the Objective numerical aperture and the excitation wave-
length). Thus, a pixel size of 70–150 nm (three times lower
than the instrumental waist) can be usually set. Furthermore,
please consider that the minimum size of the image should be
at least 3-times larger than the maximum displacement of
interest (plus the instrumental waist). This is needed to reach
a good convergence of the fitting algorithm and a statistically
significant sampling of molecular displacements. Concerning
the camera-based system used here, please note that the pixel
physical size on the chip is fixed. Therefore, decreasing the pixel
size inevitably decreases the signal in each pixel (that depends
on the square of the pixel size), makes the field of view smaller,
and demands for a higher magnification power.- A typical frequency distribution of camera background yields a
peak at a certain value of Digital Levels (DL) (due to the
camera response to no photon) that represents the contribu-
tion of Analog Digital (AD) converter. This contribution can
be approximated as a Gaussian distribution to estimate the
offset and the variance introduced by the signal recording. At
higher DL values, the distribution becomes exponential and
represents the average camera response to a single photon. The
center and the variance of the Gaussian function represent the
camera offset and error, respectively, while the decay constant
of the exponential part affords an estimate of the DL assigned
by the camera to each single photon. The higher is the ratio
between the average DL of a single photon and the AD con-
verter error, the lower will be the noise in the calculated corre-
lation function. For more details please refer to ref.23. - It is suggested to extensively sonicate the solution for 20 min to
avoid the presence of beads aggregates. - Particular attention is required in order to avoid art factual
correlations. In fact, as previously shown for similar techniques
[27], cell borders as well as out-of-focus vesicles could intro-
duce a strong correlation. If the inspection of the average image
reveals cell borders or out of focus vesicles, try to exclude the
region involved, otherwise discard the acquisition. To correct
the effect of this immobile structures subtract the average
temporal intensity from each pixel [28]. - If the data are too noisy, try to increase the number of acquired
frames, increase the laser power, average more G(ξ,χ,τ)
together. - If theσ 02 and PSF values are comparable, it means that the
dynamics of isolated fluorophores (not large aggregates) is
extracted. By contrast, ifσ 02 >>PSF, it means that either
large aggregates are present or hidden dynamics are present
(i.e., a faster time resolution is needed) [11].
288 Francesco Cardarelli