Development of a Two-photon 3-D Laser Scanning Cytometer

DESCRIPTION (provided by applicant): This proposal describes the construction and testing of a multi-foci multiphoton laser scanning cytometer which will have superior performance in terms of speed and sensitivity in comparison to current commercial instruments, and possess the added ability to image arbitrarily large volumetric tissue samples. Image cytometry is a technique that images a population of cells on a solid substrate. It is similar to flow cytometry in that it can quickly obtain multivariate parameters data on cells and classify how these parameters are distributed across a cell population. This type of analysis is valuable in obtaining detailed information about subpopulations and in identifying rare events, and has found a number of medical and clinical applications in fields such as oncology and hematology. For the instrument in this proposal, the excitation optics will incorporate a 64 lenslet array which will allow the cytometer to scan large regions quickly and with high sensitivity. The emission optics will incorporate a dual channel detection system with a 64 PMT array on each detection channel. The laser excitation source consists of a pulsed Ti-Saph laser system that can generate 1.8 W of mode-locked power at a repetition rate of 80 MHz. The sample will be on a translation stage that is capable of movement on the range of centimeters in the x-y plane of the cytometer. This will allow the imaging of areas on the range of centimeters. Axial resolution is provided by piezo translation stage for the microscope objective. In order to extend the axial imaging depth a microtome will be incorporated into the system. This will allow imaging arbitrarily deep into tissue blocks. The performance of this system will be tested in model systems consisting of cell dilutions. Furthermore, the instrument will be used to characterize in situ tumor metastasis in a mouse model by imaging an organ that has been injected with cells transfected with GFP.