Wide-Field Multi-Parameter FLIM: long-term minimal invasive observation of proteins in living cells

Abstract

Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes.

Figure 1. Test of the data-analysis algorithm on real FLIM data.

a) Mean and b) standard deviation values of the normalized amplitude of the fast component in the fluorescence decay of a Rhodamine 6G n-butanol solution as a function of the number of photons per pixel. The measured values are shown in black, the values expected by an unbiased estimator which reaches the theoretical limit of the CRLB are given in red. c) Measured standard deviation of the average lifetime (black) and expected one by a mono-exponential fit assuming a F-value equal to 1 (red).

Figure 2. Non-linear photobleaching and ROS production.

Dependence of the eGFP (a) and tagRFP (b) fluorescence emission signal as a function of the energy dose at several excitation intensities. In case of the eGFP molecules, the higher the intensity, the faster was the bleaching of the signal in fixed HeLa cells. (c) Long-term illumination of living HeLa cells loaded with to monitor the production of ROS species in the 473GFP and (d) 532RFP channels. No increase of the fluorescence due to the illumination-induced oxidation of the molecules was observed during 45 minutes. Few seconds of strong illumination by a Hg-lamp ( for 5 seconds) were sufficient to generate ROS and increase the fluorescence signal. The intensity of the fluorescence signals is given in arbitrary units [a.u.].

Figure 3. Long-term WFMP-FLIM experiment on HeLa cells.

(a) 180 minutes of continuous WFMP-FLIM exposure of a living HeLa cell expressing a tagRFP-5AA-eGFP binary construct by pulsed interleaved excitation does not induce any detectable photobleaching. The two image streams are pseudo colored: eGFP (green) and tagRFP spectral detection channels (red). (b) Fluorescence decays in the 473GFP (green), 473RFP (red) and (c) 532GFP (green) and 532RFP (red) detection channels. The 473GFP/473RFP and the 532GFP/532RFP channels have been merged in pseudo-color images.

Figure 4. Spatial distribution of the average lifetimes.

WFMP-FLIM image of tagRFP-5AA-eGFP transfected HeLa cells. (a) Intensity image. (b) Spatial distribution of the average lifetimes in the 473GFP, (c) 473RFP and (d) 532RFP channels. Spatial distribution of (e) and (f) . (g) Histogram of the parameters in the 473GFP and 473RFP channels. In the 473RFP channel the pre-exponential factor assumes negative values.

Figure 5. Two-color and lifetime kymogram.

(a) Double transfected neuronal process: (red) tagRFP marked mitochondria and (green) eGFP in cell cytoplasm. (b) Distribution of the average lifetimes of eGFP and tagRFP molecules in the 473GFP and 532RFP FLIM channels. (c)The movement of the mitochondria and the cytoplasm flux are simultaneously measured and displayed in form of a kymogram. The sample was continuously measured for about 270 minutes. (d) Average lifetime kymogram of the tagRFP-labeled mitochondria. For each pixel of the kymogram, the average lifetime in the 532RFP channel was estimated. The average lifetime of the tagFRP-marked mitochondria were nearly constant during the measurement time and did not vary significantly.

Figure 6. Scheme of the WFMP-FLIM experimental setup.