Histopathological examination typically involves tissue resection or biopsy, fixation, sectioning, and staining protocols. A non-contact high-resolution photoacoustic remote sensing microscopy system is presented which is capable of depth-resolved imaging of cell nuclei in fixed and fresh tissues without the need for stains or labels. The reflection-mode system is based on a 0.5 numerical aperture reflective objective and enables fast optical scanning using a 600 kHz repetition rate fiber laser to produce histological-like images with 0.39 µm resolution and with close agreement to traditional H&E and fluorescence staining.
Histopathology of lipid-rich tissues is often a difficult endeavor, owing to the limited tissue processing workflows that can appropriately preserve tissue while keeping fatty deposits intact. Here, we present the first usage of near-infrared (NIR) photoacoustic remote sensing (PARS) to achieve imaging contrast from lipids without the need for exogenous stains or labels. In our system, the facile production of 1225 nm excitation pulses is achieved by the stimulated Raman scattering of a 1064 nm source propagating through an optical fiber. PARS-based detection is achieved by monitoring the change in the scattering profile of a co-aligned 1550 nm continuous-wave interrogation beam in response to absorption of the 1225 nm light by lipids. Our non-contact, reflection-mode approach can achieve a FWHM resolution of up to 0.96 µm and signal-to-noise ratios as high as 45 dB from carbon fibers and 9.7 dB from a lipid phantom. NIR-PARS offers a promising approach to image lipid-rich samples with a simplified workflow.
Histological evaluation of tissues is currently a lengthy process that typically precludes intraoperative margin assessment. While numerous approaches have aimed to address the need for intraoperative virtual histology, none have yet proved sufficiently efficacious. We demonstrate the use of a new all-optical imaging modality, photoacoustic remote sensing (PARS), capable of virtual histopathological imaging, while simultaneously providing visualization of microvasculature in both freshly resected tissues and live animal subjects. We demonstrate high resolutions of 0.44µm and 1.2µm for 266-nm and 532-nm excitation wavelengths, respectively, as well as the characterization of maximum permissible exposure limits for both excitation wavelengths.
Hematoxylin and eosin (H&E) staining is the gold standard for most 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 1310-nm microscopy system capable of virtual H&E 3D imaging of tissues. Virtual hematoxylin staining of nuclei is achieved with UV-PARS, while virtual eosin staining is achieved using the already implemented interrogation laser from UV-PARS for scattering contrast. We demonstrate the capabilities of this dual-contrast system for en-face planar and depth-resolved imaging of human tissue samples exhibiting high concordance with H&E staining procedures and confocal fluorescence microscopy. To our knowledge, this is the first microscopy approach capable of depth-resolved imaging of unstained thick tissues with virtual H&E contrast.
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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
, respectively, with 266-nm light. CLSM lateral and axial resolution was measured as 0.97 and
, 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) is a novel all-optical imaging modality that allows for non-contact detection of initial photoacoustic pressures. Using 266-nm excitation pulses, ultraviolet PARS (UV-PARS) has previously demonstrated imaging contrast for cell nuclei in histological samples with
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