_____________________________________________________________________________________
Rolly Wiegand – CALM Leica SP5 manual
21
Fig. 28
Scan parameters
4.7.2. Pinhole size
The pinhole size is given in Airy units (AU) and the diameter defines the thickness of the
optical section to be imaged and is depending on the excitation wavelength and the
objective lens properties. The default setting is 1 Airy unit, which is recommend and
collects about 80% of the light. Opening of the pinhole might collect more light from the
sample, but most of it is out-of-focus light and thus degrades the confocality of the images.
If ‘super resolution’ is a requirement, the pinhole can be closed below the size of 1 Airy
unit to get thinner optical sections and exclude most of the out-of-focus light. However, this
will also significantly decrease the amount of light for detection and thus is only possible
with sufficiently bright samples.
4.7.3. Scan speed
The scan speed is defined by the laser dwell time per pixel. The default of 400 Hz is
recommended. Live cell imaging might require higher scan speeds, for poor fluorescent
labels the scan speed might have to be reduced. However, longer dwell times, i.e. slower
scan speed, also increases the bleaching and this might become a problem with dim
samples that are at the same time prone to quick bleaching. If, as in the case of live cell
imaging, the fastest scanning is desirable, this can be achieved by bidirectional scanning.
Click the ‘Bidirectional X’ box to activate this function, which doubles the scan speed.
However, this also requires a phase adjustment and if you want to use this function,
please ask the CALM staff in advance.
4.7.4. Zoom
By changing the scanning angle, a laser scanning confocal microscope provides the
unique option to zoom in on part of the sample. This defines the pixel size and has a
profound effect on the resolution of the image. The zoom can be used to adjust the system
to achieve a sampling rate that fulfils the Nyquist criterion. A sampling rate calculator is
provided by the facility to calculate the accurate sampling rate from the image resolution
(pixels per line), the NA and magnification of the lens and the excitation wavelength.
Based on the zoom, resolution settings and lens properties, the system automatically
calculates the pixel dimensions, which are shown in the same panel together with the
Image resolution
Scan speed
Zoom
Image and pixel dimensions
Averaging and accumulation mode
Scan field rotation
