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Rolly Wiegand – CALM Leica SP5 manual
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it by pressing the arrow next to the ‘End’ position display field. Now you have defined the
Z-stack and the software automatically calculates the total Z-distance of the scan as given
in the field termed ‘Z-Volume’ at the bottom of the window, which is a misleading term and
more accurately should be called ‘Z-distance’.
To complete the programming you have to define the Z-interval at which optical sections
should be acquired between the defined first and last section. This axial sampling rate
should fulfil the Nyquist criterion and can be calculated using the appropriate Nyquist
calculator available in the CALM facility and the computed value can be typed into the field
’Z-step’. The software then automatically indicates the number of steps to be acquired for
the given settings. As a thumb rule, the Z-step should be approximately 2.5 times the XY-
pixel dimension, and for a given XY-pixel dimension of 45 nm the Z-interval should be
approximately 130 nm. Alternatively, you can use the option ‘System optimised’, which
should automatically give you the Z-interval and number of steps according to the Nyquist
sampling rate.
A widespread misperception is that the Z-interval (or Z-step) defines the thickness of the
actual optical slice that is being acquired, but this is not correct. The thickness of the
optical section is defined by the pinhole size and at a setting of 1 Airy unit it is
approximately 400–500 nm thick. Thus, with a Z-interval of e.g. 130 nm, image information
from different adjacent optical sections will be overlapping. However, all software
packages capable of handling 3D data sets will calculate the actual 3D object from this
given information.
For the use of line or frame averaging whilst acquiring Z-stack, the bleaching effects of the
whole specimen caused by the repeated scanning should be taken into account. If
bleaching is a problem, avoid averaging and rather rely on removing the increased noise in
the image by post-acquisition image deconvolution.
4.8.1. ‘Set’ and ‘Go to’ plane function
You can define any Z-position within your define Z-stack to revisit this optical section. For
instance, if you want to mark a plane in the middle of your sample, move there by using
the Z-stepper on the control console. Then press the button ‘Set plane’ (see Fig. 31) and
the software will store this Z-position. To revisit this marked Z-position, click the ‘Go to’
button and the Z-stepper will automatically move to this position.
4.8.2. Starting and switching off the Z-stack function
After completion of all necessary settings, press the ‘Start’ button to commence the
complete Z-scan (see Fig. 26). Depending on the image size, averaging, sequential
acquisition and number of Z-steps, a scan can easily take up to 20 minutes or more. The
progress of the scan is indicated at the bottom of the main window in percentage and by a
progress bar. It is also displayed in the image display window on the right screen by the Z-
slicer on the right hand side, including start and end plane.
After completion of the entire Z-scan, this function can be inactivated by pressing the
arrows ‘Begin’ and ‘End’, which turn from red to black in the inactivated state (see Fig. 31).
4.9. Using the ROI tool
If you have set your zoom to the required factor and do not wish to scan the entire field,
you can use the ‘Region-of-Interest’ (ROI) tool (see Fig. 32) to decrease the size of the
scanned field but retaining the zoom factor. This will help to avoid scanning of unwanted
areas and will save time, if fewer lines are scanned, and it also helps to reduce file sizes.
To define a ROI, activate the function by clicking the box as shown in Fig. 32. In the
display window on the right monitor, generate a frame defining the ROI by left-clicking the
top left corner of the position of the frame and pulling the frame to the desired size, i.e. the
