An Infra-red Light-transmitting Aperture Controller for Use in Single-cell Fluorescence Photometry

Photometric techniques are commonly used to monitor the output from fluorescent indicators during the study of cellular signalling. At the single-cell level, the region of interest is normally set by a variable aperture placed within the microscope emission pathway. The present study reports an improved aperture controller which adjusts the area for fluorescence measurement, whilst allowing objects throughout the field of view to be continuously monitored using infra-red illumination. A rectangular aperture is selected by four 715-nm long-pass glass filters which block > 99.9% of the fluorescence emission at 480-600 nm. A 780-nm long-pass glass filter is used to provide infra-red illumination which does not interfere with the fluorescence signal, yet is detectable by a standard CCD camera. This allows detection of morphological events throughout the field of view and facilitates manipulation of extracellular pipettes, without interruption to a single-cell fluorescence recording. The infra-red light-transmitting controller is suitable for use with a range of other fluorescent indicators, including those routinely 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.