Combined transmission, dark field and fluorescence microscopy for intact, 3D tissue analysis of biopsies.

Journal of Biomedical Optics
Marius I BoamfaPieter J van der Zaag

Abstract

Currently, tissue biopsies are sectioned into 3- to 5-μm-thick slices that are used for conventional pathology analysis. Previous work by confocal microscopy and light-sheet microscopy has shown that analyzing biopsies intact in three-dimensions (3D) is possible and may lead to a better understanding of cancer growth patterns. Although accurate, these methods require fluorescent staining of the tissue, in addition to tissue clearing. If the 3D biopsy analysis could be done sufficiently swiftly, this approach may be used for on-site assessment of the adequacy of a biopsy taken. We aim to show that, by transmission microscopy of optically cleared tissue punches, the tissue architecture can be determined without the need for fluorescent staining. Transmission microscopy is used by combining bright field microscopy with dark field and epifluorescent microscopy to compare samples that have also been analyzed by fluorescent confocal microscopy. With increasing distance to the focal plane, the higher-frequency part of the spatial frequency spectrum of transmitted light is attenuated increasingly. This property is exploited for tissue segmentation, detecting whether tissue is present at a certain position in the focal plane image. Usin...Continue Reading

Methods Mentioned

BETA
fluorescence microscopy
confocal microscopy
biopsies
biopsy
confocal
Fluorescence
transmission microscopy
light-sheet
transmission

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