Fluorescence lifetime imaging of calcium using Quin-2.
We describe the use of a new imaging technology, fluorescence lifetime imaging (FLIM), for the imaging of the calcium concentrations based on the fluorescence lifetime of a calcium indicator. The fluorescence lifetime of Quin-2 is shown to be highly sensitive to [Ca2+]. We create two-dimensional lifetime images using the phase shift and modulation of the Quin-2 in response to intensity-modulated light. The two-dimensional phase and modulation values are obtained using a gain-modulated image intensifier and a slow-scan CCD camera. The lifetime values in the 2D image were verified using standard frequency-domain measurements. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra, which may allow Ca2+ imaging using other Ca2+ probes not in current widespread use due to the lack of spectral shifts. Fluorescence lifetime imaging can be superior to stationary (steady-state) imaging because lifetimes are independent of the local probe concentration and/or intensity, and should thus be widely applicable to chemical imaging using fluorescence microscopy.
Lakowicz
J R
JR
Department of Biological Chemistry, University of Maryland, Baltimore.
Szmacinski
H
H
Nowaczyk
K
K
Johnson
M L
ML
D000634:Aminoquinolines; D002118:Calcium / Q000378:metabolism*; D005456:Fluorescent Dyes; D008027:Light; D008962:Models, Theoretical; D013050:Spectrometry, Fluorescence / Q000379:methods; D013223:Statistics as Topic
D000634:Aminoquinolines; D005456:Fluorescent Dyes; C035739:Quin2; D002118:Calcium