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Rolly Wiegand – CALM Leica SP5 manual
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dimensions of the entire image. The approximate XY-pixel dimensions at maximum
resolution using a sampling rate that fulfils the Nyquist criterion is 45 nm and further
zooming results in over-sampling.
Fig. 29
‘Zoom’ navigator
This function will zoom in on the centre of the entire field. However, if you want to image
part of the entire field that is not in the centre, you can use the arrow buttons (see Fig. 29,
circle) to shift the area you have zoomed in to the desired position within the entire field.
4.7.5. Averaging
This function allows scanning the same optical section several times, as can be defined by
the user, and automatically calculates the average image of several scans, which
significantly reduces the background noise, and thus improves the signal-to-noise ratio.
For instance, if a pixel from a ‘real’ signal in the image records the intensities of 120, 125,
115 and 120 in four consecutive scans, the average would be 120 and would constitute a
bright feature in the image, whereas a single random photon hit in a different pixel in only
one of the four scans of the same image with an intensity of 40 would have an average of
10 and thus a low intensity feature. However, more averaging obviously causes more
photobleaching, in particular if large Z-stacks are acquired. It is therefore recommended
not to increase averaging to more than four scans unless the fluorescent label is very
stable. If photobleaching still is a problem, averaging should be reduced or avoided at all.
Bear in mind that the remaining noise in the image can be removed by post-acquisition
image deconvolution and appropriate hardware and software is available in the CALM
facility. Line averaging is the faster option, but for fixed samples, averages between
frames might provide a very short recovery time from photobleaching.
4.7.6. Accumulation
Similar to the averaging function, this mode allows accumulating the signal from several
scans into one image without averaging and can be used if the photon count from weak
fluorophores is very low. However, bear in mind that unlike in the averaging mode, in the
accumulation mode the noise will be added up as well and will multiply by the number of
scans.
4.7.7. Scan field
By tilting the scanning mirrors of the system, the scan field can be rotated by +/- 180
˚
. This
is a useful tool if the object to be imaged can only be fully fitted into the scan field by
rotating it. This function will slightly increase the overall scanning time.
4.8. Acquisition of Z-stacks
For post-acquisition image deconvolution and accurate quantitation, it is always
recommended to image entire cells or tissue sections in 3 dimensions rather than
representative single sections. Programming the acquisition of a Z-stack requires to define
the start and end section to be imaged as well as the Z-step (or Z-interval), which defines
the stepping of the Z-galvo between the different optical sections.
