Zerene Stacker

[OzRay]

Whilst I await the arrival of my microscope, I've been researching the other side of the business and that's the actual taking and post-processing side of things. It's patently evident that one of the most ubiquitous tools in use here is Zerene Stacker and I've read all the pages in the Zerene Systems web site, searched here, but still remain a little perplexed about the whole deal.

I'm not so concerned, yet, about DMap vs PMax and the like, but I'm completely at a loss as to how one decides the optimum number of shots you should take to get good results. Images posted here vary from a few dozen to hundreds of stacked shots and, for the life of me, I can't tell what the deciding factor was to use 30 shots over 200 shots.

Is it simply a matter of guestimation, the sheer grunt available in one's computer, the sophistication of one's equipment, size of one's hard drive/s or phases of the moon? Is there a rough yardstick that one could use as a reasonable starting point, subject dependent, so as to not go overboard with images?

Cheers

Ray

[AndrewC]

Two approaches:

1) you can work it out theoretically using relatively simple DOF formulae

2) Set something up - adjust focus for your start position, see how much dof you've got for your particular lens - look for a fine detail and see how much stage adjustment puts it from in focus to oof, then step perhaps 50% of that distance between each slice. to check, you can quickly run a stack of only 5 slices for a particular slice depth, put it into ZS and see if you see oof bands appearing across the output image.

It is a lot easier to work out what is happening if you can tether your camera to a computer so you instantly see detail on a large screen.

Basically you want to have something like 25% overlap of focussed detail at the front and back of each slice.

If you tell us what lenses you will use we can give you best guesses.

For instance, I tend to use the following, others probably have their own favourite settings.

El-Nikkor 80mm reversed enlarging lens with 100mm extension: 204um slice
El-Nikkor 80mm reversed enlarging lens with 200mm extension: 102um slice
El-Nikkor 50mm reversed enlarging lens with 200mm extension: 51um slice
Nikon 10x objective 160mm extension: 6um slice
Nikon 20x objective 160mm extension: 3um slice

I probably over do it, but then I made an automatic stage so it doesn't hurt me to take too many images.

Hard disk space: I used to shoot NEF files, I quickly converted to shooting jpgs for stacks as otherwise your disk space vanishes very quickly !!

[OzRay]

Well, I'm going to be plonking my EP-1 on my microscope (when it arrives), focussing through the eyepiece and likely fine tuning on the camera's live view screen. I have no idea what the lenses will actually provide in terms of magnification, but I was hoping that there would be some sort of rule of thumb that would give you a pretty good approximation of 'sufficient' shots depending on subject size.

Cheers

Ray

[AndrewC]

Sufficient shots = subject_depth / step_distance

where

step_distance < 0.75 * focal_depth

I don't actually have any good examples of what is sometimes called "focus banding", if someone else doesn't post one I'll make one to show you. Focus banding is when you move focus too much between shots so you have regions in the stack which are never in focus. In other words:

step_distance > focal_depth is a no no !

Does that help ? To be honest, run a few short stacks of something like a fly eye and see what works.

[rjlittlefield]

a few dozen to hundreds of stacked shots and, for the life of me, I can't tell what the deciding factor was to use 30 shots over 200 shots.

The number required varies widely depending on 1) magnification, 2) MAGNIFICATION, 3) resolution needed, 4) subject depth, and 5) aesthetics. The number possible also varies depending on 6) photographer patience, 7) computer speed, and 8 ) time available. No doubt I've left out some other important factors.

The reason that magnification is so important is that DOF drops roughly in proportion to magnification squared. As a result, the number of frames needed to cover subjects having the same shape increases roughly in proportion to magnification. To get an equally sharp image over the same relative depth, you'll need 5 times as many frames at 10X onto sensor (micro) than at 2X onto sensor (macro),

If you look at the first four examples in the Zerene Stacker gallery, you'll see stacks with 34, 77, 6, and 83 frames. The one with 6 frames is relatively low magnification and is framed so that the main subjects all lie close to a plane. 6 shots make a shallow in-focus slab that covers those subjects, and the rest of the image is allowed to go OOF. The others are all higher magnification with deeper in-focus slabs.

how one decides the optimum number of shots you should take to get good results

Experimentation is the best way I know. When working with a new setup, shoot a stack with very fine spacing, then process it at varying increments: every frame, every 2nd frame, every 3rd frame, and so on. At some point, you'll see more focus banding than you're willing to live with. Back off a little from there and you have a reliable setting for that setup. Change the magnification or aperture, and you'll have to repeat the process. But keep good notes, and after a short while you can rely on the notes instead of repeating the experiment for every setup.

If your subject is very small, very deep, and you want very sharp images, then you're liable to find that the experiment says you need more frames than you're willing or able to shoot. In that case, you'll have to compromise on either the total depth or some aspect(s) of quality.

I was hoping that there would be some sort of rule of thumb that would give you a pretty good approximation of 'sufficient' shots depending on subject size

For microscopy, DOF in microns can be roughly estimated as 20/(m*NA). For example, 10X NA 0.25 gives DOF = 20/(10*0.25) = 8 microns = 0.008 mm. If you want to shoot something 0.5 mm thick with that setup, it'll take around 0.5/0.008 = 63 frames.

--Rik

[OzRay]

Thanks all, I think it's fallen into place now. Not having used a microscope for a long, long time, it just didn't click that the depth of field was going to be magnitudes less with the microscope than with conventional or macro photography. And with fixed lens apertures, there's no way to increase depth of field.

Cheers

Ray

[elf]

Whilst I await the arrival of my microscope, I've been researching the other side of the business and that's the actual taking and post-processing side of things. It's patently evident that one of the most ubiquitous tools in use here is Zerene Stacker and I've read all the pages in the Zerene Systems web site, searched here, but still remain a little perplexed about the whole deal.

I'm not so concerned, yet, about DMap vs PMax and the like, but I'm completely at a loss as to how one decides the optimum number of shots you should take to get good results. Images posted here vary from a few dozen to hundreds of stacked shots and, for the life of me, I can't tell what the deciding factor was to use 30 shots over 200 shots.

Is it simply a matter of guestimation, the sheer grunt available in one's computer, the sophistication of one's equipment, size of one's hard drive/s or phases of the moon? Is there a rough yardstick that one could use as a reasonable starting point, subject dependent, so as to not go overboard with images?

Cheers

Ray

You could do the math or just download the Excel spreadsheet here: http://www.photomacrography.net/forum/v ... php?t=8054

[OzRay]

You could do the math or just download the Excel spreadsheet here: http://www.photomacrography.net/forum/v ... php?t=8054

Thanks, I'll have a look at that, but it seems, judging by the last comment in the thread, that it may not be ideal for microscopy.

Cheers

Ray

[elf]

That limit was for my custom dial The spreadsheet can handle any level of magnification for which the formulas are valid. They're the same formulas that others have referenced in this thread.

[rjlittlefield]

it may not be ideal for microscopy.

For what it's worth, I don't have much faith in any of the formulas for DOF in microscopy.

If you match up the table of DOFs provided at Nikon's MicroscopyU HERE with the formula given later on that same page, and also with the numbers computed by Nikon's DOF calculator HERE, which supposedly uses the same formula, what you'll discover is that they don't match up.

For example, the table lists 4X NA 0.1 as DOF = 55.5 microns. But the lowest number the calculator will produce is 65 microns. That's with e=4, which is the smallest value that the calculator allows. If you go direct to the formula and try to match the table, what's required at 4X NA 0.1 is e = 0.2, which is physically unrealistic by a factor of 20 or so. Even worse, the table says 10X NA 0.25 gives 8.5 microns; the smallest value from the calculator is 10.4, and the value of e needed to match 8.5 is e = -0.75, a negative number, completely impossible. At higher magnifications, the discrepancies are even worse. The table lists 40x NA 0.65 as 1.0 micron; the calculator's minimum is 1.46; the value with e=0 is 1.30; the value with a more plausible e=10 is 1.69.

I'm pretty confident that Nikon's calculator is correctly implementing the formula they list. I am not so confident that the formula reflects reality, and it certainly does not reflect the table that appears just above the same formula on a different page.

Of course all the numbers that I've quibbled about here lie within a range of 2X for a specified magnification and aperture. So as ballpark estimates, they're fine. Just don't take 'em too seriously. If the number matters, measure it for your own application.

--Rik

[AndrewC]

Like we said - suck it and see !

Shoot a stack at the highest resolution (smallest step) you can. Then stack it subsets until you see banding: every slice, every 2nd, every 3rd, etc

Andrew

[OzRay]

Yeah, I think the suck it and see method will be the easiest at the start. I don't even know how good the microscope will be, so I may not have the finesse available to be very selective. What I'll do is start with subjects that don't need huge numbers of shots and build up from there. I reckon that will give a 'feel' for how all of this will come together. Can't wait for this thing to arrive.

Cheers

Ray

[Charles Krebs]

Ray,

You may find that, once you get above a 10X objective with a "biological" microscope you can do an excellent job simply "by eye". Not only that, but the stacks are often made from fewer frames than, say, an insect head photographed with a tabletop setup.

This is because (with the vast majority of biological objectives) once you go above a 10X the subject should be under a 0.17mm coverglass. So although DOF gets more shallow, the subjects tend to be prepared to be much "flatter". And above a 40X the DOF is so shallow that, even with an indexed fine-focus dial, the steps are so small that they are hard to do by looking at the fine-focus dial and moving it a fixed amount. This is one reason I emphasized the need for a trinocular head that allowed you to view at the same time the trinocular tube was in use. With a pair of 10X eyepieces it is very easy to see the "slice" that is in focus and move it between shots.

[OzRay]

Thank Charles, I was hoping that would be the case.

Cheers

Ray