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Terapixel hyperspectral whole-slide imaging via slit-array detection and projection.

ABSTRACT: Digital pathology via whole-slide imaging (WSI) systems has recently been approved for the primary diagnostic use in the US. Acquiring whole-slide images with spectral information at each pixel permits the use of multiplexed antibody labeling and allow for the measurement of cellularly resolved chemical information. Here, we report the development of a high-throughput terapixel hyperspectral WSI system using prism-based slit-array dispersion. We demonstrate a slit-array detection scheme for absorption-based measurements and a slit-array projection scheme for fluorescence-based measurements. The spectral resolution and spectral range in the reported schemes can be adjusted by changing the orientation of the slit-array mask. We use our system to acquire 74 5-megapixel brightfield images at different wavelengths in ∼1 s, corresponding to a throughput of 0.375 gigapixels / s. A terapixel whole-slide spatial-spectral data cube can be obtained in ∼45 min. The reported system is compatible with existing WSI systems and can be developed as an add-on module for whole-slide spectral imaging. It may find broad applications in high-throughput chemical imaging with multiple antibody labeling. The use of slit array for structured illumination may also provide insights for developing high-throughput hyperspectral confocal imaging systems.

SUBMITTER: Liao J

PROVIDER: S-EPMC8357323 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundIn osteosarcoma survival rates could not be improved over the last 30 years. Novel biomarkers are warranted to allow risk stratification of patients for more individual treatment following initial diagnosis. Although previous studies of the tumor microenvironment have identified promising candidates, novel biomarkers have not been translated into routine histopathology. Substantial difficulties regarding immunohistochemical detection and quantification of antigens in decalcified and heterogeneous osteosarcoma might largely explain this translational short-coming. Furthermore, we hypothesized that conventional hot spot analysis is often not representative for the whole section when applied to heterogeneous tissues like osteosarcoma. We aimed to overcome these difficulties for major biomarkers of the immunovascular microenvironment.MethodsImmunohistochemistry was systematically optimized for cell surface (CD31, CD8) and intracellular antigens (FOXP3) including evaluation of 200 different antigen retrieval conditions. Distribution patterns of these antigens were analyzed in formalin-fixed and paraffin-embedded samples from 120 high-grade central osteosarcoma biopsies and computer-assisted whole-slide analysis was compared with conventional quantification methods including hot spot analysis.ResultsMore than 96% of osteosarcoma samples were positive for all antigens after optimization of immunohistochemistry. In contrast, standard immunohistochemistry retrieved false negative results in 35-65% of decalcified osteosarcoma specimens. Standard hot spot analysis was applicable for homogeneous distributed FOXP3+ and CD8+ cells. However, heterogeneous distribution of vascular CD31 did not allow reliable quantification with hot spot analysis in 85% of all samples. Computer-assisted whole-slide analysis of total CD31- immunoreactive area proved as the most appropriate quantification method.ConclusionStandard staining and quantification procedures are not applicable in decalcified formalin-fixed and paraffin-embedded samples for major parameters of the immunovascular microenvironment in osteosarcoma. Whole-slide imaging and optimized antigen retrieval overcome these limitations.

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Terapixel hyperspectral whole-slide imaging via slit-array detection and projection.

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