Here, we have described novel approaches for high-throughput site-selective cytometry to study clonal expansion of cells within intact tissue. In a previous study, we demonstrated that the number of recombinant cells can be measured in FYDR mice; in this study, we show that quantitative morphological analysis of these recombinant cells and their clusters can also be performed. Importantly, these methods are some of the first permitting quantitative analysis over a size scale spanning four orders of magnitudes. We apply this powerful approach for the rapid detection of rare fluorescent cell clusters throughout the entire organ over centimetre length-scales while quantifying the three-dimensional morphology of each fluorescent cell clusters and their constituents' cells on the micrometre scale. The frequency, brightness and the sizes of the both recombinant foci and nuclei inside the clusters have been quantified.
In three-dimensional site-selective tissue cytometry, a large area is imaged rapidly with WFM, and specific regions of interest are imaged at high-resolution with TPM. The current implementation of this site-selective tissue cytometry uses a standard, lower speed TPM. While it is sufficient for many site selective studies, it is not suitable for whole organ imaging where higher data acquisition speed is needed. There is potential for a similar site selective scheme to be implemented with video rate TPM (Bahlmann et al. 2007
; Kim et al. 2007a
; Ragan et al. 2007
) providing even greater throughput. Using a video-rate (30 frames
) TPM imaging would decrease the imaging acquisition time by 210 times reducing the total acquisition time to merely minutes per specimen.
Although, powerful in its present implementation, it may be desirable in the future to make the imaging process more automated. For example, the results from WFM imaging can be more rapidly and objectively evaluated using automated imaging processing. More specifically, all regions of interest in the WFM image can be segmented and identified automatically. In addition, the centroids of these regions can be computed and compiled into a list. From this list of the positions of interest in the wide field image, appropriate x–y coordinates can be calculated and fed to a robotic x–y positioning stage to provide automatic guidance for high-resolution TPM imaging at the selected sites.
In conclusion, we have described a novel imaging platform that provides new avenues for in depth analysis of nuclear, cell and tissue morphometry. In this example, we examined rare fluorescent recombinant cells, but there are many potential applications for this new imaging platform. For example, there is a great interest in being able to track and monitor the behaviour of stem cells used in regenerative medicine. This platform provides a novel and rapid methodology for assessing the location and the morphology of such rare cells.