Selective photoreactions in a programmable array microscope (PAM): Photoinitiated polymerization, photodecaging, and photochromic conversion

Abstract

BackgroundInnovative 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.MethodsIncluded 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 ConclusionThis 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. © 2005 International Society for Analytical Cytology

Fluorescence recovery after photobleaching and photoconversion in multiple arbitrary regions of interest using a programmable array microscope

“…fluorescence recovery after photobleaching (FRAP) constitute important tools in cell biology for studying the kinetics of macromolecular motion, interactions and activity (for reviews, see McNally (2008); Rabut and Ellenberg (2005). We have reported previously experiments with the PAM involving photoconversion (Fulwyler et al, 2005; Hagen et al, 2007). One application explored with the newer LCoS-based PAM is the measurement of lateral diffusion of fluorescent protein-membrane receptor fusion proteins by FRAP.…”

Unsupervised Fluorescence Lifetime Imaging Microscopy for High Content and High Throughput Screening

“…In addition to the "inventory" use of the platform in cell screening, the platform can therefore also be used to "hunt" for rare events with the aim of sample retrieval. Single colonies, cells, or cellular subpopulations could be isolated, for instance, by photogelation procedures (22) or laser microdissection and pressure catapulting (23) techniques. The protein or genetic content of the objects with specific lifetime properties can then be analyzed by the relevant techniques.…”

“…These two modes of operation are generally known as image cytometry for analysis and sorting (ICAS) (22). ICAS is suitable for adherent cells and tissues where flow cytometric techniques cannot be used.…”

Trends in Biological Optical Microscopy

“…Its use is particularly helpful when temporal changes of a certain fluorophore distribution induced by photobleaching or photoactivation in an image are observed and analyzed to study transport processes. [9] H.-U. Dodt presented an approach for large-field-of-view microscopy using the detection of autofluorescence of biological molecules or the emission of green fluorescent protein (GFP) perpendicular to an applied excitation light sheet.…”