Iron Man User Guide v. 1.4
8
5)
In the Online Acquisition window
–
-
The objective should be the one you’ve selected (don’t recommend changing this here, go back to
the ocular tab to change objectives and refocus)
-
The
frame size
indicates how many pixels in each
dimension you want your image to be – more
pixels = more scanning time and larger file. For
most applications, 512x512 or 1024x1024 is
sufficient.
-
Speed
is the scan speed. Faster scan speed will
expose the sample to less light (as it does not
dwell as long per pixel) and a shorter total scan
time, but may produce a less bright image. For
fixed samples, you can try dropping this to 3-5 as
a starting point. If you find your signal bleaching
during Z series, increase scan speed to reduce
pixel dwell time.
-
Averaging
– number of times the sample will be
scanned to produce an average image.
Increasing this number can help clean up noisy
images and can allow you to reduce your scan
speed.
-
Bit Depth
– the number of grey levels the image will contain. Higher bit depth will result in greater
signal dynamic range (greater discrimination between pixel intensity values). 12 or 16 bit.
-
Scan Area – you can control the region to scan and
zoom
here. This is true optical zoom and
should be used to optimize sampling/pixel size to correctly gather required data. Ideally, you want
2.5-3 pixels per smallest resolvable object. So the Abbe limit of resolution is about 200-250 nm, so
the pixels should be about 65-100 nm each. Larger pixel size might allow you to collect the image
faster, but will not allow you to measure small features with high accuracy. Be consistent in your
choice of zoom to ensure your image pixel sizes are the same for multiple days and samples.
-
Other features in this area that I would not mess with: scan mode (leave at frame, you want the
whole frame not just a single line); line step (leave at 1, changing this causes the system to skip
scanning lines to go faster and interpolate the data in between); direction (leave at -->).
