Thank you John B.
That was excellent information.
It seems that we are in the same boat in many ways as far as Toupview is concerned.
Do you see the ability to stack images as a way to overcome the depth of field problem?
My young life was filled with astronomy where we took PICTURES with FILM. Still had some DOF problems but not like the micro world.
You were a big help!
Mike aka Mars1
Hi Mike, incidentally I like your handle!
As far as the DOF problem goes, it's a very different scenario when imaging microtome-cut and stained slides such as mine, which are only about 4-10 (maybe 12 but rarely) microns thick, and to make matters significantly more complicated, translucent. When stacking opaque and incidentally-illuminated objects it's easy - simply working through the depth as required, where no two stack-slices will focus con the same X,Y position of the finished image.
This isn't the case with my and similar slides however, as at one depth into the section there may for example be a nice image of a nucleus to focus on for a stack slice, and directly beneath (or indeed above) this (on the same X,Y but different Z position) there may lie a very nice detail of a cytoplasmic strand, cell-wall or even another nucleus (in the case of tissue-depth covering two cell nuclei at once).... This would not stack well at all - one detail covering or confusing the other. When stacking such translucent subjects as this with coincident X,Y details, focus-points must be chosen to avoid this. In short the purpose of the stack must be considered, in terms of 'what information do I require from this stack?' - are the nuclei of a particular area of interest?, is an overall structural image the aim?...
I often choose not to stack or to stack only with 2-3 images as I wish to see the perspective
of say a cell's side-wall (that is perpendicular to the coverslip's surface) to tell me which way a vein is oriented in the section. For an overall view a lower power is used, which of course has a far larger DOF and presents no problems, for that particular view. I often use my x70 for fine detail as it has an N.A. of 0.9 and a coverslip correction collar, and of course doesn't require immersion. The DOF of this objective is truly tiny, but the resolution it offers makes it's use worthwhile. This objective will require stacking of a single nucleus!
The other method I use is to employ 'optical sectioning' - especially useful for pollen-grain analysis as although stained the whole grain will, if stained well, still be translucent and therefore amenable to transmitted illumination. This technique involves selecting focus depths to image details that are coincident at an X,Y coordinate or will confuse or even give a false impression if stacked. For example the 'polar view' and the 'equatorial view' of such a pollen-grain is used for images to identify and catalogue pollen. The definitive view would not necessarily be a SEM image as this would show exclusively surface detail. With optical sectioning structures such as pores that allow passage of pollen tubes through the pollen's outer 'shell' may be imaged in section even with an un-sectioned (microtome section that is) whole pollen-grain.
Sooo, stacking is a very useful tool indeed, in transmitted brightfield also, but needs to be used in this context with some consideration of the above and other factors I find with experience....
The first question then to ask before stacking is - 'what do I want to see in this image?'.
Sorry to be so long-winded!
Hope this helps a little to explain how I use stacking.....