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.
Highlights
Histological visualizations are critical to clinical disease management and are fundamental to biological understanding
Imaging was conducted on two photoacoustic remote sensing (PARS) imaging platforms (Fig. 2): (I) A single-color system which employs a single 266 nm excitation laser for targeting DNA contrast which operates at a repetition rate of 50 kHz
This work presented the efficacy of PARS microscopy in visualizing a wide variety of tissue preparations including human formalin-fixed paraffin embedded (FFPE) slides, FFPE tissue blocks, frozen pathology sections and unprocessed murine kidney
Summary
Histological visualizations are critical to clinical disease management and are fundamental to biological understanding. A microscope which can disrupt the standard histopathology workflow and provide H&E-like contrast directly within the resection site could radically change the clinical pathology paradigm Such a device would need to meet several key requirements: (I) The device must be capable of emulating common existing. Pathologists are accustomed to assessing stained tissues and such a device must be capable of producing comparable visualizations of cellular structures with appropriate resolution and chromophore-specific contrast Such visualizations may be leveraged by existing AI recognition systems which have been previously trained on conventional histology preparations for use in cancer detection and surgical guidance. This would enable parallel integration into existing workflows at hospitals and encourage adoption These capabilities, when combined, would result in a microscope that is suitable for intraoperative environments and would facilitate diagnostic quality H&E-like contrasts in fresh tissue specimens or directly within resection sites
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