Automated Whole Slide Imaging for Label-Free Histology Using Photon Absorption Remote Sensing Microscopy.

OBJECTIVE: Pathologists rely on histochemical stains to impart contrast in thin translucent tissue samples, revealing tissue features necessary for identifying pathological conditions. However, the chemical labeling process is destructive and often irreversible or challenging to undo, imposing practical limits on the number of stains that can be applied to the same tissue section. Here we present an automated label-free whole slide scanner using a PARS microscope designed for imaging thin, transmissible samples. METHODS: Peak SNR and in-focus acquisitions are achieved across entire tissue sections using the scattering signal from the PARS detection beam to measure the optimal focal plane. Whole slide images (WSI) are seamlessly stitched together using a custom contrast leveling algorithm. Identical tissue sections are subsequently H&E stained and brightfield imaged. The one-to-one WSIs from both modalities are visually and quantitatively compared. RESULTS: PARS WSIs are presented at standard 40x magnification in malignant human breast and skin samples. We show correspondence of subcellular diagnostic details in both PARS and H&E WSIs and demonstrate virtual H&E staining of an entire PARS WSI. The one-to-one WSI from both modalities show quantitative similarity in nuclear features and structural information. CONCLUSION: PARS WSIs are compatible with existing digital pathology tools, and samples remain suitable for histochemical, immunohistochemical, and other staining techniques. SIGNIFICANCE: This work is a critical advance for integrating label-free optical methods into standard histopathology workflows.

Label-Free Non-Contact Vascular Imaging using Photon Absorption Remote Sensing.

OBJECTIVE: Functional vascular imaging is a critical method for early detection and prevention of disease. Established non-contact vascular imaging techniques capture predominantly structural information. In this study, a novel non-contact label-free in vivo Photon Absorption Remote Sensing (PARS) microscope is developed for structural and functional vascular imaging. METHODS: The presented in vivo PARS microscope captures the endogenous absorption of green (532nm) light to form a complete picture of vasculature and surrounding tissues. Imaging system repeatability is enhanced through robust transient absorption signal extraction, and state-of-the-art real-time alignment methods. RESULTS: Detailed imaging of vascular structure is demonstrated through in vivo microscopy of two established animal models: mouse ear and chicken embryo. Preliminary functional contrast is realized through video rate imaging of red blood cell dynamics in the capillary networks of chicken embryos. CONCLUSION: The presented in vivo PARS microscope successfully captures detailed structural and functional vascular contrast. SIGNIFICANCE: This innovative non-contact label-free imaging technique holds promise as a tool for preventative medical care, as functional change often precedes structural change.

Multi-channel feature extraction for virtual histological staining of photon absorption remote sensing images.

Accurate and fast histological staining is crucial in histopathology, impacting diagnostic precision and reliability. Traditional staining methods are time-consuming and subjective, causing delays in diagnosis. Digital pathology plays a vital role in advancing and optimizing histology processes to improve efficiency and reduce turnaround times. This study introduces a novel deep learning-based framework for virtual histological staining using photon absorption remote sensing (PARS) images. By extracting features from PARS time-resolved signals using a variant of the K-means method, valuable multi-modal information is captured. The proposed multi-channel cycleGAN model expands on the traditional cycleGAN framework, allowing the inclusion of additional features. Experimental results reveal that specific combinations of features outperform the conventional channels by improving the labeling of tissue structures prior to model training. Applied to human skin and mouse brain tissue, the results underscore the significance of choosing the optimal combination of features, as it reveals a substantial visual and quantitative concurrence between the virtually stained and the gold standard chemically stained hematoxylin and eosin images, surpassing the performance of other feature combinations. Accurate virtual staining is valuable for reliable diagnostic information, aiding pathologists in disease classification, grading, and treatment planning. This study aims to advance label-free histological imaging and opens doors for intraoperative microscopy applications.

Photon Absorption Remote Sensing Imaging of Breast Needle Core Biopsies Is Diagnostically Equivalent to Gold Standard H&E Histologic Assessment.

Photon absorption remote sensing (PARS) is a new laser-based microscope technique that permits cellular-level resolution of unstained fresh, frozen, and fixed tissues. Our objective was to determine whether PARS could provide an image quality sufficient for the diagnostic assessment of breast cancer needle core biopsies (NCB). We PARS imaged and virtually H&E stained seven independent unstained formalin-fixed paraffin-embedded breast NCB sections. These identical tissue sections were subsequently stained with standard H&E and digitally scanned. Both the 40× PARS and H&E whole-slide images were assessed by seven breast cancer pathologists, masked to the origin of the images. A concordance analysis was performed to quantify the diagnostic performances of standard H&E and PARS virtual H&E. The PARS images were deemed to be of diagnostic quality, and pathologists were unable to distinguish the image origin, above that expected by chance. The diagnostic concordance on cancer vs. benign was high between PARS and conventional H&E (98% agreement) and there was complete agreement for within-PARS images. Similarly, agreement was substantial (kappa > 0.6) for specific cancer subtypes. PARS virtual H&E inter-rater reliability was broadly consistent with the published literature on diagnostic performance of conventional histology NCBs across all tested histologic features. PARS was able to image unstained tissues slides that were diagnostically equivalent to conventional H&E. Due to its ability to non-destructively image fixed and fresh tissues, and the suitability of the PARS output for artificial intelligence assistance in diagnosis, this technology has the potential to improve the speed and accuracy of breast cancer diagnosis.