Time To Learn Stacking

[Mitch640]

I have spent the last 6-7 months just learning to use a microscope and take pictures and video though it. I could be wrong, but I think I am at the limits of the scope, lenses and camera I have now, for single image quality. I have to learn stacking to get to the next level.

I have Combine ZP and have used earlier viersions on a sporadic basis for the last 4 or 5 years, so I am a little bit familiar with the concept. I downloaded the trial of Zerene, but could not get a decent stack out of it, and looking back, I think it was because my images were probably not in any really close focus distance. Meaning I don't have a motorized focus setup. I do have a vernier on one of the fine focus dial, which is what I used. Combine didn't seem to mind if each stack image was exactly the same or not.

So, my questions are, can I ever get decent stacks just using the dial on the fine focus knob? Should I start at the top and work down, or bottom up? Should I take a bunch of small adjusted shots, or fewer wide distance shots?

Theres probably a lot more I need to know, but I don't know where to start.

[DrLazer]

can I ever get decent stacks just using the dial on the fine focus knob?

Mitch, I wish I had a device with "fine focus", still looking around for a solution. All my stacks are done by moving the rear standard on my bellows unit ... by hand. The subject wobbles around so much when I do this as my rear standard has a locking mechanism and not a wheel. I use the positioning reticle on the viewfinder to roughly get the subject in the same place with each step. Zerene has no problems handling these movements between images (my zerene stacks so far). If the stacks you are trying are not working with fine focus I would suggest the problem may be something else, for example, changes in exposure between one image and the next. Best thing would be to post one of your failed attempts and Rik and the gang will be all over it.

[Mitch640]

I'm sure it was something I did. The trial download is now expired, and it's been months since I tried a stack with Zerene, and I didn't save any of them, or the images. I can't even remember the lighting i used, although that long ago, it was probably just the light hitting the sensor from transmitted light from the scope. Shouldn't have changed much if at all.

I'll try and get some stacks this afternoon. I have a wasp wing mounted that I want to try.

[rjlittlefield]

Mitch, to retry Zerene, just download a copy of the current StackShot beta version from http://zerenesystems.com/cms/stacker/do ... ckshotbeta. It belongs to a different "trial group" from the standard release, so it will run even though your old trial has expired. That will get you back in business without needing to ask for an extended trial key.

Searching here at photomacrography.net, I can't find that you posted any of your previous results, so I have no idea what went wrong for you.

Regarding focus steps, if you're shooting through a microscope then just turning the fine focus knob by hand should work great. It doesn't matter if you work from the top down or bottom up, but going back and forth is a bad idea. The best focus step gives a little overlap between the in-focus portion of one image and the in-focus portion of the next. You can figure out a good focus step by eyeballing the change in focus. If you're working top down, then pick some feature that's just barely in focus at the bottom of the in-focus slab. Keep your eye on that feature as you focus lower, and stop when the selected feature approaches the top of the in-focus slab. Once you get a good step size from eyeballing, then you can look at the index marks to see what the step corresponds to there.

As DrLazer says, show us what you get and we'll help you work past whatever problems you have. With a thoroughly static subject like a dead fly, I doubt you'll have much trouble, but it'll be interesting to see what comes up.

--Rik

[Mitch640]

Thanks for that link. I didn't keep any of the original images or trials, because they were probably so bad that they weren't worth keeping. It was when I was still trying to get any decent image from my scope, before I cleaned it. LOL

I'll be trying to get some images this afternoon.

[Mitch640]

Here is a 128 image stack of a Violet flower pollen. I wanted to do one of a wasp wing, but after looking at it again, I realized that I could focus on the tips of the hairs, then dial down to the bases of the hairs, then I saw other hairs that if I kept dialing the focus, went back out to the tips. It took me a minute to realize that I was going right on through the wing and out the other side. I fugured that wouldn't work well.

This has less color and I am seeing echos of some of the pollen grains, and not sure what that's from. Not a good image, but the individual images weren't much better.

[rjlittlefield]

Echos are usually caused by some sort of movement. I suggest taking a look at your stack as described in the Zerene Stacker FAQ:

How can I detect movement in my stack?

The easiest way is to “play” the stack as it were a filmstrip. To do this, just press-and-drag within the list of input files. File selection will track the cursor, while the image shown in the source window will update to match. This makes it easy to quickly find any portion of the stack that you are interested in, and at the same time makes it easy to joggle between two or three frames to identify the exact frame where some problem turns up.

If you have already processed the stack, then you can also put a checkmark on “Show as adjusted” in the input files panel. This will show the source images as they ended up after alignment. This makes it much easier to spot movement of the subject. Of course if you are interested in seeing how stable and well aligned your setup is, you will want to leave “show as adjusted” unchecked so that you're seeing the source images before alignment.

By default, processing the stack also generates a set of highly compressed preview-quality images that can be played more quickly than the original high quality source images. For this reason, it's usually better to put off “playing” your stack until after it's been processed once, unless you have some reason up front to suspect that subject movement may be an issue.

I have also seen cases where echoes resulted from having images out-of-order in the stack, or equivalently, from accidentally including some test shots that were focused in the middle of the subject, added to the regular stack that was shot from one end to the other.

The fly wing probably would have worked fine, though the image might have ended up confusing because without the depth cues provided by focus, it could be hard to tell what's in front and what's behind. Transparent structures like that can be great in stereo or motion, so keep that in mind for future work.

--Rik

[Mitch640]

Do you happen to remember that discussion awhile back where I said that EOS utility was lagging behind in updating the preview window of the image? I noticed it doing that again. I made two sets of stacks, one at 10x, which is this one above, and a shorter one at 20x, with maybe 25 images. I noticed that things seemed to move in the preview, like the objective was hitting the coverslip, [of course, it wasn't], but the preview would jump, after I had tripped the shutter. I thought the camera would still record what it was actually seeing, and not what the preview was showing me, but that appears not to be the case. In the 20x stack, the last 5 images look like something altogether different, like it was missing 10 or 12 images in the stack. Very crazy. Maybe you can see why this journey has been so frustrating for me. LOL

I didn't save the stack file, so I would have to redo part of it to preview the images. I think I will just do another stack of something else tomorrow when I'm not so at the end of my day.

[rjlittlefield]

To minimize your frustration, I suggest a couple of mods to your workflow.

First, set EOS Utility to record to the camera's card in addition to transferring to the computer. That will let you figure out whether there's a problem with just the transfer part of EOS Utility, or something more general happening with the camera.

Second, make it a habit to Save Project before exiting. That will cost you some disk, at least for however long you keep the project around, but it'll make much easier the job of investigating what went wrong. Stacks take enough time to shoot and process that it's good to wring quite a bit of information out of them before tossing.

--Rik

[Mitch640]

This is the second one from today. It's starting to look better, but I think pollen or anything that is thin enough to be in focus behind another object that is in focus at a different depth, probably isn't a good subject for stacking.



I think I will do some of my aquarium plants next. The lighting is good and the focus goes around the plant.

[Mitch640]

This is a shot of a mix of salt and sugar and water, the liquid put on a slide and allowed to dry. It was quite granular after it dried, with a lot of depth, probably due to the super saturated mix. This is a stack of 109 images, using Pmax.

Magnified about 160x.

[rjlittlefield]

Excellent. No obvious artifacts of any sort.

--Rik

[Mitch640]

Thanks Rik, it's starting to make sense. Not sure where that color came from though. I mean, it's in the stack shots, but the dried crystals are white.

I think there is something wrong with the camera too. Or EOS Utility. Sometimes I hit the shutter button on the screen panel and it would click off two shots. Today, it actually clicked off three shots several times and I had to go delete two of them. Did yours ever do that?

[rjlittlefield]

Thanks Rik, it's starting to make sense. Not sure where that color came from though. I mean, it's in the stack shots, but the dried crystals are white.

I thought about asking, but assumed that the color was deliberate. This is just a white balance problem someplace in the shooting. It's common to get effects like this by having the camera set on daylight or flash, but illuminating with tungsten. My personal favorite way to mess up is to set a custom white balance by shooting a gray frame, then forget to tell the camera to actually use the custom balance that I just set. (You might think that the camera would automatically switch to custom balance when one gets set, but no, it's a separate step. On my old 300D, it was a completely separate step with no reminder. On my newer T1i, at least there's a reminder although it's still a separate step.)

I think there is something wrong with the camera too. Or EOS Utility. Sometimes I hit the shutter button on the screen panel and it would click off two shots. Today, it actually clicked off three shots several times and I had to go delete two of them. Did yours ever do that?

Check to see whether your camera and/or EOS Utility has gotten set into continuous shooting mode, where the camera keeps shooting as long as the shutter release is "held down".

--Rik

[Mitch640]

Ah, good call. It was on Continuous. I moved it to One Shot. I wasn't actually holding the button down for long enough to get more than one shot if I was using the button on the camera. I guess the one on the panel connected to the mouse isn't as positive.

WB was on Custom, and I used the eyedropper. It might be CS4 changing things when I load the RAW's though. I'll have to check that.