Journal: Scientific Reports

Article Title: Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation

doi: 10.1038/s41598-018-36112-w

Figure Lengend Snippet: Fixation time of cytoplasmic protein and development of autofluorescence by aldehyde-fixation in HeLa cells. ( A ) Shown is a representative FRAP experiment during fixation with 2% glutaraldehyde (GA). A circular area with 2 μm diameter (white arrow) was bleached in a HeLa cell, which expressed cytosolic mCitrine, at the indicated time points after changing the medium to 2% GA. Bleaching was achieved by scanning the corresponding area with 5 laser lines of a white light laser at 100% transmission and a 405-nm laser diode at 100% intensity. The upper row shows fluorescent micrographs directly before each bleaching. The lower row shows images directly after bleaching. Scale Bar: 10 μm; ( B ) Fixation time for cultured HeLa cells as determined by consecutive bleaching and fluorescence recovery of mCitrine during chemical fixation using the indicated aldehyde concentrations. Depicted are the time points after which no further diffusion of mCitrine was observed except for 4% FA, 0% GA ( #) , where diffusion was measurable during the whole course of the 20-min experiment after fixation and also in separate experiments after more than 60 min (Figs and ). ( C ) Increase in autofluorescence of HeLa cells upon fixation with the indicated concentrations of aldehydes in 3 different fluorescence channels corresponding to blue (DAPI; excitation (ex.) 360–370 nm/emission (em.) 420–470 nm), green (EGFP; ex. 460–480 nm/em. 495–540 nm) and red (RFP; ex. 535–550 nm/em. 570–625 nm) fluorescence measured by widefield microscopy normalised to living cells. Data is shown for single cells (coloured symbols) and mean (black lines). All autofluorescence measurements in EGFP and RFP channels are significantly higher (p < 0.001 using student's t-test) than in living cells. All measurements in DAPI channel, except after fixation with 2% GA alone (n.s.), are significantly lower (p < 0.001 using student´s t-test) than in living cells. n = 5 independent experiments (40–83 cells).

Article Snippet: Cells were imaged on a widefield microscope (Olympus IX-81) with a 20 × 0.7 NA objective (Olympus GmbH, Hamburg, Germany).

Techniques: Transmission Assay, Cell Culture, Fluorescence, Diffusion-based Assay, Microscopy