AN INFRARED LIGHT-TRANSMITTING APERTURE CONTROLLER FOR USE IN SINGLE-CELL FLUORESCENCE PHOTOMETRY

Photometric techniques are commonly used to monitor the output from fl uorescent indicators during the study of cellular signalling. At the s ingle-cell level, the region of interest is normally set by a variable aperture placed within the microscope emission pathway. The present s tudy reports an improved aperture controller which adjusts the area fo r fluorescence measurement, whilst allowing objects throughout the fie ld of view to be continuously monitored using infra-red illumination. A rectangular aperture is selected by four 715-nm long-pass glass filt ers which block >99.9% of the fluorescence emission at 480-600nm. A 78 0-nm long-pass glass filter is used to provide infra-red illumination which does not interfere with the fluorescence signal, yet is detectab le by a standard CCD camera, This allows detection of morphological ev ents throughout the field of view and facilitates manipulation of extr acellular pipettes, without interruption to a single-cell fluorescence recording. The infra-red light-transmitting controller is suitable fo r use with a range of other fluorescent indicators, including those ro utinely used to detect Ca2+, Cl-, Na+ and pH. Data are presented which demonstrate the use of this controller to measure ADP-evoked [Ca2+](i ) increases in single human erythroleukaemia cells loaded with the Ca2 + indicator fura-2,