Fist attempt at stacking

Starting out in microscopy? Post images and ask questions relating to the microscope and get answers from our more advanced users on the subject.

Moderators: rjlittlefield, ChrisR, Chris S., Pau

xys
Posts: 49
Joined: Thu Apr 21, 2011 11:42 am

Fist attempt at stacking

Post by xys »

This is from a prepared slide (came with toy microscope) labeled "Silver berry scaly hair". Olympus CHA-P student microscope, achromat PO10, NA 0.25, 5x projection lens, micro-4/3 (E-P!) camera. 11 sections at 12.5 um depth increments. First picture is bright field, second is polarized light. I used CombinZP and let it do whatever it does by default (I'm not even sure I used it correctly, however, the results appear to my uneducated eye credible). These are from out-of-camera jpegs with no processing (except that the stacked images were resized). Any comments, critique, or suggestions would be much appreciated.

Rashid

Image
Image

rjlittlefield
Site Admin
Posts: 23561
Joined: Tue Aug 01, 2006 8:34 am
Location: Richland, Washington State, USA
Contact:

Post by rjlittlefield »

These certainly appear credible, but often it is hard to tell if something is missing until you compare against a more complete rendering of the same stack.

If these images were created by Do Stack (first stacking method in the list), then I suggest to run Pyramoid Maximum Contrast on the same stack. That method hardly ever misses anything. Then you can layer the two outputs in Photoshop or some similar tool, and click the top layer invisible/visible to flash between them. The flashing makes differences obvious.

One quirk of CombineZP is that it gives a "mirrored margin" around the outside of the image. You can see this at the left side of your images where there are chevron shapes that result from mirroring slanted fibers. Usual practice is to just crop off the mirrored margins.

--Rik

xys
Posts: 49
Joined: Thu Apr 21, 2011 11:42 am

Post by xys »

Rik, many thanks for your comments. What I did was, indeed, Do Stack. I tried your suggestion of comparing this with the results of Pyramoid Maximum Contrast, and yes, there were (subtle) differences, as you indicated. A couple of questions if I may: (1) Was the number or spacing of the sections that I used (11 sections, about 12.5 um spacing) a reasonable number? (I just did that arbitrarily). (2) Is it critical that the first and last sections are taken at the closest and farthest point of the subject that are in focus, or can one start a bit before and end a bit beyond the respective focus planes without introducing serious problems?

Many thanks, again.

Rashid

rjlittlefield
Site Admin
Posts: 23561
Joined: Tue Aug 01, 2006 8:34 am
Location: Richland, Washington State, USA
Contact:

Post by rjlittlefield »

Yes, the spacing is reasonable. Exactly what's required depends in part on the subject. Finer spacing may be needed for subjects with very sharp detail and regular structures that make it easy to spot focus banding. A good example is the compound eye of a small fly. Subjects with detail that is less fine or less regular might look OK with even coarser spacing.

It is OK to have some additional frames before and after the parts you really care about. In most cases these will not hurt the stack, and even if they do cause a problem, it is easy to reprocess the stack with the unnecessary frames left out. It is far better to shoot more than you need than not enough.

--Rik

xys
Posts: 49
Joined: Thu Apr 21, 2011 11:42 am

Post by xys »

Rik, many thanks for your very helpful comments.

Rashid

Post Reply Previous topicNext topic