We demonstrate a rapid deep ultraviolet transmission and scattering microscopy system which utilizes a single 266 nm nanosecond pulsed laser source to generate virtual histology images. By detecting both the transmitted and back-scattered 266 nm radiation, distinct contrasts of the tissue section are produced. Maximally realistic virtual histology images are then generated using each contrast as separate inputs to a CycleGAN deep neural network. This work demonstrates virtual histology images of mm-scale areas at fine resolutions (< 500 nm) within minutes. This system greatly simplifies the optics and alignment required for imaging, and could provide future utility for clinical studies.
Ultraviolet photacoustic remote sensing (UV-PARS) microscopy is a non-contact, label-free imaging modality which has demonstrated the ability to generate virtual histology images that show good concordance to gold-standard hematoxylin and eosin (H&E) stains of fixed tissue sections. However, UV-PARS microscopy requires time-consuming and complex coalignment of multiple beams for imaging. In this work, we demonstrate UV transmittance and scattering microscopy for virtual histology and compare with UV-PARS microscopy. Maximally realistic virtual histology images are generated for both UV-PARS and UV-transmittance microscopy techniques using a cycle-consistent generative adversarial network (CycleGAN) and compared to one another alongside the gold-standard brightfield microscopy of H&E stained fixed tissues.
Pathologist evaluation of brightfield microscopy images of H&E-stained tissues is time-consuming and labor-intensive, preventing intraoperative feedback to surgeons. Combined ultraviolet reflectance and photoacoustic remote sensing (PARS) microscopy was recently introduced, providing a label-free way to obtain cell nuclei and cytoplasm images. Additionally, utilizing deep learning and fast voice-coil scanning, maximally realistic virtual histology can be rapidly generated with this technique. Here, we introduce combined ultraviolet confocal reflectance, autofluorescence, and PARS microscopy, utilizing a single 266 nm excitation beam. This system now allows us to generate sectioned virtual histology in thick tissues well suited to the intraoperative setting.
Ultraviolet photoacoustic remote sensing microscopy provides label-free optical absorption contrast comparable to hematoxylin staining. This has been combined with 266 nm optical scattering microscopy offering eosin-like contrast. Here, we use unsupervised deep learning-based style transfer using the CycleGAN approach to render these pseudo-colored virtual histological images in a realistic stain style comparable to the H&E gold standard in unstained human and murine tissue specimens. A multi-pathologist diagnostic concordance study found a sensitivity of 89%, specificity of 91%, and accuracy of 90%. A blinded subjective stain quality survey suggested virtual histology was preferred over frozen sections at the 95% confidence level.
We present photoacoustic remote sensing and autofluorescence microscopy with surface excitation (PARS-AMUSE) system that combines several methodologies of optical imaging including absorption, scattering and radiative emission. We generate virtual hematoxylin and eosin (H&E) images from absorption and scattering data using a generative adversarial network for margin assessment and cancer grading with simultaneous metabolic measurements from the autofluorescence of NADH and FAD. This system utilizes a single excitation source for all three imaging modalities to minimize complexity and cost. Using this system, we aim to profile specific cancers and predict cancer aggression.
We demonstrate a rapid photoacoustic remote sensing (PARS) microscopy system with voice-coil stage scanning, capable of acquiring 1cm2 virtual histology images at 800nm x 800nm lateral sampling density in 3.75 minutes, high-resolution (300nm x 300nm sampling density) images at 1mm2 in 20 seconds and 300μm x 300μm in 3 seconds. The combined resolution and scanning speed that can be achieved by this system may pave way for clinical translation to rapidly assess resected tumor margins in the surgical suite.
We present a novel microscopy technique called photoacoustic remote sensing combined with autofluorescence with surface excitation (PARS-AMUSE) to generate virtual hematoxylin and eosin (H&E) images with simultaneous metabolic measurements to predict cancer aggression. PARS uses absorption and scattering contrast for the virtual H&E images to visualize structural information, perform margin assessment and grade cancers. AMUSE utilizes the same light source used in PARS to excite NADH and FAD autofluorescence for quantifying the optical redox ratio (ORR), an important indicator of tissue metabolism. Using our system, we aim to differentiate receptor status and predict cancer aggression.
A multimodal approach for margin assessment combines fast, cross-sectional imaging using 1050 nm spectral-domain optical coherence tomography (SD-OCT), offering sensitivity to highly-scattering malignant tissues, with complementary sub-cellular resolution and molecular specificity provided by ultraviolet photoacoustic remote sensing virtual histology. The interferometric synthetic aperture microscopy (ISAM) extension to OCT was implemented to achieve spatially-invariant lateral resolution, reducing the resolution disparity between each modality by avoiding numerical aperture trade-offs. System validation included carbon fiber and TiO2 particle phantom imaging, and ex vivo biological imaging was demonstrated in fresh, thick murine tissues, and benign and malignant human breast samples.
A combined synthetic aperture optical coherence microscopy and ultraviolet photoacoustic remote sensing system is presented, capable of fast scanning of tissues for tumor margin inspection. It provides a fast 3D OCT mode for imaging tissues to depths of ~1mm, and a superficial virtual histology mode provided by absorption contrast UV-PARS for virtual hematoxylin contrast and coherence-gated scattering microscopy for virtual eosin contrast. Breast lumpectomy specimens are scanned in each mode to evaluate the extent of features in depth and generate en-face images with histological detail and realism, providing results accurately interpretable by pathologists.
An estimated 20-40% of patients who undergo breast-conserving surgery require repeat operations due to the length of time required post-surgery to analyze the resected sample. Recently, a novel non-contact microscopy technique known as ultraviolet photoacoustic remote sensing (UV-PARS) has been developed, which can produce virtual hematoxylin and eosin (H&E) stained images. By using a voice-coil scanning stage in conjunction with an on-demand pulsed laser, we demonstrate 1cm x 1cm gross UV-PARS virtual H&E scans in approximately 50 seconds, along with full-resolution 1cm x 1cm scans in approximately 8 minutes. With widefield high-speed scanning, UV-PARS shows promise for future translation to clinical application.
Currently, there is an inability to obtain fast realistic label-free virtual histopathological images of tissues. We previously introduced ultraviolet photoacoustic remote sensing microscopy as a method to obtain virtual hematoxylin contrast albeit without the ability to obtain virtual eosin contrast. By utilizing UV scattering as a high-resolution eosin channel we are able to produce complete H&E-like virtual histology of unstained human breast lumpectomy specimen sections. By further leveraging a novel colormap matching algorithm with this UV scattering, we generate H&E-like output that is shown to have strong concordance with true H&E-stained adjacent sections, showing promising diagnostic utility.
Photoacoustic remote sensing (PARS) microscopy suffers from slow imaging speeds as a result of so far being an exclusively laser scanning microscopy-based technique. Here we introduce a camera-based PARS approach using a 10 million frames-per-second camera together with oblique 532nm excitation and white-light interrogation. 2mm x 1.2mm images of 20µm diameter gold bonding wires are obtained in fractions of a second albeit with lower resolution. Using these wide-field images, regions-of-interest can be established. Additionally, the observation of supersonic wavefronts suggest the generation of shockwaves. This observation is used to derive an empirical model for the time evolution of PARS signals.
Previous photoacoustic remote sensing virtual histology approaches were too slow to use intraoperatively. We present a new scanning methodology with simultaneous galvanometer mirror and constant velocity mechanical scanning to greatly increase image acquisition speed. Human breast and prostate samples are imaged over an area of 4mm x 4mm in 40s with a 0.5μm resolution resolving both cancerous and healthy tissue. Histological detail is clearly visible in our images where tissue organization and subcellular nuclei density can be observed to aid histologists in determining margin status and cancer grading.
We develop a dual-modality imaging system for virtual histology in breast tumor specimens, augmenting depth-resolved scattering contrast from OCT with sub-cellular resolution and label-free molecular specificity from UV photoacoustic remote sensing.
We demonstrate the ability to perform fast ultraviolet photoacoustic remote sensing microscopy of tissue margins using a mosaic scanning approach where a camera-based system is used to determine scan coordinates within a 2-mm margin.
We demonstrate the use of Photoacoustic Remote Sensing (PARS) and scattering microscopy capable of acquiring virtual depth-resolved images of tissues with virtual contrast of hematoxylin using PARS & eosin using scattering microscopy.
Significance: Complementary absorption and fluorescence contrast could prove useful for a wide range of biomedical applications. However, current absorption-based photoacoustic microscopy systems require the ultrasound transducers to physically touch the samples, thereby increasing contamination and limiting strong optical focusing in reflection mode.
Aim: We sought to develop an all-optical system for imaging cells and tissues using the three combined imaging modalities: photoacoustic remote sensing (PARS), epifluorescence, and confocal laser scanning microscopy (CLSM).
Approach: A PARS subsystem with ultraviolet excitation was used to obtain label-free absorption-contrast images of nucleic acids in ex vivo tissue samples. Co-integrated epifluorescence and CLSM subsystems were used to verify the 2D and 3D nuclei distribution.
Results: Complementary absorption and fluorescence contrast were demonstrated in phantom imaging experiments and subsequent cell and tissue imaging experiments. Lateral and axial resolution of ultraviolet-PARS (UV-PARS) is shown to be 0.39 and 1.6 μm, respectively, with 266-nm light. CLSM lateral and axial resolution was measured as 0.97 and 2.0 μm, respectively. This resolution is sufficient to image individual cell layers with fine optical sectioning. UV-PARS images of cell nuclei are validated in thick tissue using CLSM.
Conclusions: Multimodal absorption and fluorescence contrast are obtained with a non-contact all-optical microscopy system for the first time and utilized to obtain images of cells and tissues with subcellular resolution.
Photoacoustic remote sensing (PARS) microscopy with multi-wavelength excitation and spectral unmixing was recently reported for non-contact imaging of hemoglobin oxygen saturation. In this work, we aim to extend this technique to provide additional functional information on oxygen metabolic activity. Stimulated Raman scattering provides multi-wavelength PARS excitation for SO2 estimation, and is integrated with Doppler OCT for quantitative blood flow rate determination. Structural imaging is demonstrated in phantoms and in vivo angiography. To evaluate system performance, tube phantoms containing blood of varied oxygenation and flow rates were imaged. This represents progress towards deploying the technique for in vivo oxygen consumption monitoring.
Hematoxylin and Eosin (H and E) staining is the gold standard for the majority of histopathological diagnostics but requires lengthy processing times not suitable for point-of-care diagnosis. Here we demonstrate a 266-nm excitation Ultraviolet Photoacoustic Remote Sensing (UV-PARS) and Scattering Microscopy system capable of virtual H and E 3D imaging of tissues in conjunction with with confocal fluorescence microscopy (CFM) for validation in thick tissues. We demonstrate the capabilities of this dual-contrast system for en-face planar and volumetric imaging of human tissue samples exhibiting high concordance with the gold standard of H and E staining procedures as well as confocal fluorescence microscopy. To our knowledge, this is the first near real-time microscopy approach capable of volumetric imaging unstained thick tissues with virtual H and E contrast.
Rapid, accurate intraoperative assessment of excised specimens and in vivo resection sites is a critical need in tissue-conserving cancer surgeries. We develop a multimodal imaging system integrating ultraviolet photoacoustic remote sensing (UV-PARS) microscopy with spectral-domain optical coherence tomography (SD-OCT) to provide comprehensive, co-registered information for identifying atypical tissue features. 1050-nm SD-OCT generates a widefield, depth-resolved view of subsurface tissue morphology, with 9-μm axial resolution. UV-PARS produces virtual histological C-scan images of cell nuclei, at 390-nm lateral resolution. Dual-contrast in vivo mouse imaging, and ex vivo imaging of breast and prostate specimens is demonstrated, and correlated with conventional H and E staining.
We report the use of frequency decomposition with ultraviolet photoacoustic remote sensing (UV-PARS) microscopy that enables contact-free and label-free virtual H and E staining. Along with principal component analysis of frequency information, this image reconstruction technique allows for the incorporation of scale-specific information encoded in the frequency spectrum of generated PARS signals. The performance of this image reconstruction technique as demonstrated on histological samples achieves contrast-to-noise ratios of over 55-dB between nuclei and nearby cytoplasmic structures. This technique allows for the rapid and facile production of virtual H and E stained images without the need for additional excitation wavelengths.
KEYWORDS: Microscopy, In vivo imaging, Signal to noise ratio, Photoacoustic spectroscopy, Absorption, Ultraviolet radiation, Tissue optics, Image resolution, Remote sensing
Photoacoustic remote sensing (PARS) is a non-contact imaging modality that is based on the optical absorption contrast of endogenous molecules. PARS has shown promise in vascular imaging, blood oxygenation estimation, and virtual biopsy without the need for exogenous labels. Here we demonstrate simultaneous imaging of cell nuclei and blood using UV and visible excitation wavelengths. This is important for decoupling blood signals from cell nuclei signals in removed tissue and resection beds. A 532nm fiber laser is split with one light path frequency doubled using a CLBO crystal to 266nm. These two wavelength lasers are co-aligned and co-focused with a 1310nm interrogation beam and using a reflective objective to image microvasculature and cell nuclei with intrinsic optical absorptions at 532nm and 266nm, respectively. These images are taken serially and co-registered with lateral resolutions of 1.2μm and 0.44μm respectively. Co-alignment using multiple wavelengths is demonstrated using carbon fiber phantoms. We imaged both paraffin embedded tissue and in vivo mouse ear. Cell nuclei in sectioned tissues were clearly visualized with a SNR of 42dB while hemoglobin demonstrated an SNR of 39dB. In vivo cell nuclei and vasculature images produced an SNR up to 40dB and 35dB, respectively.
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