7. Achieving the best quality images with the AF51 –
avoiding artefacts
The AF51 can be used in a simple ‘point-and-shoot’ mode but this will often not yield the
best quality results or results suitable for scientific quantitative processing and analysis.
This is because the image may be subject to artefacts with certain control settings and
under certain conditions. Also the CMOS sensor will have some imperfections such as hot
pixels and local variations in amplifier characteristics that only show up in very low light
level conditions and need to be corrected for to get properly calibrated quantitative
information from the images. This chapter will describe those artefacts and give pointers
as to how to avoid or minimise them.
Uneven illumination – flat field correction
This can occur if the camera is not properly fitted to the eyepiece – see figures 5.6 and
5.7. Following the advice in chapter 5 will minimise this. In some circumstances and with
some illumination systems on some microscopes you will not be able to completely flatten
out the illumination across the field using the correct camera fitting procedure but only
minimise it – the residual uneven illumination may still be significant for your purposes.
Also, even if the illumination appears flat and good enough for making illustration pictures,
there will nearly always be some subtle variations in sensitivity across the field due to dust
particles on lenses, etc. which need to be corrected if you are doing various kinds of
quantitative photometric image processing and image comparisons
Under these circumstances a master flat field image (a ‘
master flat
’) should be prepared
and applied as flat field correction to the main image frames when you capture them. The
PARD Capture software provides the facilities to both make a master flat field image and
apply flat field correction during image capture. If you are using other software to capture
your images from the camera you may have to perform flat field correction using multiple
software packages over several imaging sessions to achieve the same effect.
When preparing a master flat field image a multi-frame average is advised to minimise
noise in your master flat because any residual random noise pattern in the master flat will
be transmitted to your main images that you will be correcting with it. In general you
should first acquire a master dark field image (see below) then subtract this for every
frame in your master flat average as you acquire it (using the ‘Apply dark field
subtraction?’ option). You should also enable the ‘Scale mean of each frame to first?’
feature and, optionally, supply a corrections mask if there is dead space in the image
(such as when imaging the circular field of view down an eyepiece). An example of a
‘master flat’ produced in this way using the AF51 on a PUMA microscope is shown in
figure 7.1
I advise that you capture your master flat with a degree of illumination that will be typical
for the actual picture image capture session. So if you are going to capture an scene in full
bright field then make your master flat with full bright field illumination but if you are going
to be capturing a low light level scene (e.g. fluorescence) then use a low light level for your
master flat. Also use the same constant manual exposure setting (and other camera
settings) for taking the master flat series as you will be using for the main image capture.
OptArc AF51 Camera Page 88 of 99 User Guide v1.02
