A Choanoflagellate and Lacrymaria

Images made through a microscope. All subject types.

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dy5
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A Choanoflagellate and Lacrymaria

Post by dy5 »

A couple of photos of creatures living in my neighbor's backyard pond.

The first is a group of choanoflagellates, possibly Salpingoeca sp., sitting on a spherical algae, Golenkinia sp. (I think). A quick bit of research turned up that the choanoflagellates share a common ancestor with all multi-cellular creatures making them well-studied by evolutionary biologists. If anyone's interested, there's basic information here: https://www.cell.com/current-biology/fu ... 05)00142-9 The creatures are very common, but I hadn't noticed them until they started showing up sitting on the algae spheres. Is it weird that I think they are really cute?
Choanoflagellates 3-1.jpg
The second image is from my first encounter with Lacrymaria. There are fascinating videos of this creature, including the award-winning video by Charles Krebs. However, they are even more spectacular and bizarre in person. Given the animal's thrashing around, it was sheer luck to get a frame with the mouth and almost all of the neck in focus.
Lacrimaria copy 3.jpg
Both photos taken with DIC and high-speed flash.

dy5
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Re: A Choanoflagellate and Lacrymaria

Post by dy5 »

Adding a photo to show the length of the choanaflagellate flagellum (hence the odd orientation). Also this one had just ingested its lunch.
Attachments
Choanoflagellate long flag.jpg

rjlittlefield
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Re: A Choanoflagellate and Lacrymaria

Post by rjlittlefield »

These are lovely!

--Rik

dy5
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Re: A Choanoflagellate and Lacrymaria

Post by dy5 »

Thanks!

Cheers, David

Gopal42986
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Re: A Choanoflagellate and Lacrymaria

Post by Gopal42986 »

Amazing....

tjlmicro
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Re: A Choanoflagellate and Lacrymaria

Post by tjlmicro »

These are really great shots. Could you give a little information on the equipment used, plesae (microscope, objective, flash or not)? Thanks, Tim

dy5
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Re: A Choanoflagellate and Lacrymaria

Post by dy5 »

Tim -

Sure, but with two exceptions, there's nothing exotic: Nikon scope with plan apo objectives and DIC, and a Nikon D850 camera. The camera is tethered to a Mac M1 mini that displays the image on a monitor. The control software is Capture One.

Exception 1 - based on some discussions here several years ago, I put together a high-speed flash system for the microscope. A beam-spitter allows use of both the flash (a Profoto D2) and the normal lamp. You lose a bit over an f-stop of light, but that hasn't been a problem. Also no problem setting up Köhler illumination. With the D2 intensity at my usual setting 6 (out of 10), I measured a half-amplitude flash duration of 60µs = approximately 1/16,600. That eliminates any vibration concerns and is plenty fast enough to freeze cilia and flagella. A downside of the short flash durations is the need for higher ISO settings with the accompanying increase of noise in the images.

Exception 2 - when trying to photograph something moving quickly, I use a foot pedal to trigger the exposure. It is a huge help. The hardware is nothing fancy (for instance https://www.amazon.com/gp/product/B0755 ... UTF8&psc=1), and it's easy to set up. Something like the Lacrymaria photo would have been much harder to get without the foot pedal.

Ultimately, however, the most important determinants of success for me are practice, patience, and luck.

Cheers, David

tjlmicro
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Re: A Choanoflagellate and Lacrymaria

Post by tjlmicro »

Thanks for that useful information, David. Very helpful. I need to look into a foot pedal for the exposure for my Canon 1300D camera. I'm sure it would make my photomicrography life easier!

tjlmicro
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Re: A Choanoflagellate and Lacrymaria

Post by tjlmicro »

David, I have another question. I am not familiar with how a foot pedal is hooked up. I have my camera directly connected to my computer so that I can use my computer screen to focus and take the pictures. Is the foot pedal connected to the computer and essentially acts like a mouse which I use to take the exposure? Thanks for any info.

Tim

dy5
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Re: A Choanoflagellate and Lacrymaria

Post by dy5 »

Yes, that's right. I this context, it's simply providing a mouse click. Or you could have it do a keyboard command. There is configuration software to assign functions to the pedals. If you use a Mac, the configuration is clunky to set up, but you only have to do it once. A single pedal model would be fine for just triggering an exposure.

viktor j nilsson
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Re: A Choanoflagellate and Lacrymaria

Post by viktor j nilsson »

Awesome images.
Have you shown your beamsplitter setup somewhere? I'd be interested to see it. That Profoto D2 looks like a beast for this purpose, but I'm struggling to see how you'd adapt it.

dy5
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Re: A Choanoflagellate and Lacrymaria

Post by dy5 »

Viktor -

Yes, the D2 is a beast. Almost seven pounds and awkwardly big. However, the high light output, short flash durations, and several other features make it a good choice for the job.

The attached photos will give you an idea of the setup, which was inspired by the discussion here http://www.photomacrography.net/forum/v ... 25&t=31359, particularly the post by nathanm at the bottom of the second page.

Beyond the flash unit and the heavy-duty supports necessary for it (2nd photo), there are two major components:

1. The beam-splitter assembly utilizes Thorlabs 60mm cage components. The stock lamp has a condenser lens, and there is a second lens plus diffuser in the base of the microscope. I played with lenses in the flash's light path. Ultimately, the system worked well without those lenses.

2. The D2 is coupled to the beam-splitter assembly with a metal cone that was part of a Nikon photomicrography setup from olden times. I tried various snoots, but this worked best. The cone is lined with reflective foil. The flash tube of the D2 is circular, so the direct+reflected light at the bottom of the cone is reasonably uniform.

I can’t see any difference illumination-wise between photos taken with the two light sources.

Cheers, David
Backroom lab A cropped copy.jpg
D2 setup C copy.jpg
D2 set-up B copy.jpg
D2 set-up A copy.jpg

Pau
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Re: A Choanoflagellate and Lacrymaria

Post by Pau »

Excellent setup, Thanks for posting it. I've linked it at the FAQ:How (and why) to use electronic flash at the microscope?
https://www.photomacrography.net/forum/ ... 63#p161563
Pau

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Re: A Choanoflagellate and Lacrymaria

Post by Pau »

...The stock lamp has a condenser lens, and there is a second lens plus diffuser in the base of the microscope. I played with lenses in the flash's light path. Ultimately, the system worked well without those lenses.
Are you still able to focus the lamp filament at the rear focal plane after Kölher?
I'm asking it due to the increased distance of the light path.
Pau

dy5
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Re: A Choanoflagellate and Lacrymaria

Post by dy5 »

Good point, Pau. I worried about that and also the unequal length of the light paths for the two sources. It doesn't seem that my system should work given those distances, but, in fact, it does. My knowledge of the detailed optics is rudimentary or less, so I can't explain why.

The 80i uses what Nikon calls 'fly eye' illumination. The normal lamp is 'prefocused,' so there is no control of that available. When I look at the back focal plane of the objective, I see the facets of the fly eye in focus when the condenser is adjusted properly. I've attached photos of the internal light path and a diagram. Maybe that can help someone figure out what is going on optically.

In any event, I do get uniform, flare-free illumination.

Cheers, David

Added: I just noticed that the two interior photos are not quite the same. One might be a 90i instead of an 80i. The photos came from the installation manual for an LED conversion (looks nice, but seems too expensive = $675).
80i fly eye light path copy.jpg
80i interior copy.jpg
80i internal light path copy.jpg

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