Fluorescence lifetime imaging of Ca2+ using visible wavelength excitation and emission

摘要

Fluorescence lifetime imaging (FLIM) is a new methodology in which the image contrast is derived from the fluorescence lifetime, not the local concentration and/or intensity of the fluorophore, at each point in a two-dimensional image. In our apparatus, the lifetime images are created from a series of phase-sensitive images obtained with a gain-modulated image intensifier. The phase-sensitive images obtained with various phase shifts of the gain-modulation signal are used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. Pixel-to-pixel scanning is not required to obtain the images. As an example of biochemical imaging we created lifetime images of the calcium concentration based on Ca$+2$PLU$/-induced lifetime changes of calcium green (CaG), which is shown to be highly sensitive to $LB@Ca$+2$PLU$/$RB@. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra, which allows Ca$+2$PLU$/ imaging using Ca$+2$PLU$/ probes which do not display spectral shifts. The concept of fluorescence lifetime imaging has numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence, the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples.