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Background: Innovative thinking and experimentation were the hallmarks of Mack Fulwyler's approach to research. This report summarizes some of the ideas and their early realizations that he pursued in the field of imaging cytometry, work that was not published before his untimely death, although he composed the initial draft of this report., Methods: Included are related experiments implemented in the programmable array microscope (PAM) devised for patterned illumination and detection, the instrument that Mack Fulwyler employed during a sabbatical leave in Göttingen in 1998. Despite being the originator of instrumentation for flow cytometry and sorting, Mack Fulwyler was intensely interested in imaging systems, recognizing their ability to resolve cellular details obscured by the whole cell signals generally acquired in flow. At one point, these interests merged with those of two other authors (I.T.Y. and T.M.J.), leading to the Image Cytometry and Sorting (ICAS) strategy and project. A major goal was uncomplicated rare cell detection and isolation using a sequential process of cellular labeling via suitable probes, whole field imaging, and selective area-restricted photoinduced reactions designed to encapsulate and/or chemically or physically tag cells in a manner permitting subsequent fractionation by bulk techniques., Results and Conclusion: This publication features photoinduced polymerization, photodecaging, photoactivation, and photochromic conversion reactions carried out by Fulwyler and/or the other authors with the PAM, employing operator designated patterns and locations in various samples. Photopolymerization of polyethylene glycol-diacrylate to a gel-like structure allowing the specific selection of objects (cells) for further analysis and processing techniques was the approach explored personally by Mack Fulwyler in relation to the ICAS concept.

We have implemented continuous-wave two-photon excitation of near-UV absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 647-nm emission of an Ar-Kr mixed gas laser was used to excite the UV-absorbing DNA dyes DAPI, the bisbenzimidazole Hoechst 33342, and ethidium bromide in a shared aperture SNOM with uncoated fiber tips. Polytene chromosomes of Drosophila melanogaster and the nuclei of 3T3 Balb/c cells labeled with these dyes were readily imaged. The fluorescence intensity showed the expected nonlinear (second order) dependence on the excitation power in the range of 8-180 mW. We measured the fluorescence intensity as a function of the tip-sample displacement in the direction normal to the sample surface in the single- and two-photon excitation modes (SPE, TPE). The fluorescence intensity decayed faster in TPE than in SPE.

We report on scanning far- and near-field two-photon microscopy of cell nuclei stained with DAPI and bisbenzimidazole Hoechst 33342 (BBI-342) with the 647-nm laser line of a cw ArKr mixed-gas laser. Two-photon-excited fluorescence images are obtained for 50-200 mW of average power at the sample. A nearly quadratic dependence of fluorescence intensity on laser power confirmed the two-photon effect. The nonlinearity was further supported by evidence of three-dimensional sectioning in a scanning far-field microscope. We find that the cw two-photon irradiation sufficient for imaging within typically 5 s does not significantly impair cell cycling of BBI-342-labeled live cells. Finally, high-resolution imaging in scanning near-field microscopy with good contrast is demonstrated.