Quantitative tissue cytometry (Tissomics): multimodal slide-based cytometry, confocal imaging, and volume rendering is the key

Multiplexed high-content cytometric analysis of cells is a prerequisite for Cytomics and Systems Biology. Slide Based Cytometry (SBC) analysis yields quantitative cell related data on various cell constituents. It allows to measure and identify in high-throughput hundred-thousands of objects and obtain cytometric data on light absorption, scatter and fluorescence signals. Selected cells of interest can be rescanned and morphologically evaluated. To be cytometric SBC measurement needs high focal depth in order to acquire the fluorescence of the whole cell. For tissue analysis section thickness of >30μm is needed to reduce cell sectioning leading in multiple labelled specimens to an overestimation of multiple stained cells due to stereology, mimicking co-expression or elevated expression that is in fact due to coincidences in the z-axis direction. By confocal sectioning and 3D-reconstruction these overlays could be eliminated but confocal 3D imaging is slow and the resulting data are not cytometric. To overcome this obstacle, we combined SBC analysis with confocal imaging using a Laser Scanning Cytometer (iCys, Compucyte Corp., MA). Single to triple labelled 30-120μm thick human brain sections were scanned cytometrically (up to three laser 405nm, 488nm, 633nm) and double and triple labeled cells were identified. In the second step these objects were relocated, scanned confocally and 3D-reconstructed (Mathematica®, MathGL3d). This combination of high-throughput SBC and high-resolution confocal imaging enables for unequivocal identification of multiple labelled objects and is a prerequisite for Cytomic tissue analysis, Tissomics. (Support: HBFG 036/379-1)