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Site selective two-photon tissue image cytometry has previously been successfully applied to measure the number of rare cells in three-dimensional tissue specimens up to cubic millimetres in size. However, the extension of this approach for high-throughput quantification of cellular morphological states has not been demonstrated. In this paper, we report the use of site-selective tissue image cytometry for the study of homologous recombination (HR) events during cell division in the pancreas of transgenic mice. Since HRs are rare events, recombinant cells distribute sparsely inside the organ. A detailed measurement throughout the whole tissue is thus not practical. Instead, the site selective two-photon tissue cytometer incorporates a low magnification, wide field, one-photon imaging subsystem that rapidly identifies regions of interest containing recombinant cell clusters. Subsequently, high-resolution three-dimensional assays based on two-photon microscopy can be performed only in these regions of interest. We further show that three-dimensional morphology extraction algorithms can be used to analyse the resultant high-resolution two-photon image stacks providing information not only on the frequency and the distribution of these recombinant cell clusters and their constituent cells, but also on their morphology.

Quantitative morphometric measurements using site selective image cytometry of intact tissue