Fig. 5.

Living cerebellar granule cells (CGCs) were stained with FM1-43 and imaged using 800 nm TPE. The dye partitioned into membrane structures as expected (a). (b) The fluorescence spectra for membrane-intercalated FM1-43 and FM4-64, predicting that FM4-64 would act as an acceptor for FM1-43 energy; FM4-64 normalized absorbance, green filled circles, FM1-43 normalized emission, red filled circles, FM4-64 normalized emission, open circles. The line plot is the integrand, namely the product of the acceptor absorption and the donor emission, multiplied by the donor wavelength, λ⁴. (c) Decay curves from a 128 × 128 pixel FLIM image, using a 435-485 nm BP filter to dissect FM1-43 emission (filled circles, two-binned pixels underneath the cross in a), fit to a bi-exponential curve (black line). The same pixel was sampled after the same cells were counter-stained with FM4-64 (open filled circles) and the normalized data fit to a bi-exponential decay (red line, six-binned pixels). (d) The resulting donor mean fluorescence lifetime, $\bar{\tau }$, frequency distributions before (black filled circles and line) and after (red filled circles and line) FM4-64 counter-staining. (e,f) The FLIM maps generated from the distribution data for non-FRET and FRET images, respectively. In these FLIM maps, the image data brightness values have been equalized to reveal a donor-specific decrease in $\bar{\tau }$ without a loss of image clarity due to intensity quenching. 800 nm TPE was used as before, with a 500-550 BP emission filter to resolve FM1-43 (i.e. donor) fluorescence. Images were made using a Zeiss Plan NeoFLUAR 1.3 NA 40× oil-immersion objective lens.