maintenance Petri dish. Higher accuracy can be reached by
counting the detached cells before plating them.- The “Cropped Mode” technology allows shifting lines from
the ROI only instead of the whole frame, with a practical
effective reduction of the exposed chip size (called Cropped
Sensor Mode in our EMCCD). For this configuration to be
effective, a couple of slits mounted in the optical path must
cover the chip outside of the ROI. Thanks to this setup, a time
resolution down to 10^4 s can be achieved. When working in
Cropped Mode, please remember to remove the first and the
last few horizontal lines (3–10 depending on the size of the
ROI) for each frame as the camera background is usually biased
close to borders. - To accurately fit the correlation function a spatial sampling
(pixel size) lower than the waist of the PSF is suggested. The
PSF waist is typically in the 200–500 nm range (depending on
Fig. 5Analysis of GFP-GPI dynamics in living cells. (a) A representative TIRF image of GFP-GPI. (b) Time
evolution of the correlation function (first row) and the corresponding fitting (second row). Residuals (third row)
show that a one-component Gaussian model does not afford a satisfactory description of the system. Fitting
with a two-Gaussian model (fourth row) better describes the experimental function, producing random
residuals (fifth row). (c) the two calculatediMSD curves are qualitatively in keeping with the theoretical
predictions, in the sense that they show an almost constantiMSD component (trapped molecules) and a
linearly increasing one (diffusing molecules) (Reproduced from ref. 11 with permission)
Fluctuation-Based Diffusion Laws 287