First attempt with Nikon CF E 10x 0.25 - fly genitalia

viktor j nilsson · Post by **viktor j nilsson** » Sat Mar 23, 2013 4:53 am

Hi all,
My name is Viktor Nilsson, and I am an amateur entomologist / professional evolutionary ecologist from Sweden.

This is my first post here, but I've been reading quite a lot of threads for quite a long time.
I've been doing some quick and dirty stacking for a couple of years now, using Combine ZM and a P&S camera fixed against the eyepiece of my Leica StereoZoom 7. That has fulfilled the purpose of documenting my findings, but I've increasingly felt the urge to take things a step further. Here is my first attempt to get more "serious" about stacking. I appreciate all feedback to refine my technique.
The setup is an Wild M20 microscope, Nikon CF E 10x 0.25, an home-made dovetail adapter, cheap extension tubes and a Canon 40D:

Let's jump right to the final pictur:

Zerene Stacker, PMax. I have edited away some debris, set white point using Curves in PS, sharped it a bit and pulled down the saturation of magentas to 0 using "replace color" to get rid of some (mild) CA.
Completely unaltered output from Zerene Stacker is here:

Hi-res: http://img818.imageshack.us/img818/8863 ... zspmax.jpg

The subject is the male genitalia of the 6th ever (!!!) known individual of a recently described Lonchaeid fly, Earomyia netherlandica MacGowan 2004. This species has been found in England (twice) Netherlands (twice), Hungary (once) and most recently, by me, in Sweden. Wanting to write a nice article about this find was what inspired me to get serious about stacking.

Positioning the genatiala was difficult! I placed a steel spacer on a microscope slide, filled it with glycerine and placed a cover glass over it so that it covered half the "hole". I then placed the genitalia into the glycerine throug the open part of the "hole", pushed the genitalia in under the cover glass and positioned it using a bent needle. I did the positioning under the dissecting microscope at relatively modest magnification and then moved the slide over to the microscope setup to shoot it. It was hard to get rid of several tiny bubbles, and I do not know how the glycerine managed to suck up that much dust in such a short time. Maybe the dust was already in the pipette I used. 10x magnification is really unforgiving! Photo of the arrangement:

The lighting was done with a single Yongnuo YN-465 flash, diffused with paper towels around and below the subject. The Jansjö focusing light was on while shooting, but I don't think it delivered very much ligh. The flash was set at about 1/2 or 2/3 power, the shutter speed was set at 1/250. "Flash" white balance on the Canon 40D Camera. I used a 3 micron step size (the resolution of the M20 fine focus is 1 micron).

Fine focus travel on the M20 is only about 2 mm, but for this subject that was not a problem. I am also building a horizontal rig (with a newport stage) for low-mag shooting. The table is not the sturdiest. I will try to improve that, but at 1/250 shutter speed, I do not think that vibrations are a huge concern.

I am really happy with the results. But I'd love to hear your thoughts on what could be improved!

rjlittlefield · Post by **rjlittlefield** » Sat Mar 23, 2013 9:26 am

This is looking very good.

The next thing I would do is to make a stereo pair. You can read about how to do this at http://zerenesystems.com/cms/stacker/do ... eticstereo. Very briefly, in Zerene Stacker all you have to do is go to Options > Preferences > Stereo/Rocking, check the box for "Generate stereo pair or rocking sequence", set the number of output images (2), set the first and last shifts (I suggest -2 and +2 to start), then click OK followed by Stack > Align & Stack All (PMax). This will produce two Output Images showing the stack as it would appear viewed from slightly to the left and right of center. You can confirm the results with Stereo > Start Preview in Zerene Stacker, but ultimately you will want to assemble a high quality cropped version using 3rd party tools such as StereoPhoto Maker or even just Photoshop.

I am very eager to see the stereo version. I am completely ignorant about the structure of this type of fly, and I expect that the immediate 3D perception will help me a lot to sort things out.

--Rik

viktor j nilsson · Post by **viktor j nilsson** » Sat Mar 23, 2013 11:54 am

Thank you, Rik! Your kind words are truly encouraging.

I probably hadn't ventured into generating stereo images at this point myself, as I am still not able to "see it". I used to be good at seeing 3D images when I was a kid, but I haven't been practising a lot during the last 15 years!

Anyway, here is my first attemp. I used the settings you suggested (2, +2, -2). Just by rocking the two output images, I am amazed at the information Zerene is able to extract from a simple z-stack.

I think that I placed the two images correctly. But please correct me if they should be switched.

rjlittlefield · Post by **rjlittlefield** » Sat Mar 23, 2013 12:08 pm

Thank you very much! Now I understand the structure instantly.

The pair as posted is correct for cross-eye viewing. That is the usual way to post because it can be viewed without aids by people who have developed the knack.

If I simply launch StereoPhoto Maker, drag the image onto the SPM window, and press the "Swap Left/Right" button, then the pair becomes correct for parallel viewing with the aid of a viewer device such as described HERE.

With the viewer, no special eye exercises are required. All the training you need, you have already gotten just by walking around looking at the world. There will be a few seconds of learning about how to point the viewer and turn the knob to align the images.

For projected presentation, such as in a conference talk, it works well to make several slides at different angles and just scroll back and forth through them using Powerpoint's control keys. This allows you to control the speed of rocking to match audience reaction, and to pause the motion at any time to point out specific features.

--Rik

NikonUser · Post by **NikonUser** » Sat Mar 23, 2013 4:49 pm

Image seems a little too dark.
Compare with this genital capsule of a male Caricea alma
http://www.photomacrography.net/forum/v ... highlight=

viktor j nilsson · Post by **viktor j nilsson** » Sat Mar 23, 2013 5:48 pm

Thanks both!

NikonUser, I agree now that I see your photo that my image is a bit dark. It is strange how hard it was for me to see that without you pointing it out. Before that, I was actually really happy with my exposure! I tested several different combinations of shutter speeds and flash settings, but was really terrified that I would blow out the highlights, so I took a few step back. Which felt comfortable at the time, but I guess I stepped back too much. I will try to push the exposure a bit higher next time.

Your photo of the Caricea genitalia definately has a lot more oomph too it!

rjlittlefield · Post by **rjlittlefield** » Sat Mar 23, 2013 7:36 pm

viktor j nilsson wrote:Your photo of the Caricea genitalia definately has a lot more oomph to it!

One other thing you can improve is to sharpen your images pretty heavily. When shooting through a microscope objective, some optical sharpness is always being lost to diffraction. (Your 10X NA 0.25 objective acts like a regular lens at f/20.) In Photoshop, try Unsharp Mask filtering with some settings that would ordinarily be ridiculous, like 130% at 1.5 pixels. You'll be surprised how much additional detail you can pull out before obvious sharpening artifacts start to appear.

--Rik