Abstract
Current immunofluorescence protocols are limited as they do not provide reliable antibody staining within large tissue volumes (mm3) and cannot localise and quantify multiple antigens or cell populations in the same tissue at high resolution. To address this limitation, we have developed an approach to three-dimensionally visualise large tissue volumes (mm3) at high resolution (<1 µm) and with multiple antigen labelling, for volumetric and quantitative analysis. This is made possible through computer reconstruction of serial sectioned and sequentially immunostained butyl-methyl methacrylate (BMMA) embedded tissue. Using this novel immunofluorescent computed tomography (ICT) approach, we have three-dimensionally reconstructed part of the murine lower eyelid that contains the meibomian gland and localised cell nuclei (DAPI), Ki67 and cytokeratin 1 (CK1), as well as performing non-linear optical (NLO) microscopy imaging of collagen, to assess cell density, cell proliferation, gland keratinisation and gland volume respectively. Antigenicity was maintained after four iterative stains on the same tissue, suggesting that there is no defined limit to the number of antigens that can be immunostained for reconstruction, as long as the sections remain intact and the previous antibody has been successfully eluted. BMMA resin embedding also preserved fluorescence of transgenic proteins. We propose that ICT may provide valuable high resolution, three-dimensional biological maps of multiple biomolecules within a single tissue or organ to better characterise and quantify tissue structure and function.
Highlights
Recent advances in optical microscopy and digital image processing has greatly expanded our ability to visualise cellular structure and function
Antibody Staining of Single butyl-methyl methacrylate (BMMA) Tissue Sections To demonstrate iterative antibody staining, a single 2 mm section of a 2month old mouse meibomian gland was sequentially probed for the filamentous proteins cytokeratin (CK) 1, 6 and 10 as well as the cell proliferation marker Ki67
Through immunofluorescent computed tomography (ICT), we have demonstrated a method for highly specific localisation and quantification of multiple antigens and cell populations in a large tissue volume that, in theory, can cover entire organs
Summary
Recent advances in optical microscopy and digital image processing has greatly expanded our ability to visualise cellular structure and function. The reintroduction of single plane illumination microscopy (SPIM) with wide-field imaging combined with high-speed camera detection has extended our temporal resolution in detecting rapid cellular and intracellular events, as well as our ability to image large scale structures and probe events during development [8,9]. Whole mount staining and confocal microscopy provide three-dimensional information, antibody penetration is unreliable through large tissues (.0.3 mm3) and the imaging depth is limited by attenuation of light and the working distance of the objective. Light-sheet microscopy removes the problem of an objective’s working distance and provides high-resolution deep tissue imaging, on the centimetre scale [9]. Light sheets are optimally used for detection of transgenically expressed fluorophores, such as GFP, as whole mount immunostaining is subject to the same limitations found using confocal microscopy. The axial resolution of confocal microscopy is rarely better than 700 nm, except when using STED [6,7], and is lower in comparison to imaging thin physical sections individually
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