Nuclearia and Mayorella ?

discomorphella · Post by **discomorphella** » Fri Dec 30, 2011 2:30 am

I am not sure if I have these two typed to the correct genus, but I am certain that some of you can correct my taxonomic errors....The putative Mayorellae were stuffed with colorful blue-green and green algae, in the process of being digested, which altered their colors. Both of these came from a local pond. I am still getting my D300 integrated with my Varioorthomat shutter and the BX60. There's an annoying hot spot which I have tracked down to the shutter module (all 5 that I have tried have it) but never-the-less I thought I'd post these while I am in the middle of eliminating it. Standard wet mount, BX-60 with UPlanApo 40/0.85 UIS objective, Variozoom eyepiece, 0.32X projection lens relaying to D300.

David

Mitch640 · Post by **Mitch640** » Fri Dec 30, 2011 7:42 am

Very nice images, especially the last two. I don't often see colors like that in my images.

discomorphella · Post by **discomorphella** » Fri Dec 30, 2011 11:42 am

Thanks Mitch,

I had not seen a collection of partially digested algal cells in an amoeba like this one either. I just realized that I have the wrong scale bar on the lower 2 images as well, I'll have to re-edit.

David

Charles Krebs · Post by **Charles Krebs** » Fri Dec 30, 2011 2:26 pm

I just realized that I have the wrong scale bar on the lower 2 images as well

Well it does look like it has a good appetite, but that would be an amazing growth spurt

Very mice images. DIC looks so much better when there are some natural color components to these otherwise colorless subjects.

Fill us in on the "hardware" used here just a little.

discomorphella · Post by **discomorphella** » Fri Dec 30, 2011 3:50 pm

Hi Charlie--

Ok, I edited in the correct scale bar on the "Mayorella". Although I would not put good odds on it being an actual Mayorella, its what I keyed it out to be, but I am a p-chemist, not a protistologist...
Hardware is a bit of a deviation from the old Orthoplan...The optical path for these shots is a BX-60, with a 40/0.85 UIS PlanApo objective and the standard transmitted DIC slider (for biological specimens, but not the ultra-thin one), a UCA mag changer, and then a Leitz Vario-orthomat 2 shutter/ lens system, followed my homebrew D300 adapter, which I had a picture of a while ago (see http://www.photomacrography.net/forum/v ... highlight=). This takes the vario-orthomat's 0.32X projection lens out of the old film holder and holds it at the correct distance from the CCD on the D300. It's working well, except for a REALLY annoying hot spot which is coming from the optics inside the shutter module (a simpler adapter which only has the zoom eyepiece and the 0.32X lens is free of it). The Vario-orthomat 2 has a complex series of beamsplitters and lenses to project the boundaries of the film into the user's FOV, and also to let you spot meter from within the actual film FOV. I suspect that the CCD surface is more reflective than the originally designed-for 35 mm film and this is causing some internal reflection that gets back to the camera. However, the optics are complex, and I like being able to use the spot meter, so rather than just ripping them all out and retaining only the shutter I am trying out various more complicated solutions. If/when I get it all done I will put drawings etc into the equipment forum. I usually just keep the camera in bulb mode (I dont need to use mirror up mode with the leitz vibration-free shutter) and adjust the vario-orthomat shutter time to dial in the exposure. It can do a reasonable job of calculating the exposure if you play with the "apparent" film speed.

David

Charles Krebs · Post by **Charles Krebs** » Fri Dec 30, 2011 6:29 pm

David,
I'll be interested to learn if you are able to overcome the "hotspot" issue. I've had this with one 0.5X adapter and could not defeat it. It is a strange thing... I made up a T-mount that had a clear filter within it to keep out dust. Thought I would have all sorts of flare/hotspot issues... but nothing! Yet some optics just don't play nicely with digital sensors in this regard.

Ferry · Post by **Ferry** » Sat Dec 31, 2011 12:39 pm

Hello David,

Very nice pictures! The first organism is indeed a Nuclearia species, possible N. moebiusi. The second organism is not a Mayorella, but a Thecamoeba species.

Ferry

curt0909 · Post by **curt0909** » Sat Dec 31, 2011 2:05 pm

Excellent photos. What is your equipment?

discomorphella · Post by **discomorphella** » Sat Dec 31, 2011 3:16 pm

Thanks all,

Ferry, thanks much for the ID. Could you briefly describe the characteristics you used to decide these were Thecamoebae? The more I learn about which characteristics are important the better I'll be with my keys....
The equipment is described in the thread above in detail, but its basically a stock Olympus BX-60 with the "standard" DIC lower and upper Nomarski prisms and a UplanApo 40X / 0.85 NA objective. This 40X has a spherical abberation correction collar on it, making it very useful for looking at creatures in wet mounts. The only modifications are to the camera, which actually started out its life as a Leitz Varioorthomat 2 35mm film apparatus (a Variable Zoom eyepiece, shutter/exposure meter module and a 0.32X projection lens and film back). I removed the 35 mm film back and used the 0.32X lens to relay to my D300. There's a picture in the link in the thread above.

Happy New Years and Good Hunting under your scopes to all....

David

curt0909 · Post by **curt0909** » Sat Dec 31, 2011 8:28 pm

Thanks, I don't know how I missed that post. That unique set up is giving you great results.

Ferry · Post by **Ferry** » Sun Jan 01, 2012 1:57 pm

Happy New Year to all of you!!!!

David, you can see the visual differences between Mayorella and Thecamoeba here: http://www.arcella.nl/mayorella and here: http://www.arcella.nl/thecamoeba.

Mayorella amoebae do have mammilliform subpseudopdia. Crystals or crystalline inclusions are usually present, often attached to a spherical body or paired.

Thecamoebae are usually oblong, with a pellicle-like layer. This 'skin' is rather stiff with wrinkles and striae. No crystals.

I have an Orthoplan and could buy a Variable Zoom Eyepiece on eBay two weeks ago. I don't have a 0.32X projection lens, but use an old Olympus 50 mm camera objective between the Eyepiece and my Canon 550D, which I changed for my Nikon D300 after reading a paper from Charles . It works excellent.

Ferry

Pau · Post by **Pau** » Sun Jan 01, 2012 3:27 pm

Ferry wrote:...I don't have a 0.32X projection lens, but use an old Olympus 50 mm camera objective between the Eyepiece and my Canon 550D, which I changed for my Nikon D300 after reading a paper from Charles . It works excellent.

I don't know how to calculate it, but I think it will be approx. the same: I use the Zeiss 0.25X T2 mount camera coupler an in fact it is just a 63mm lens focused at infinite.

discomorphella · Post by **discomorphella** » Sun Jan 01, 2012 3:51 pm

Hi Ferry--

Thanks for the links to the amoeba genera. After you corrected my ID I went back through my keys ("free Living Fresh Water Protozoa by Patterson, and "The Illustrated Guide To The Protozoa" from the Society of Protozoologists mostly) and found where I made my error. Many thanks. Did you get the Variozoom eyepiece? I find it very useful, although with the DX sensor on the D300 I tend to use the 5 and 6.3X magnification more than the higher (8,10, 12.5). Its also highly useful with a 0.1X lens and a cheap video camera for doing microscope demonstrations; you can zoom in/out while standing by the scope.

Pau, if there's any way to get the prescription data for your and Ferry's lenses I can try to ray trace it, at least crudely. Of course if its all working well you can figure out the actual magnification rather easily, so perhaps we don't need to bother. The only reason I used the 0.32X lens is that it was already there in the 35mm film back, and my reasoning was to try and replace the 35mm film back with my D300 back. I have found a very annoying hot spot, which happens on ALL the shutter modules I have tested, which I can attenuate greatly by dividing out a carefully taken background shot using ImageJ. A friend of mine (who is also a p-chemist with an orthoplan...) and I are now trying to eliminate the hot spot, which is almost certainly due to a reflection from the CCD getting back into the spot metering optics in the shutter module. I am about to make an optical isolator that will go between the 0.32X lens and the shutter. That will miscalibrate the exposure a bit, but that can be compensated for once the hot spot is vanquished.
By some good fortune, the variozoom / shutter / 0.32X / D300 combination is parfocal on my Orthoplan and the BX-60. Maybe this bit of good luck has to be paid for by having a hot spot...

David

Pau · Post by **Pau** » Mon Jan 02, 2012 4:53 am

discomorphella wrote: Pau, if there's any way to get the prescription data for your and Ferry's lenses I can try to ray trace it, at least crudely. Of course if its all working well you can figure out the actual magnification rather easily, so perhaps we don't need to bother. The only reason I used the 0.32X lens is that it was already there in the 35mm film back, and my reasoning was to try and replace the 35mm film back with my D300 back. I have found a very annoying hot spot, which happens on ALL the shutter modules I have tested, which I can attenuate greatly by dividing out a carefully taken background shot using ImageJ. A friend of mine (who is also a p-chemist with an orthoplan...) and I are now trying to eliminate the hot spot, which is almost certainly due to a reflection from the CCD getting back into the spot metering optics in the shutter module. I am about to make an optical isolator that will go between the 0.32X lens and the shutter. That will miscalibrate the exposure a bit, but that can be compensated for once the hot spot is vanquished.

David, the only data I have from Zeiss literature is 0.25X and 63mm. Used in the camera out of the microscope as taking lens it is a very poor one fix focussed to infinite (approx.). In fact its quality as relay lens is very good but not better than an Olympus OM 50mm 1.8 I use sometimes over the eyepiece. Its main advantage is that it provides an easy way to couple the camera and it works nicely with all my eyepieces (6.3X, 8X and 10X). I mostly use it with the 6.3X to have an adequate FOV with a Canon APSc camera.
The bright spot problem is usual when the rear lens element is almost flat, like with the otherwise excellent Tamron macro 90mm 2.5.
I'm a bit puzzled about why yo do use the Ortomat shutter module and photometer, in particular if the problem rely in its optics: the Canon EOS camera has a good photometer an with continuous light the EFSC function must be even quiter than a central shutter.

discomorphella · Post by **discomorphella** » Mon Jan 02, 2012 6:10 am

Hi Pau--

Since I have (mostly as a matter of personal history, I got my first Nikkormat when I was a youngster) all Nikon equipment, it was much less expensive to modify a Vario-orthomat 2 than to get a Cannon back. At least that was the situation when I started this project...I haven't seen a firmware modification for a D300 to mimic the Cannon EFSC "electronic shutter" yet, and the vario shutter lets me use the Nikon while in "live view" mode without having to use "mirror up" mode to eliminate the mirror shake. I do have to run the Nikon in "bulb" mode but that's not too inconvenient (the other alternative is to use flash for everything...).
The hot spot is not derived from the 0.32X lens, it appears to have a decent anti-reflection coating. In fact, I made a simpler adapter which joined the 0.32X lens, Nikon bayonet F mount and the variozoom eyepiece and there's no hot spot at all. The reflection appears to be coming from within the shutter/exposture module. We removed the entire spot / FOV optics from one shutter module, and it also has no hotspot. What I do like about the vario shutter module is that it will spot meter from within the Field Of View and calculate an exposture based on that spot. I can do that with the Nikon directly but only if I am looking into the camera's viewfinder, which is not convenient at all...so IF we can, we are trying to add an optical isolator (quarter wave plate and linear polarizer) or another antireflection coated window in the right place to suppress the reflection. I haven't found a way to use the camera's own spot metering remotely using Nikon Camera Control Pro 2 (while the camera is in maual mode too, there's no CPU-lens attached...). If anyone knows how to do that it would be very useful.

David