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What is the appropriate increment of stacked pictures

Posted by Stephan Wolfsried  
Stephan Wolfsried January 30, 2011 11:38AM

An often raised question.
Here my approach: I bought an object micrometer (fine grid mm by mm, line is 1/100 mm wide, see photo)
The tested objective is for example the Luminar 25 with shortest object distance possible (bellows max shrinked)
FOV is here 4,8 mm.
Put the Objectmicrometer with a minor slope under the assembly and measure the total depth of view of the whole picture.
In this example it is 0,58 mm.
The line of the grid is sharp over a distance of appr. 0,2 mm in the picture.
The rest is the german Dreisatz: DOV is 0,2/4,8*0,58 mm which is appr. 0,024 mm.

So the appropriate increment of stacks with this parameters is 0,024 mm or better a little bit less.


Edited 1 time(s). Last edit at 01/30/2011 11:39AM by Stephan Wolfsried.
open | download - Lum 25 0,58 DOV Balgen eingezogen (2).JPG (57 KB)
Branko Rieck January 31, 2011 09:14AM
Hi Stephan,

Thank you for posting this! Did I trigger this as a reaction to my post,13,211527,211579#msg-211579 where I mentioned you specifically?

One of the points you are making is a very important one: microphotography is much more than buying an (expensive) equipment, it is a very demanding “science” to really get it right, and you are getting it right as anyone can see !

What you show us in your post is one of the basic parameters you absolutely have to know, so if you have a zoom objective, you have to make this calculation for the whole zoom range (in steps, you can interpolate the rest), or if it is fixed, then you have to do it for every objective you have.

An additional step that can help you to get the “sharp” DOF right (for the software you use) in your example would be to create a stack of 5 copies of the picture you showed us of the grid, and let your software process it with the Depth Map algorithm. At a certain point the phenomenon called “stacking mush” will appear, and this will tell you exactly where the software no longer considers the image sharp. And that is basically more important than what your eye considers sharp, because it is the software that processes the image, and not the eye (I tried it with your example image of the grid, and got the same result as you have, but it is nevertheless important to know). Of course this is a time-consuming procedure, but it really pays off.

You wrote: “So the appropriate increment of stacks with this parameters is 0,024 mm or better a little bit less.” I would like to stress the last part of this sentence! Most software products need an overlap in the “sharp” areas to really work well, so I have found that 80% of that calculated value is a good compromise between the number of images taken and the quality achieved. There is also (depending on the program and the algorithm you use) a number of images that is too much. Looking at Chritian Auer’s post,13,202989,212095#msg-212095 the picture with 59 images is worse than the two others (look at the “halos” that are more pronounced - I’ll do a post comparing his pictures once I get my feet on the ground after a busy weekend).

Stephan, thank you for giving us this help. It is important that people that have proven their abilities (something I have yet to do) to lead us in the right direction and to share their “trade secrets”. I do have a request for you, and basically all other people that post their stacked images: for educational purposes it would be very interesting for all (but especially the beginners in this field) to know some technical details of the final images. I have started to add a line “Stacking: composed of xx pictures with Zerene Stacker, Pyramid algorithm” to every picture I upload. Would you (and all others) mind adding this info in the future as well?

Stephan Wolfsried January 31, 2011 05:03PM

first thanks for the compliments!

Staying in my example even an increment of 0,03 mm wouldn't lead to a visible difference in my experience. Try taking only every second picture in the stack and compare results-often You see really no difference. So the 80% are very theoretically, my goal was to get a feeling for the appropriate increment with the bellows, I expected higher resolution leads into more stack images.
The ratio of about 4 was surprising, however, because the resolution isn't that higher with the bellows. I tested this with control structures on wafer fragments. Its about double or so.

Since I only use helicon with Method B the declaration to every picture seems to be superfluous to me, and the number of stacks tells something about topology of the specimen and not necessarily only about photo quality.

The necessary accuracy of stacking increments from my point of view is determined as described. The more critical issue is for me how to handle the transition from sharp to unsharp areas. Here one determines if a picture looks natural or not. Those objectives with higher magnification suffer from unsufficient DOF. Its even a difference to take a photo of the identical specimen with the identical FOV with the Luminar 25 mm and the Luminar 16 mm. You can parametrize the same FOV with the extension of the bellows. I tried that with the Uranosphaerite from Hagendorf uploaded yesterday, because its a sphere more or less. Results with the 16 mm are remarkable worse. And the Nikon M Plan 40x ELWD has a fantastic resolution of 0,35...0,5 micron, but even with stack increments of 0,001 (!) mm I didn't succeeed in taking a reasonable picture.

So let us avoid making a too exact science about stacking increments, it is one neccesary parameter among several. Writing down all of them is too time consuming for me and the community has for sure enough info available to practice successsfully. A little bit of know how advantage must remain, I hope You understand that. So much I can say: The most important criterion is the lighting. With this I do experiments since several years. Surpises come even after years. That is one of the attractions of taking micro photos. Describing lighting and even for Yourself only is a tricky thing.


Edited 2 time(s). Last edit at 02/01/2011 08:32AM by Stephan Wolfsried.
Volker Betz February 04, 2011 09:21AM
Hello Stephan,

my two cents to the subject. Since a few weeks I use a Ortholux I Microscope body with a Lumix D2 attached. The Phototube is equipped with a turret and 4 Lenses:

15 mm Mikrotar - steps around 10 µm
20 mm Canon Macro Lens - steps around 20 µm
25 mm Zeiss Luminar - steps around 20-30 µm
40 mm Zeiss Luminar - steps around 50-100µm

This numbers are not final, I am still experimenting. For the use of additional software ( Helicon 3 d view, interesting !)
the steps should be in identical increments within a picture)

The extention of this setup is always 22.5 cm and field of view is given by the selection of lens on the turret. There is a 1.25 X lens in the turret, I have not found out its better to use it or not. The camera is almost confocal with the oculars, which are very heplful to optimize to adjust the light and give a superb (non stereo) view.

Important to mention the the steps are dependent from the N/A of the lens, not from the focal lenght !

The Ortholux object table has a scale with 1 µm steps, so steps of a few µm a easy to do.

I found that the most important point is that the camera body must be separately fixed, additional to the lens bajonet connection and long expsure times (3 sec.) give some more sharpness.

The Ortholux I is a rugged 9 kg construction useeul for short focal lenghts. I like also to use the 63 mm Zeiss luminar (150-200 µm steps) for the larger field of view . This and a 40 mm Leitz photar (100 µm steps) I use on bellows with a Canon 450 D.


Stephan Wolfsried February 06, 2011 03:45PM

yes I also fixed the bellows a second time beside the camera to get the setup more stiffness.
Your ranges for the luminars fit to my experience. The Luminar 40 mm is also best with 50 to 100 µm in my setup.

Cheers Stephan
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