Biology Reference
In-Depth Information
1.3.5.1
Analog detector calibration procedure
For a given PMT voltage and pixel dwell time, time series are acquired in the spot
selection mode.
1.
Set pinhole to 1 Airy unit and amplifier offset to 0.
2.
Repeated acquisitions are done at increasing laser intensities. The initial
acquisition is carried out with a low-laser-intensity setting to avoid detector
saturation. Increase the laser intensity until reaching around 90% of detector
saturation and then systematically ramp down the laser intensity. A final
acquisition is also collected with the laser turned off. Typically, 10-20
acquisitions are performed for each PMT gain and pixel dwell time.
3.
Open the different acquisitions in ImageJ and ask software to extract
standard deviation of values collected. Variance corresponds to this value
squared. Plot variances versus mean intensities. Slopes of the variance
versus the mean intensity of curves are calculated only for the linear regime of
the PMT. An example is shown in
Fig. 1.9
. Calculate slope, which corresponds to
detector variance number needed to be entered in the GUI.
It is important to note that the slope can depend on many parameters (dwell time, PMT
voltage, scan speed, temperature, etc.) and that, for each set of parameters, the detector
variance should be determined. Similar calibration can be performed for a CCD cam-
era by generating image time series of constant stable light source and generating a
graph of the variance of each pixel as a function of the mean intensity. If the CLSM
has photon-counting detectors, then this calibration is not necessary.
1.3.6
Data interpretation and pharmacological analysis
Oligomer density can be expressed as a number of protomers/beam areas. As
beam area can be converted to
m
2
, protein density can be expressed as
m
m
2
(i.e., monomer or dimer/
m
2
). In the case of RTKs, when imaging
protomers/
m
m
FIGURE 1.9
Example of detector broadening determination for different pixel dwell time.
