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We developed a 3-D image cytometer based on two-photon scanning microscopy. The system keeps the inherent advantages from two-photon scanning microscopy: (1) The ability of imaging thick tissue samples up to a few hundred micrometers, (2) The ability to study tissue structures with subcellular resolution, (3) The ability to monitor tissue biochemistry and metabolism, and (4) The reduction of specimen photobleaching and photodamage. Therefore, 3-D image cytometer has the ability to characterize multiple cell layer specimens, in contrast with 2-D image cytometer where only single cell layer samples can be imaged. 3-D image cytometry increases its frame rate by adapting a polygonal mirror scanner and high-speed photomultiplier tubes. The current frame rate is 13 frames per second. High throughput rate is achieved by imaging multiple cell layer specimens in 3-D at a high frame rate. The throughput rate of this system is dependent on the choice of objective lenses, specimen properties, and the speed of computer-controlled specimen stage. It can be up to approximately 100 cells per second which is comparable with that of 2-D image cytometers. With the high throughput rate and deep tissue imaging capability, 3-D image cytometer has the potential for the detection of rare cellular events inside living, intact tissues. A promising application of this 3-D image cytometer is the study of mitotic recombination in tissues. Mitotic recombination is a mechanism for genetic change. Therefore it is one of causes for carcinogenesis. However, the study of this process is difficult because recombination event is rare and it occurs at a rate of one cell in 105 cells. The new method for the study is (1) to engineer transgenic mice whose cells will express fluorescence in the presence of mitotic recombination, (2) to detect cells which have undergone mitotic recombination with 3-D image cytometry. The estimated time required to quantify spontaneous recombination rate is approximately within a few hours in the case that the mutation occurs at a rate of 1/105. The ability of this 3-D image cytometer to resolve tissue structures at video rate was demonstrated in the study of ex vivo human skin dermal structure. 25 X 25 section images were taken by shifting the acquisition region with computer- controlled specimen stage. Wide area images were reconstructed by combining each image sections. The size of complete wide area images is approximately 25 mm X 25 mm. We further performed experiments to verify this cytometer's ability for population statistics measurement. We prepared cell cultures containing a mixture of cells expressing cyan and yellow fluorescent proteins. These cell cultures with mixing ratio ranging from 1/10 up to 1/105 were imaged. Experimental results show that the presence of a few rare cells in large pool of the other cells can be quantitatively measured. We also imaged a punched ear specimen from a transgenic mouse which carries green fluorescent protein. The data demonstrates that this system can resolve a single green fluorescent cells in the tissue.
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