A Novel Immunofluorescent Computed Tomography (ICT) Method to Localise and Quantify Multiple Antigens in Large Tissue Volumes at High Resolution

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.

Ocular surface inflammation impairs structure and function of meibomian gland

“…Marked dilation with decreased number of acini are observed in a day 20 sample (a, reproduced from Parfitt et al, 2012). …”

“…Three-dimensional reconstruction image shows that the unaffected meibomian gland consists of a duct and many acinar portion, and shows the loss of acini and dilation of ductal portion in day 20 specimen following an alkali exposure (Fig. 7) as compared with an uninjured gland (Parfitt et al, 2012). …”

Immunofluorescence Tomography of Mouse Ocular Surface Epithelial Stem Cells and Their Niche Microenvironment

“…The protocol for sequential immunostaining and image acquisition has been described previously. 29 All immunostaining steps were done using a TedPella BioWave microwave (Ted Pella) for antigen retrieval as well as rapid and consistent staining under vacuum and at regulated temperatures.…”

Differentiation Patterns of Immortalized Human Meibomian Gland Epithelial Cells in Three-Dimensional Culture

“…The holocrine turnover was investigated in mouse meibomian glands by tracking several different CK progenitors and reconstructing them three-dimensionally using a novel technique called immunofluorescent computed tomography. 53 To our knowledge, there have been no previous reports of establishment of 3D cell culture using human immortalized meibocytes; however, molecular biomarkers as described above should be incorporated into future 3D studies to ensure utility.…”