A SIMPLE OPTICAL FIBER DEVICE FOR QUANTITATIVE FLUORESCENCE MICROSCOPY OF SINGLE LIVING CELLS

A simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical fiber. After passage through appropriate filters the light is measured using a photomultiplier tube. The optical fiber is mounted in one of the microscope outlets. Signals derived from the photomultiplier are converted to voltage, amplified, and displayed on a recorder. In the excitation pathway a shutter control unit is mounted. With this control unit the period that the excitation pathway is 'opened' and 'closed' can be adjusted, to reduce cell damage and/or bleaching of the probe. This option allows time-lapse recording of experiments up to 1 h. We have used this set-up with a single and dual emission fluorescent probe to determine intracellular calcium concentrations and pH, respectively. In Fluo-3-loaded K562 target cells bound to natural killer cells, a temporary rise in [Ca2+]i was accompanied by bleb formation. The simple construction of this set-up is interchangeable between different types of fluorescence microscopes and can easily be combined with other microscopy techniques, e.g., patch clamp.