Abstract

Intracellular ion concentration images are essential for studying the biochemical processes inside a cell and among the ions Ca²⁺ and Na⁺ are at the top oflist in terms of importance. The ions become "visible" when they bind to their specific fluorescent dyes. Each fluorochrome has its own distinct absorption and emission spectra and therefore observation of two ions in a conventional epifluorescence setup would entail switching the filter sets. A mechanical change of the filter sets can be cumbersome in imaging a dynamic phenomenon in an "in vivo" experiment. Also it can lead to registration errors when overlaying the images of different ions. Here we show how the use of an electrically tunable emission filter (Varispec, CRI, USA) can help solve the problem. We describe our setup based on an inverted microscope, Varispec and a cooled high resolution CCD camera. First we show the test results on fluorescent microspheres (Molecular Probes) and then the results obtained by using Sodium Green and Fluo-4 as indicators of Na and Ca ions respectively. We also show the results obtained with different excitation sources viz. mercury lamp, super luminescent LEDS and lasers.

© 2003 SPIE