PAM design disclosed herein can be modified such that a single UV pulse is replicated into first and second parts, the first part serving as a baseline UV pulse, and the second part passing through a long optical fiber delay line to delay the second part to just after a MIR pulse such that the second part serves as a probing UV pulse (thus reducing or avoiding inter-pulse fluctuations). As another example, the imaging speed can be limited when the selected UV and MIR lasers have low pulse repetition rates. For example, if the lasers both have a pulse repetition rate that maxes out at 1 kHz, it could take over two hours to scan a 1 mm by 1 mm area with a 500 nm step size. Thus it can be desirable to use IR and UV sources with higher repetition rates. For example commercially available OPO and QCL IR sources can have repetition rates as high as 1 MHz or higher, up to 100× faster than the Ekspla OPOs described above. Frequency quadrupled Nd-YAG lasers can also operate at rates of 100 kHz or higher. Femtosecond broadband sources can operate with pulse repletion rates as high as tens of MHz and can produce MIR or UV pulses or in some cases both. As another example, multiple scans may be need to be collected and averaged together, to compensate for inter-pulse fluctuations and other noise sources, further slowing imaging speed. However, multi-focal photoacoustic imaging using a linear array of ultrasonic transducers can improve the imaging speed significantly, even more than 100 times faster. Additional aspects of multi-focal embodiments are described and disclosed in connection with
Two conventional vibrational imaging microscopies provide far-field and label-free imaging at sub-cellular resolution: Raman scattering microscopy and photothermal MIR microscopy. Examples of Raman scattering microscopy are described by Evans, C. L. & Xie, X. S., “Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu. Rev. Anal. Chem. 1, 883-909 (2008) and Zhang, C., Zhang, D. & Cheng, J.-X., “Coherent Raman scattering microscopy in biology and medicine,” Annu. Rev. Biomed. Eng. 17, 415-445 (2015), which are hereby incorporated by reference in their entireties. Examples of photothermal MIR microscopy are described by Furstenberg, R., Kendziora, C. A., Papantonakis, M. R., Nguyen, V. & McGill, “R. A. Chemical imaging using infrared photothermal microspectroscopy,” In Proceedings of SPIE Defense, Security, and Sensing (eds Druy, M. A. & Crocombe, R. A.) 837411 (SPIE, 2012) and Zhang, D. et al., “Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution,” Sci. Adv. 2, e1600521 (2016), which are hereby incorporated by reference in their entireties.
Compared with Raman scattering microscopy, the detection sensitivity of the ULM-PAM system, determined by direct MIR photon absorption, is much higher, since the fundamental MIR cross-section is several orders of magnitude larger than that of the Raman scattering cross-section. In addition, this method not only exhibits the same features as Raman scattering microscopy, such as high resolution and low water background, but also enables the direct imaging of various vibrational bonds in the mid-MIR spectral range. Compared with photothermal MIR microscopy, this method is essentially a new way to exploit the MIR photothermal effect, but photoacoustic detection is used instead to retrieve the MIR-absorption induced local temperature rise, enabling MIR imaging in thick, highly optically absorbing and scattering biological samples. Perhaps more importantly, photoacoustic temperature sensing is based on the Grüneisen relaxation effect, which gives a photoacoustic signal change of about 3%/° C. in the physiological temperature range. This change is two orders of magnitude greater than the photothermally induced refractive index change (˜10−4/° C.), making ULM-PAM more sensitive than photothermal MIR microscopy. Furthermore, since water is almost transparent at UV wavelengths from 200 nm to 300 nm (see
If desired, the ULM-PAM system can be modified to attain nanoscale far-field chemical imaging by shortening the wavelength of the probe beam (e.g., laser 410) to the X-ray wavelength regime. Current X-ray microscopy for biological samples operates either in the soft X-ray regime for water transparency (2.33-4.40 nm) or the hard X-ray regime, using imaging contrast arising from natural X-ray absorption, to provide nanometer scale resolution. Imaging specific chemicals or structures inside biological samples requires exogenous labeling, e.g., silver-enhanced immunogold labeling. X-ray acoustic imaging has also been demonstrated with a pulsed X-ray source as described, for example, by Lasch, P., Boese, M., Pacifico, A. & Diem, M., “FT-IR spectroscopic investigations of single cells on the subcellular level,” Vibr. Spectrosc. 28, 147-157 (2002), which is hereby incorporated by reference in its entirety. By combining the MIR-absorption induced Grüneisen relaxation effect and X-ray-acoustic imaging, the ULM-PAM system can achieve X-ray-acoustic imaging with MIR-absorption contrast, utilizing acoustic signals generated by pulsed X-rays to report MIR absorption contrast in materials, thereby achieving far- field and label-free imaging at nanometer scale resolution.
In some embodiments, the ULM-PAM system 400 can scan and collect photoacoustic measurements from multiple locations within a sample 10 simultaneously, e.g., in order to speed up imaging and spectroscopic operations. As an example, the ULM-PAM system 400 can include one or more optical components that spread the pulses from ultraviolet light source 410 and mid-infrared light source 412 into sheet-like beams and a microlens array, where each microlens focuses a portion of the laser light onto a corresponding portion of the (such as along a scan axis, which is aligned with the x-axis in the example of
Although some examples of ULM-PAM systems and methods describe a two-step measuring scheme with a first step of initiating (e.g., triggering by a pulser) delivery of a first ultraviolet laser pulse to a sub-region within a region to generate a first (baseline) photoacoustic signal and a second step of initiating delivery of a UV probing pulse to the region, followed by initiating delivery of a second ultraviolet laser pulse to the sub-region to generate a second photoacoustic signal, it would be understood that the order of these steps can be reversed according to another aspect. For example, according to one aspect, the technique initiates delivery of the first ultraviolet laser pulse to a sub-region within a region to generate a first (baseline) photoacoustic signal where the initiation (e.g., triggering) occurs after (e.g., a predetermined time period after the initiation of the delivery of the first ultraviolet laser pulse) the step of initiating delivery of a UV probing pulse to the region, followed by initiating delivery of a second ultraviolet laser pulse to the sub-region.
Modifications, additions, or omissions may be made to any of the above-described embodiments without departing from the scope of the disclosure. Any of the embodiments described above may include more, fewer, or other features without departing from the scope of the disclosure. Additionally, the steps of the described features may be performed in any suitable order without departing from the scope of the disclosure.
It should be understood that the present invention as described above can be implemented in the form of control logic using computer software in a modular or integrated manner. Based on the disclosure and teachings provided herein, a person of ordinary skill in the art will know and appreciate other ways and/or methods to implement the present invention using hardware and a combination of hardware and software.
Any of the software components or functions described in this application, may be implemented as software code using any suitable computer language and/or computational software such as, for example, Java, C, C#, C++ or Python, LabVIEW, Mathematica, or other suitable language/computational software, including low level code, including code written for field programmable gate arrays, for example in VHDL. The code may include software libraries for functions like data acquisition and control, motion control, image acquisition and display, etc. Some or all of the code may also run on a personal computer, single board computer, embedded controller, microcontroller, digital signal processor, field programmable gate array and/or any combination thereof or any similar computation device and/or logic device(s). The software code may be stored as a series of instructions, or commands on a CRM such as a random access memory (RAM), a read only memory (ROM), a magnetic medium such as a hard-drive or a floppy disk, or an optical medium such as a CD-ROM, or solid stage storage such as a solid state hard drive or removable flash memory device or any suitable storage device. Any such CRM may reside on or within a single computational apparatus, and may be present on or within different computational apparatuses within a system or network.
Although the foregoing disclosed embodiments have been described in some detail to facilitate understanding, the described embodiments are to be considered illustrative and not limiting. It will be apparent to one of ordinary skill in the art that certain changes and modifications can be practiced within the scope of the appended claims.
One or more features from any embodiment may be combined with one or more features of any other embodiment without departing from the scope of the disclosure. Further, modifications, additions, or omissions may be made to any embodiment without departing from the scope of the disclosure. The components of any embodiment may be integrated or separated according to particular needs without departing from the scope of the disclosure.
This application claims priority to and benefit of U.S. Provisional Patent Application No. 62/726,860, titled “High-Resolution, High-Contrast Mid-Infrared Imaging Of Fresh Biological Samples With Ultraviolet-Localized Photoacoustic Microscopy” and filed on Sep. 4, 2018, which is hereby incorporated by reference in its entirety and for all purposes.
This invention was made with government support under Grant No(s) CA186567, EB016986, NS090579 and NS099717 awarded by the National Institutes of Health. The government has certain rights in the invention.
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| Number | Date | Country | |
|---|---|---|---|
| 20200073103 A1 | Mar 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62726860 | Sep 2018 | US |
