Reflective objective-based ultraviolet photoacoustic remote sensing virtual histopathology

Haven, Nathaniel J. M.; Kedarisetti, Pradyumna; Restall, Brendon S.; Zemp, Roger J.

doi:10.1364/ol.382415

Cited by 37 publications

(17 citation statements)

References 13 publications

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“…These capabilities, as highlighted earlier, lead to a device suitable for intraoperative and clinical environments. Previously, PARS has demonstrated the ability to visualize nuclear contrast in chicken embryos and animal tissue 32,33 . However, these reports were limited in their field of view and did not reveal significant structural information.…”

Section: Introductionmentioning

confidence: 99%

Non-contact reflection-mode optical absorption spectroscopy using photoacoustic remote sensing

Bell

Reza

2020

Opt. Lett.

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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 labor intensive and contribute to delays in clinical feedback that can extend to two to three weeks for standard paraffin-embedded tissue preparation and interpretation. Here, we present a label-free reflectionmode imaging modality that reveals cellular-scale morphology by detecting intrinsic endogenous contrast. We accomplish this with the novel photoacoustic remote sensing (PARS) detection system that permits non-contact optical absorption contrast to be extracted from thick and opaque biological targets with optical resolution. PARS was examined both as a rapid assessment tool that is capable of managing large samples (>1 cm 2) in under 10 minutes, 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 paraffinembedded 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.

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Section: Introductionmentioning

confidence: 99%

Non-contact reflection-mode optical absorption spectroscopy using photoacoustic remote sensing

Bell

Reza

2020

Opt. Lett.

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“… 15 – 21 These techniques have subsequently provided hematoxylin-like visualizations of nuclear structures in tissue blocks, slides, and fresh tissues. 15 – 21 However, these techniques do not provide full H&E emulation, as they lack the extranuclear tissue structures captured by eosin staining. Here, we propose approaching this problem by leveraging, for the first time, PARS unique dual scattering and absorption contrast.…”

Section: Resultsmentioning

confidence: 99%

“… 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.…”

Section: Introductionmentioning

confidence: 99%

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

2021

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Significance: Histopathological analysis of tissues is an essential tool for grading, staging, diagnosing, and resecting cancers and other malignancies. Current histopathological imaging techniques require substantial sample processing, prior to staining with hematoxylin and eosin (H&E) dyes, to highlight nuclear and cellular morphology. Sample preparation and staining is resource intensive and introduces potential for variability during sample preparation.Aim: We present a method for direct label-free histopathological assessment of formalin-fixed paraffin-embedded tissue blocks and thin tissue sections using a dual-contrast photoacoustic remote sensing (PARS) microscopy system.Approach: To emulate the nuclear and cellular contrast of H&E staining, we leverage unique properties of the PARS system. Here, the ultraviolet excitation PARS microscope takes advantage of DNA's unique optical absorption to provide nuclear contrast analogous to hematoxylin staining of cell nuclei. Concurrently, the optical scattering contrast of the PARS detection system is leveraged to provide bulk tissue contrast reminiscent of eosin staining of cell membranes. Results:We demonstrate the efficacy of this technique by imaging human breast tissue and human skin tissues in formalin-fixed paraffin-embedded tissue blocks and frozen sections, respectively. Salient nuclear and extranuclear features such as cancerous cells, glands and ducts, adipocytes, and stromal structures such as collagen are captured. Conclusions:The presented dual-contrast PARS microscope enables label-free visualization of tissues with contrast and quality comparable to the current gold standard for histopathological analysis. Thus, the proposed system is well positioned to augment existing histopathological workflows, providing histological imaging directly on unstained tissue blocks and sections.

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“…(2019). 28 To briefly summarize, a 1310-nm continuous-wave interrogation beam (Thorlabs, SLD1018PXL) is co-aligned with a 266-nm pulsed excitation beam, generated by frequency doubling the output of a 40-kHz 532-nm nanosecond pulsed fiber laser (IPG Photonics, GLP-10) using a caesium lithium borate (CLBO) (Eksma Optics) crystal. These two beams are then directed through a galvanometer scanning mirror system then a 0.5 NA dry reflective objective (Thorlabs, LMM-40X-UVV).…”

Section: Methodsmentioning

confidence: 99%

“…To date, one missing comparison is that UV-PARS histology has previously demonstrated absorption-contrast imaging of cell nuclei but has yet to be validated in thick tissue sections. 20 , 27 , 28 The validation of the 3D virtual histology capabilities of UV-PARS is an important step toward label-free tissue imaging both ex vivo and in vivo. The combination of confocal laser scanning microscopy (CLSM) and the absorption contrast gained with UV-PARS could help augment intraoperative surgery by spot checking margin status in areas of interest in fluorescence guided surgeries as well as replace the hematoxylin and eosin (H&E) staining histological analysis post-surgery with a much faster and less labor-intensive imaging technique that could be employed intraoperatively.…”

Section: Introductionmentioning

confidence: 99%

Multimodal 3D photoacoustic remote sensing and confocal fluorescence microscopy imaging

et al. 2021

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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.

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Reflective objective-based ultraviolet photoacoustic remote sensing virtual histopathology

Cited by 37 publications

References 13 publications

Non-contact reflection-mode optical absorption spectroscopy using photoacoustic remote sensing

Non-contact reflection-mode optical absorption spectroscopy using photoacoustic remote sensing

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

Multimodal 3D photoacoustic remote sensing and confocal fluorescence microscopy imaging

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