Histology
Histology & The Ideal Microscope
Cancer represents a diverse group of diseases with highly variable clinical behaviors and disparate responses to therapy. Microscopic assessment of biopsies is the mainstay to determining the cancer classification, disease progression, tumor grade and the intrinsic biology of the disease, all of which are necessary to formulate a complete treatment plan. Evaluating surgical margins to ensure to no cancerous cells have been left behind is also highly reliant on microscopic assessment of the resected tissue. To this end, histopathology plays a central role in the management and treatment of cancerous diseases.
Presently, tissue specimens are examined under standard bright-field microscopes which illuminate the sample from underneath and enable assessment from above. The specimens must therefore be thin sections such that light can transmit through. Additionally, as most biological tissue is colorless, the thin sections must be stained with chromogenic dyes such as hematoxylin and eosin (H&E) prior to examination. These stains reveal the microscopic tissue structure and enable the histological assessment of tissue. However, preparing histology slides is a time consuming and resource intensive process that can sometimes take several days before a pathological assessment can be presented, potentially delaying the formulation of a treatment plan and leading to poorer patient outcomes. In intraoperative settings, frozen sectioning is frequently employed to examine tissue pathology. Although this method has demonstrated improved patient outcomes, the technical quality of the slides produced is lower than post-operative histopathological analysis, leading to a variability in diagnosis and often requiring patients to undergo re-excisions or additional therapy after the surgery. Ideally, an intraoperative tool could circumvent the existing histopathological workflow and visualize tissue structure freshly resected specimens or even directly on the patient’s body.
Such an ideal microscope would require several features to be feasible in hospitals:
Our technology, photon absorption remote sensing (PARS®), is a non-contact method that can visualize the optical absorption contrast present within tissue without the use of any staining or dyes. Additionally, PARS® operates based on the reflection of light and is hence suitable for imaging thick tissue. These capabilities enable PARS® to be a powerful tool that can visualize tissue structure on directly on the patient’s body or on freshly resected tissue, potentially saving weeks long tissue processing. This leads to several benefits for the patient and the hospital. For example, by imaging tissue pathology directly on biopsies, patient management can be fast tracked, and treatment plans can be formulated much more quickly, resulting in improved patient outcomes. Similarly, by imaging excised tissue intraoperatively, cancer margins can be visualized while the patient is still under anesthesia and further tissue can be excised if necessary, leading to superior oncological outcomes. PARS® has been rapidly developed as a histological tool and has demonstrated visualization of tissue structure in unstained tissues, frozen sections, tissue blocks and even freshly excised tissue. In aggregate, these results position PARS® to challenge existing histopathological workflows, augment existing techniques or potentially replace them entirely.
Cancer represents a diverse group of diseases with highly variable clinical behaviors and disparate responses to therapy. Microscopic assessment of biopsies is the mainstay to determining the cancer classification, disease progression, tumor grade and the intrinsic biology of the disease, all of which are necessary to formulate a complete treatment plan. Evaluating surgical margins to ensure to no cancerous cells have been left behind is also highly reliant on microscopic assessment of the resected tissue. To this end, histopathology plays a central role in the management and treatment of cancerous diseases.
Presently, tissue specimens are examined under standard bright-field microscopes which illuminate the sample from underneath and enable assessment from above. The specimens must therefore be thin sections such that light can transmit through. Additionally, as most biological tissue is colorless, the thin sections must be stained with chromogenic dyes such as hematoxylin and eosin (H&E) prior to examination. These stains reveal the microscopic tissue structure and enable the histological assessment of tissue. However, preparing histology slides is a time consuming and resource intensive process that can sometimes take several days before a pathological assessment can be presented, potentially delaying the formulation of a treatment plan and leading to poorer patient outcomes. In intraoperative settings, frozen sectioning is frequently employed to examine tissue pathology. Although this method has demonstrated improved patient outcomes, the technical quality of the slides produced is lower than post-operative histopathological analysis, leading to a variability in diagnosis and often requiring patients to undergo re-excisions or additional therapy after the surgery. Ideally, an intraoperative tool could circumvent the existing histopathological workflow and visualize tissue structure freshly resected specimens or even directly on the patient’s body.
Such an ideal microscope would require several features to be feasible in hospitals:
- Since pathologists are accustomed to interpreting tissues stained with H&E, any virtual histopathology method must aim to visualize the same information and produce H&E-like diagnostic quality images.
- Many modalities transmit light through the sample and detect it at the opposite end to visualize contrast. However, this technique would not be suitable for a device that aims to image thick unprocessed tissue or even visualize cancer margins on the patient’s body. Therefore, any device that aims to image thick tissue must operate based on the reflection of light from the sample.
- The modality must not require any contact with the target. Infections from surgery are a leading cause of patient morbidity and any microscope that requires contact with the target would increase the risk of infection and may mandate additional control measures at hospitals. Therefore, such a device would be impractical.
- The device must not require any staining as dyes can be potentially toxic and also impose additional control measures at hospitals.
- The device must be capable of visualizing tissue structure with high resolution sensitivity and contrast in real time.
- The device must be capable of visualizing cancer deep within tissue.
- Finally, it would be desirable if the method can image the specimens at each intermediate step during the standard histopathological workflow. This has several benefits. First it would be make it easier to augment such a device into existing workflows at hospitals. Second, there presently over 300 million specimens of human tissue preserved in paraffin molded in the shape of tissue blocks. Thus, a microscope that can visualize tissue morphology directly on FFPE blocks would not only save valuable time and resources in clinical settings, it would also make it feasible to tap into the large amount of stored tissue specimens for translational research.
Our technology, photon absorption remote sensing (PARS®), is a non-contact method that can visualize the optical absorption contrast present within tissue without the use of any staining or dyes. Additionally, PARS® operates based on the reflection of light and is hence suitable for imaging thick tissue. These capabilities enable PARS® to be a powerful tool that can visualize tissue structure on directly on the patient’s body or on freshly resected tissue, potentially saving weeks long tissue processing. This leads to several benefits for the patient and the hospital. For example, by imaging tissue pathology directly on biopsies, patient management can be fast tracked, and treatment plans can be formulated much more quickly, resulting in improved patient outcomes. Similarly, by imaging excised tissue intraoperatively, cancer margins can be visualized while the patient is still under anesthesia and further tissue can be excised if necessary, leading to superior oncological outcomes. PARS® has been rapidly developed as a histological tool and has demonstrated visualization of tissue structure in unstained tissues, frozen sections, tissue blocks and even freshly excised tissue. In aggregate, these results position PARS® to challenge existing histopathological workflows, augment existing techniques or potentially replace them entirely.
Overview of PARS® histologic imaging workflow as compared to conventional light microscopy. (a) Conventional imaging of H&E-stained slides is performed on a bright-field microscope where the Hematoxylin (purple hues) and Eosin (red hues) stains block light from a white source. PARS® may image (b) unstained FFPE slide preparations, (c) unstained FFPE blocks and (d) unprocessed tissues by taking advantage of the intrinsic optical absorption provided by the cell nuclei (DNA) and the surrounding cytoplasm (cytochrome). We image each intermediate step along the FFPE process in this paper using a single system configuration to show the versatility of PARS. No other reported technique has reported all of these capabilities in a single modality. [Sci Rep 10, 19121 (2020). https://doi.org/10.1038/s41598-020-76155-6].
Several comparisons between PARS® and conventional bright-field images of FFPE slides of human brain tissues. a) A WFOV scan using 266 nm excitation with b) a matching wide field image of the adjacent slide which has been H&E stained. c) A two-color (250 nm and 420 nm) PARS® with a false-colour map applied to match d) the adjacent H&E region. Finally e) and f) likewise show a two-color PARS® and bright-field image respectively in higher detail. [Sci Rep 10, 19121 (2020). https://doi.org/10.1038/s41598-020-76155-6
Several PARS® images of a human skin sample mounted as a frozen section slide. (a) A WFOV PARS® acquisition of the sample using the single color 266 nm system. The two-color PARS® was then used over smaller field the views in (b) and (c) focusing on the outer tissue layers. Still smaller field of views are shown in (d-g) highlighting the details available within the epidermal layers. These layers are annotated in (d). [Sci Rep 10, 19121 (2020). https://doi.org/10.1038/s41598-020-76155-6