- Dilute 10μl of 30 nm fluorescent beads solution (about 5μM)
in 90μl of distilled water (seeNote 6). Cut a squared piece
(1 cm1 cm) of agarose gel (3%) and deposit 10μl of the
solution on the top of the gel. Turn over the piece of gel on the
bottom glass of a 35 mm Glass-bottom Patri dish and squeeze
the drop on the glass. - Put the sample in the microscope holder, set the camera expo-
sure and EMgain (100 milliseconds and 1000, respectively, are
appropriate parameter values according to the system proposed
here), and eventually wait for the camera to cool down. Find a
field of view with isolated beads, accurately focus on a single
bead and acquire 100 frames (Fig.3a). Five to six repetitions of
the same measurement are recommended, in order to acquire
enough statistics. - Import the acquired image series to a data processing program
(e.g., MatLab) and average the stack in time to identify easily
isolated fluorescent spots. Take care of selecting the smallest
ones to avoid aggregates (Fig.3b). Fit the selected intensity
distribution with a Gaussian function (e.g., by Matlab “gauss-
fit” function) and verify the goodness of the fit by inspecting
the associated fit residuals (Fig.3c). The half width at half
maximum retrieved from fitting is a good estimate of the
characteristic PSF size.
Fig. 2Calibration of camera response to single photons. The figure shows the Digital Level (DL) distribution for
camera background in a 32X128 ROI, exposure 0.5 ms, in Cropped Sensor Mode. The peak at about 180 DL
represents the camera response to no photons. Particularly, it represents the contribution of the Analog Digital
(AD) converter and can be approximated by a Gaussian function to estimate the offset and the variance
introduced by the signal recording. Above 200 DL the distribution of digital levels becomes exponential and
represents the average camera response to a single photon. The measurement of these parameters allows
estimating the density of photons recorded during the acquisition. (Reproduced from ref.24 with permission)
282 Francesco Cardarelli