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Image cytometry technology has been extended to 3D based on high-speed multiphoton microscopy. This technique allows in situ study of tissue specimens preserving important cell-cell and cell-extracellular matrix interactions. The imaging system was based on high-speed multiphoton microscopy (HSMPM) for 3D deep tissue imaging with minimal photodamage. Using appropriate fluorescent labels and a specimen translation stage, we could quantify cellular and biochemical states of tissues in a high throughput manner. This approach could assay tissue structures with subcellular resolution down to a few hundred micrometers deep. Its throughput could be quantified by the rate of volume imaging: 1.45 mm(3)/h with high resolution. For a tissue containing tightly packed, stratified cellular layers, this rate corresponded to sampling about 200 cells/s. We characterized the performance of 3D tissue cytometer by quantifying rare cell populations in 2D and 3D specimens in vitro. The measured population ratios, which were obtained by image analysis, agreed well with the expected ratios down to the ratio of 1/10(5). This technology was also applied to the detection of rare skin structures based on endogenous fluorophores. Sebaceous glands and a cell cluster at the base of a hair follicle were identified. Finally, the 3D tissue cytometer was applied to detect rare cells that had undergone homologous mitotic recombination in a novel transgenic mouse model, where recombination events could result in the expression of enhanced yellow fluorescent protein in the cells. 3D tissue cytometry based on HSMPM demonstrated its screening capability with high sensitivity and showed the possibility of studying cellular and biochemical states in tissues in situ. This technique will significantly expand the scope of cytometric studies to the biomedical problems where spatial and chemical relationships between cells and their tissue environments are important.

A two-pulse experiment is described, using stimulated emission to reduce the fluorescence of 1-ethyl-4-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-pyridinium perchlorate (Pyridine 2) in a microscope of high numerical aperture. The experiment employed a 130 fs pulse at 375 nm for excitation and a 20 ± 5 ps pulse at 750 nm for stimulated emission. The pulses were provided by a mode-locked Ti:sapphire laser. The 375 nm excitation pulse was obtained by frequency-doubling and the 20 ± 5 ps infrared pulse by optical grating dispersion. The population of the excited state was monitored by varying the temporal delay between the excitation and stimulating pulse. This method enabled the measurement of the lifetime of the dye. For Pyridine 2 in glycerol, we determined a lifetime of 0.86 ± 0.2 ns. The decrease of fluorescence was due to stimulated emission, as was established by measurements of the temporal behaviour of the fluorescence signal upon amplitude modulation of the stimulating beam. We present a theoretical analysis of the temporal behaviour of depletion by stimulated emission in a two-pulse experiment.

Summary We report the realization of stimulated emission on a microscopic scale. An experiment is presented describing significant depopulation of the excited state of the fluorophore Pyridine 2 with a mode-locked Ti: Sapphire laser. Stimulated emission is performed at 750 nm and excitation at the frequency doubled wavelength of 375 nm. The pulses are synchronized so that the stimulating pulse follows the excitation pulse after 5 ps. The set-up is a modified 4Pi-confocal microscope employing one of the objective lenses for excitation and the opposing one for stimulated emission.