RESUMO
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.
Assuntos
Tecnologia de Sensoriamento Remoto , Humanos , Animais , Camundongos , Reprodutibilidade dos Testes , Amarelo de Eosina-(YS) , Hematoxilina , Coloração e RotulagemRESUMO
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.
Assuntos
Neoplasias da Mama , Microscopia , Humanos , Microscopia/métodos , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Tecnologia de Sensoriamento Remoto/métodos , Algoritmos , Feminino , Processamento de Imagem Assistida por Computador/métodos , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/diagnóstico por imagem , Pele/diagnóstico por imagem , Pele/química , Pele/citologia , Fótons , Desenho de Equipamento , Interpretação de Imagem Assistida por Computador/métodosRESUMO
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.
Assuntos
Inteligência Artificial , Neoplasias da Mama , Humanos , Feminino , Reprodutibilidade dos Testes , Tecnologia de Sensoriamento Remoto , Neoplasias da Mama/patologia , BiópsiaRESUMO
Photoacoustic remote sensing (PARS) microscopy is an emerging label-free optical absorption imaging modality. PARS operates by capturing nanosecond-scale optical fluctuations produced by photoacoustic pressures. These time-domain (TD) variations are usually projected by amplitude to determine optical absorption magnitude. However, valuable details on a target's material properties (e.g., density, speed of sound) are contained within the TD signals. This work uses a novel, to the best of our knowledge, clustering method to learn TD features, based on signal shape, which relate to underlying material traits. A modified K-means method is used to cluster TD data, capturing representative signal features. These features are then used to form virtual colorizations which may highlight tissues based on their underlying material properties. Applied in fresh resected murine brain tissue, colorized visualizations highlight distinct regions of tissue. This may potentially facilitate differentiation of tissue constituents (e.g., myelinated and unmyelinated axons, cell nuclei) in a single acquisition.
Assuntos
Microscopia , Técnicas Fotoacústicas , Animais , Camundongos , Microscopia/métodos , Técnicas Fotoacústicas/métodos , Tecnologia de Sensoriamento Remoto , Análise EspectralRESUMO
Histopathological visualizations are a pillar of modern medicine and biological research. Surgical oncology relies exclusively on post-operative histology to determine definitive surgical success and guide adjuvant treatments. The current histology workflow is based on bright-field microscopic assessment of histochemical stained tissues and has some major limitations. For example, the preparation of stained specimens for brightfield assessment requires lengthy sample processing, delaying interventions for days or even weeks. Therefore, there is a pressing need for improved histopathology methods. In this paper, we present a deep-learning-based approach for virtual label-free histochemical staining of total-absorption photoacoustic remote sensing (TA-PARS) images of unstained tissue. TA-PARS provides an array of directly measured label-free contrasts such as scattering and total absorption (radiative and non-radiative), ideal for developing H&E colorizations without the need to infer arbitrary tissue structures. We use a Pix2Pix generative adversarial network to develop visualizations analogous to H&E staining from label-free TA-PARS images. Thin sections of human skin tissue were first virtually stained with the TA-PARS, then were chemically stained with H&E producing a one-to-one comparison between the virtual and chemical staining. The one-to-one matched virtually- and chemically- stained images exhibit high concordance validating the digital colorization of the TA-PARS images against the gold standard H&E. TA-PARS images were reviewed by four dermatologic pathologists who confirmed they are of diagnostic quality, and that resolution, contrast, and color permitted interpretation as if they were H&E. The presented approach paves the way for the development of TA-PARS slide-free histological imaging, which promises to dramatically reduce the time from specimen resection to histological imaging.
Assuntos
Microscopia , Tecnologia de Sensoriamento Remoto , Humanos , Microscopia/métodos , Microtomia , Coloração e Rotulagem , Fluxo de TrabalhoRESUMO
In the past decades, absorption modalities have emerged as powerful tools for label-free functional and structural imaging of cells and tissues. Many biomolecules present unique absorption spectra providing chromophore-specific information on properties such as chemical bonding, and sample composition. As chromophores absorb photons the absorbed energy is emitted as photons (radiative relaxation) or converted to heat and under specific conditions pressure (non-radiative relaxation). Modalities like fluorescence microscopy may capture radiative relaxation to provide contrast, while modalities like photoacoustic microscopy may leverage non-radiative heat and pressures. Here we show an all-optical non-contact total-absorption photoacoustic remote sensing (TA-PARS) microscope, which can capture both radiative and non-radiative absorption effects in a single acquisition. The TA-PARS yields an absorption metric proposed as the quantum efficiency ratio (QER), which visualizes a biomolecule's proportional radiative and non-radiative absorption response. The TA-PARS provides label-free visualization of a range of biomolecules enabling convincing analogues to traditional histochemical staining of tissues, effectively providing label-free Hematoxylin and Eosin (H&E)-like visualizations. These findings establish an effective all-optical non-contact total-absorption microscope for label-free inspection of biological materials.
Assuntos
Técnicas Fotoacústicas , Amarelo de Eosina-(YS) , Hematoxilina , Microscopia de Fluorescência , Técnicas Fotoacústicas/métodos , Tecnologia de Sensoriamento Remoto/métodosRESUMO
Histological images are critical in the diagnosis and treatment of cancers. Unfortunately, current methods for capturing these microscopy images require resource intensive tissue preparation that may delay diagnosis for days or weeks. To streamline this process, clinicians are limited to assessing small macroscopically representative subsets of tissues. Here, a combined photoacoustic remote sensing (PARS) microscope and swept source optical coherence tomography system designed to circumvent these diagnostic limitations is presented. The proposed multimodal microscope provides label-free three-dimensional depth resolved virtual histology visualizations, capturing nuclear and extranuclear tissue morphology directly on thick unprocessed specimens. The capabilities of the proposed method are demonstrated directly in unprocessed formalin fixed resected tissues. The first images of nuclear contrast in resected human tissues, and the first three-dimensional visualization of subsurface nuclear morphology in resected Rattus tissues, captured with a non-contact photoacoustic system are presented here. Moreover, the proposed system captures the first co-registered OCT and PARS images enabling direct histological assessment of unprocessed tissues. This work represents a vital step towards the development of a rapid histological imaging modality to circumvent the limitations of current histopathology techniques.
Assuntos
Imageamento Tridimensional/métodos , Neoplasias/patologia , Tecnologia de Sensoriamento Remoto/métodos , Tomografia de Coerência Óptica , Animais , Técnicas Histológicas/tendências , Humanos , Microscopia , Neoplasias/diagnóstico , Técnicas Fotoacústicas/métodos , Ratos , Realidade VirtualRESUMO
Gastrointestinal (GI) tissue biopsies provide critical diagnostic information for a wide variety of conditions such as neoplastic diseases (colorectal, small bowel and stomach cancers) and non-neoplastic diseases (inflammatory disorders, infection, celiac disease). Endoscopic biopsies collect small tissue samples that require resource intensive processing to permit histopathological analysis. Unfortunately, the sparsely collected biopsy samples may fail to capture the pathologic condition because selection of biopsy sites relies on macroscopic superficial tissue features and clinician judgement. Here, we present the first all-optical non-contact label-free non-interferometric photoacoustic microscopy system capable of performing "virtual biopsies". A modular photoacoustic remote sensing (PARS™) architecture is used facilitating imaging of unprocessed tissues providing information similar to conventional histopathological staining techniques. Prospectively this would allow gastroenterologists to assess subcellular tissue morphology in situ when selecting biopsy location. Tested on preserved unstained human and freshly resected murine tissues, the presented PARS microscope rapidly retrieves images of similar area to current biopsies, while maintaining comparable quality to the current standard for histopathological analysis. Additionally, results show the first label free assessment of subsurface cellular morphology in FFPE GI tissue blocks. Clinically relevant features are recovered including cellular details such as lamina propria within colon tissue and cell nuclear structure in resected smooth muscle. Constructed with a modular architecture, this system facilitates the future development of compact imaging heads. The modular PARS system overcomes many of the challenges with imaging unstained thick tissue in situ, representing a significant milestone in the development of a clinical microscope providing virtual biopsy capabilities.
RESUMO
Mohs micrographic surgery (MMS) is a precise oncological technique where layers of tissue are resected and examined with intraoperative histopathology to minimize the removal of normal tissue while completely excising the cancer. To achieve intraoperative pathology, the tissue is frozen, sectioned and stained over a 20- to 60-minute period, then analyzed by the MMS surgeon. Surgery is continued one layer at a time until no cancerous cells remain, meaning MMS can take several hours to complete. Ideally, it would be desirable to circumvent or augment frozen sectioning methods and directly visualize subcellular morphology on the unprocessed excised tissues. Employing photoacoustic remote sensing (PARS) microscopy, we present a non-contact label-free reflection-mode method of performing such visualizations in frozen sections of human skin. PARS leverages endogenous optical absorption contrast within cell nuclei to provide visualizations reminiscent of histochemical staining techniques. Presented here, is the first true one to one comparison between PARS microscopy and standard histopathological imaging in human tissues. We demonstrate the ability of PARS microscopy to provide large grossing scans (>1 cm2, sufficient to visualize entire MMS sections) and regional scans with subcellular lateral resolution (300 nm).
RESUMO
Malignant brain tumors are among the deadliest neoplasms with the lowest survival rates of any cancer type. In considering surgical tumor resection, suboptimal extent of resection is linked to poor clinical outcomes and lower overall survival rates. Currently available tools for intraoperative histopathological assessment require an average of 20 min processing and are of limited diagnostic quality for guiding surgeries. Consequently, there is an unaddressed need for a rapid imaging technique to guide maximal resection of brain tumors. Working towards this goal, presented here is an all optical non-contact label-free reflection mode photoacoustic remote sensing (PARS) microscope. By using a tunable excitation laser, PARS takes advantage of the endogenous optical absorption peaks of DNA and cytoplasm to achieve virtual contrast analogous to standard hematoxylin and eosin (H&E) staining. In conjunction, a fast 266 nm excitation is used to generate large grossing scans and rapidly assess small fields in real-time with hematoxylin-like contrast. Images obtained using this technique show comparable quality and contrast to the current standard for histopathological assessment of brain tissues. Using the proposed method, rapid, high-throughput, histological-like imaging was achieved in unstained brain tissues, indicating PARS' utility for intraoperative guidance to improve extent of surgical resection.