Fig. 3.

TCSPC imaging of a PC12 cell expressing ECFP. Data were acquired as described, using a 10 s recording time, a long pass 470 nm emission filter and a Zeiss C-Apochromat 1.2 NA 63× water-corrected immersion objective lens. (a) The non-descanned TPE intensity image, acquired using the TCSPC card, showed a fluorescence distribution as seen in HEK293 cells, in Fig. 2. (b) The photon count over 256 time bins plotted against the x-y coordinates. (c) The fluorescence decay data from three-binned pixels from a 128 × 128 pixel image (of a 512 × 512 pixel scan: original pixel dimensions 146 nm × 146 nm) were fit to a bi-exponential curve as described in Materials and methods. The fit residuals for a mono-exponential and a bi-exponential fit are shown below the decay curve. A frequency distribution plot revealed two lifetime populations in the image; a fast component, τ₁ (open circles), and a slow component, τ₂ (filled circles); see text, (c) The pixels containing these lifetimes were reconstructed into 2-D FLIM maps, showing the short (τ₁) lifetime and the long (τ₂) lifetimes to be distributed throughout the cells with no specific accumulations, (d,e) In these FLIM images, colour corresponds to the fluorescence lifetime indicated by the false colour scale, and brightness indicates photon count.