3.3 Data Acquisition 1. Align the TIRF laser excitation according to the procedure of
your setup. In the setup proposed here, an auto-alignment
procedure is available. Once the laser is aligned, an optimal
penetration depth of about 70 nm can be selected. Put the
sample in the holder.
- Focus on a fluorescent cell by using the eyepiece (seeNote 7),
send the light to the camera, and gently push the slits allowing
only the light from the selected ROI to pass (here a 32 32
pixels ROI). - Set the appropriate exposure time and EMGain on the camera,
select the desired number of image repetitions, then start the
acquisition. Reset the EMGain and exit from the Cropped
Mode to allow temperature stabilization before acquiring a
new cell. Repeat the last two steps in order to acquire the
desired number of cells.
3.4 Calculation
of the Mean Square
Displacement from
Imaging (iMSD)
- Import the acquired image series into a data processing pro-
gram (e.g., Matlab, appropriate scripts can be found elsewhere
[23]). - Calculate the average intensity of each image and plot it in
time. If more than 10% of photobleaching is present, it is
suggested to discard the image series. If it is lower, try to
correct the effect on the correlation function by subtracting
Fig. 3Calibration of point spread function. (a) Pseudocolor image of an isolated bead and beads aggregates.
(b) 3D plot of the intensity profile of an isolated bead shows a well-defined Gaussian profile. (c) Fit of the
intensity distribution by a Gaussian function (upper panel) with the corresponding residuals (lower panel). The
good agreement between the fitted distribution and the measured intensity profile is also a proof that the
instrumental PSF can be approximated by a Gaussian function (Reproduced from ref.24 with permission)
Fluctuation-Based Diffusion Laws 283