Open AccessA method for calibration of flow cytometric wavelength shift fluorescence measurements
Author(s) -
Kachel V.,
Kempski O.,
Peters J.,
Schödel F.
Publication year - 1990
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.990110810
Subject(s) - calibration , fluorescence , wavelength , remote sensing , flow cytometry , flow (mathematics) , environmental science , optics , materials science , biological system , physics , biology , mathematics , statistics , geology , mechanics , microbiology and biotechnology
Recently, new fluorescent dyes have been introduced into flow cytometry which alter their spectral characteristics when changes occur in certain cell features, e.g., intracellular pH or calcium ion concentration. Such changes may be determined by measuring the fluorescence intensity ratio in two different wavelength ranges (5). Here a new method is described, which simplifies the use of steadily flowing fluids for calibration. The pulse electronics of a flow cytometer cannot process the static fluorescence signals of a streaming fluid. If however, the exciting or emitted fluorescence light of a calibration fluid is made pulsating, the flow cytometer electronics can evaluate those pulses. The new calibration procedure uses measurement of two wavelength windows shown in a two‐parameter display to generate an absolute calibration scale. Measurement of the spectral shift in calibration fluids under identical instrumental settings provides absolute values that measurements of intracellular concentrations can be referred to.