Reflection-mode virtual histology using photoacoustic remote sensing microscopy

Abstract

Histological visualizations are critical to clinical disease management and are fundamental to biological understanding. However, current approaches that rely on bright-field microscopy require extensive tissue preparation prior to imaging. These processes are both labor intensive and contribute to creating significant delays in clinical feedback for treatment decisions that can extend to 2–3 weeks for standard paraffin-embedded tissue preparation and interpretation, especially if ancillary testing is needed. Here, we present the first comprehensive study on the broad application of a novel label-free reflection-mode imaging modality known as photoacoustic remote sensing (PARS) for visualizing salient subcellular structures from various common histopathological tissue preparations and for use in unprocessed freshly resected tissues. The PARS modality permits non-contact visualizations of intrinsic endogenous optical absorption contrast to be extracted from thick and opaque biological targets with optical resolution. The technique was examined both as a rapid assessment tool that is capable of managing large samples (> 1 cm2) in under 10 min, and as a high contrast imaging modality capable of extracting specific biological contrast to simulate conventional histological stains such as hematoxylin and eosin (H&E). The capabilities of the proposed method are demonstrated in a variety of human tissue preparations including formalin-fixed paraffin-embedded tissue blocks and unstained slides sectioned from these blocks, including normal and neoplastic human brain, and breast epithelium involved with breast cancer. Similarly, PARS images of human skin prepared by frozen section clearly demonstrated basal cell carcinoma and normal human skin tissue. Finally, we imaged unprocessed murine kidney and achieved histologically relevant subcellular morphology in fresh tissue. This represents a vital step towards an effective real-time clinical microscope that overcomes the limitations of standard histopathologic tissue preparations and enables real-time pathology assessment.

Single acquisition label-free histology-like imaging with dual-contrast photoacoustic remote sensing microscopy

“…In this implementation, we use a 50-kHz UV excitation, which provides emulated H&E images substantially faster than the 1-kHz tunable source used in previous studies. 17,18 For example, the image presented in Fig. 5 is captured in under 15 min with the proposed dual-contrast system, whereas it would require nearly 13 h to capture with the previously reported tunable system.…”

“… 14 Moreover, PARS may provide chromophore-specific contrast by selecting excitation wavelengths to target unique biomolecule absorption spectra. 15 – 18 Applied to histological imaging, PARS has successfully used UV excitation to capture nuclear structures. 15 – 21 This technique is efficacious in providing hematoxylin-like contrast in frozen sections and paraffin-embedded blocks and sections.…”

“… 15 – 18 Applied to histological imaging, PARS has successfully used UV excitation to capture nuclear structures. 15 – 21 This technique is efficacious in providing hematoxylin-like contrast in frozen sections and paraffin-embedded blocks and sections. 15 – 21 However, these UV-PARS visualizations lack some essential diagnostic details.…”

“…Previously, PARS has provided complete H&E emulation using a tunable excitation source to independently target the absorption peaks of DNA and cell membrane structures. 17 , 18 While effective in both thin sections and tissue blocks, this technique was largely limited in field of view, resolution, and imaging speed since it required the use of a slow (1 kHz) tunable excitation source. 17 , 18 In this paper, we propose leveraging the optical scattering microscope architecture of the PARS detection system to provide bulk tissue contrast, while using an UV excitation to capture nuclear structures.…”

“… 17 , 18 While effective in both thin sections and tissue blocks, this technique was largely limited in field of view, resolution, and imaging speed since it required the use of a slow (1 kHz) tunable excitation source. 17 , 18 In this paper, we propose leveraging the optical scattering microscope architecture of the PARS detection system to provide bulk tissue contrast, while using an UV excitation to capture nuclear structures. For the first time, we use the unique ability of PARS microscopy to concurrently provide both label-free absorption and scattering contrast.…”