New (Clueless) Member Needs Help

mfdigiro · Post by **mfdigiro** » Mon Mar 25, 2013 6:51 am

Hi all,

I've found this forum and it seems like the pot of gold I've been searching for. I've learned so much just from reading the posts here and I am blown away by some of the imagery!

So I've just started a new job, and one of my assigned (volunteered) duties is to become proficient at imaging insects and other things with our automontage system. I have a lot of desire and interest, and I'm a pretty quick learner with technological stuff, however I'm pretty clueless when it comes to technical microscope and photography stuff.

So here's the equipment we have....
A Leica M165C Stereoscope fitted with a Leica DFC420 digital camera (5mp). We're running Leica Application Suite V3.4.1 to automontage the stacks and can either put them together in that program or send them to Photoshop layers to stack. We have 4 interchangebale lenses (objectives?) that say ACHRO 1.0x, 0.63x, 0.5x, and 0.32x. I've only been using the 1.0x so far. I light up the subjects with two double armed fibre optic illuminators thru a mylar diffusion cylinder. I may be leaving something out, but hopefully that gives you an idea of what I'm working with.

I'd like to be able to get good images of insects down to about 1 to 2mm, but also larger insects, since a lot of our work involves bark beetles. Ideally, I'd like to take that a step further and get images of beetle anatomy for keys and such, however I'm not sure our equipment is made for that.

Here's a bark beetle that I stacked using the LAS software.

I feel like I can do better. I am going to experiment with background colors and lighting. I also wonder if we should consider another objective (perhaps a 2x). To frame the beetle, I had to zoom in quite a ways, which seemed to affect the image quite a bit. Would I get a better image using a 2x so I didn't have to zoom in? Would it be worth the investment if we plan on imaging more material in this size range? Also, what are the limitations of this kind of setup? What would you change? Are there better software options? Is the camera decent (5mp seems kind of low)? Am I correct calling the interchangeable lenses objectives?

I appreciate any help you can provide.

Thanks!!!!
Marc

rjlittlefield · Post by **rjlittlefield** » Mon Mar 25, 2013 11:12 am

Marc, welcome aboard!

Thank you for the description of your equipment and goals, and for providing a sample image. All this is very helpful.

Before starting to address your questions, I think I should provide some background. My own emphasis is on pushing the envelope of high quality high magnification stacked images. This stems from a background of 45+ years of macro photography, combined with a career in R&D software development for scientific applications. You'll see my name on quite a few of the technical articles here at photomacrography.net. I'm the fellow who wrote Zerene Stacker because I was frustrated trying to wrestle other software into producing the image quality I personally wanted in my own images. It caught on, and now I actually spend most of my time helping other people instead of shooting subjects myself. My experience with Auto-Montage and Leica scopes is limited to a few hours of hands-on contact, combined with discussions with people like yourself who wonder how to get better images.

Coming from that background, here are my thoughts...

In general, stereo microscopes are not the best tools for making high quality photographs. That scope you're using contains wonderful optics, but they were optimized for flexibility and long working distance. The tradeoff is a significant loss in resolution, compared to what can done using objectives that are designed for non-stereo compound microscopes. A lot of the images that blow you away at photomacrography.net were created using the optics described at FAQ: How can I hook a microscope objective to my camera?, combined with one or another of the focus arrangements described in FAQ: What's the best way to focus when stacking?.

Answering your specific questions:

Would I get a better image using a 2x so I didn't have to zoom in?

Probably. But there are some odd interactions between Leica's zoom lens and their objectives. It would take some experimenting to be sure of the details.

Would it be worth the investment if we plan on imaging more material in this size range?

That's unclear. A lot depends on how willing your shop is to consider other options.

You could, for example, make strikingly better images by pairing a consumer-grade DSLR with an inexpensive 10X microscope objective and a StackShot for focusing, total investment around $3000 if you buy everything new. To see what I'm talking about, look at Blowfly at 5X using Nikon CFI BE 10X and the higher magnification treatment at Blowfly proboscis, take #2. Check out the high mag stereo version at the top of the second page of that latter thread, HERE.

My own setups are mostly for illustrating what can be done with small budgets and some tolerance for fiddling. For a production shop you'd probably want a setup more like the BratCam.

Also, what are the limitations of this kind of setup? What would you change?

See above.

Are there better software options?

Take a look at Zerene Stacker. It's not nearly as fast as Auto-Montage, but it does a better job of handling difficult geometries such as bristly bugs.

Is the camera decent (5mp seems kind of low)?

Given the optics, the camera is fine. Using higher resolution lenses, you would appreciate the higher resolution of a typical DSLR.

Am I correct calling the interchangeable lenses objectives?

Definitely. See for example Leica's brochure HERE.

I hope this helps. I look forward to more discussion.

--Rik

mfdigiro · Post by **mfdigiro** » Mon Mar 25, 2013 12:30 pm

You could, for example, make strikingly better images by pairing a consumer-grade DSLR with an inexpensive 10X microscope objective and a StackShot for focusing, total investment around $3000 if you buy everything new.

We do have a DSLR here, and I'm sure we could find an unused scope with a 10x objective to salvage. How much does the stackshot run? Are there alternatives to stackshot?

Marc

rjlittlefield · Post by **rjlittlefield** » Mon Mar 25, 2013 12:40 pm

The StackShot's claim to fame is automation in a small self-contained package that's available off the shelf. It's about $600 delivered with a shutter cable. I don't know anything else in that niche.

However, there are lots of manual solutions. If you're doing proof-of-principle and you have a microscope handy, then one option is to pair the DSLR to the scope. Or to get the subject out of that tight space under the turret, you can use the objective-on-camera approach and still use the scope stage for focus control by cantilevering a subject platform off the side of the stage or building a box platform as shown HERE.

--Rik

mfdigiro · Post by **mfdigiro** » Wed Mar 27, 2013 5:28 am

Rik,

I found a long aluminum rail with a hand crank on one end that moves a platform. It's super sturdy and I think it will work great for a manual setup. I just need to find a suitable objective, a shutter cable, and the adapters to hook it to the DSLR body. We use the rail to analyze tree cores under a scope.

I played with our stereoscope setup yesterday with better results. Of course, these specimens are a lot bigger than the bark beetle.

The last one is about 14 stacks stitched together in Photoshop. I also tried removing the pin and made the background uniform. It's pretty sloppy, but I am also teaching myself Photoshop at the same time!

Thanks!
Marc

rjlittlefield · Post by **rjlittlefield** » Wed Mar 27, 2013 10:31 am

These look very good -- nicely done.

Bigger specimens are definitely simpler to photograph well.

That last one, "14 stacks stitched together in Photoshop", may be illustrating another limitation of the microscope: limited field of view. As an alternative, that whole beetle could have been photographed tack sharp at 15+ megapixels in a single stack using a DSLR and a conventional macro lens such as Canon's 100mm f/2.8L (infinity to 1:1) or Canon's MP-E 65 (f/2.8, 1:1 to 5:1). Stitching is great fun and you can get some incredible resolution that way, but it's pretty inefficient if you have very many specimens to do.

My avatar is a thumbnail version of a 55 megapixel image shot by stitching a bunch of 6 megapixel images. It's discussed HERE, full size image HERE, with a possibly amusing followup HERE. Again, it was great fun and I learned a lot, but for production use I would recommend other approaches.

By the way, if you do continue with stack-and-stitch, then be aware that you're likely to encounter artifacts along the seams due to what are essentially "parallax errors" between neighboring stacks. There are a couple of different ways to deal with those, some by software settings and some by nontraditional optics. If you run into problems, just ask and I'll explain further.

--Rik