My First Closterium!

[josmann]

Been microbe hunting for a couple weeks now - found my first Closterium in a freshly collected sample of local creek water. Feels like a rite of passage getting to see those crystals rattling around!

I'm using a Journey to the Microcosmos kickstarter microscope (Modified with a 7W LED source), a Varimag II adapter, and a Sony A7III to get my shots. The video was color corrected and edited in Davinci Resolve (which I highly recommend as it is free, tremendously powerful, and easy to learn).

https://www.youtube.com/watch?v=eB-WgtvgKnc

The objective here was your bargain special 40x achromat that probably comes on every low end made-in-China scope today - do you guys think I would get sharper images if I upgraded to something more sophisticated? Or is my illumination source (diffuser and convex lens) or something else limiting me? Appreciate any feedback! I'm definitely interested in pushing the limits of amateur microscopy!

[smollerthings]

Great video.
How did you get that nice blue background? Rheinberg filter? How did you make it?

[Javier]

Good for you! I love those tiny dancing crystals.

[josmann]

Great video.
How did you get that nice blue background? Rheinberg filter? How did you make it?

Thanks! Yeah it’s from a stack of rheinbergs - green/blue/purple which mixes to a nice aquarium blue. My microscope came with a set of them similar to what you can get on eBay - they’re basically just cutouts from cheap photographic gel sets. The ones I used here are ~1cm dots glued to transparent plastic. That allows you to get oblique white illumination with a different background color. Truth be told, at 40x the colors do wash out a fair bit so I’ve used some saturation here to restore what they look like at, say, 100x.

Here’s another video at 100x using a similar rheinberg stack and those colors aren’t enhanced much at all (in fact it all looks even better through the eyepieces). https://youtu.be/21ohtWE-fK8

[smollerthings]

Very nice. Love those blue background. But from 40x above, they are difficult to obtain, same thing for high magnification darkfield. I need to look deeper into that, oil condensers etc...

[josmann]

Very nice. Love those blue background. But from 40x above, they are difficult to obtain, same thing for high magnification darkfield. I need to look deeper into that, oil condensers etc...

When I set up my rheinberg illumination, I usually make the center dark and the outer ring clear or lightly tinted. It occurred to me that this is basically just a less severe dark field setup and I've definitely had more success at achieving good quality images this way than just straight up dark field filters. I'm curious as to what might happen if I created a rheinberg with something like a gaussian profile where I don't create any sharp edges and just roll off from the center dark color to clear. I wonder if I could try modelling this in zemax or something to get some predictive results - would really help my workflow out knowing what to expect going in.

By the way, one thing I do which is super useful is, when I find an illumination setup I like, I pull the eyepieces off and stare into the scope. That projects an image of what the illumination field looks like so I can replicate it later by moving the filters around until I get the same pattern. Usually the good ones are when I have the center dot slightly offset and get a nice crescent of pure light at the edge of the iris aperture. Especially at high magnifications, the difference between striking 3D contrast and muddy garbage can be like .5mm of movement on the filter

[smollerthings]

When I set up my rheinberg illumination, I usually make the center dark and the outer ring clear or lightly tinted

Can you take a pic of that?

I'm curious as to what might happen if I created a rheinberg with something like a gaussian profile where I don't create any sharp edges and just roll off from the center dark color to clear. I wonder if I could try modelling this in zemax or something to get some predictive results - would really help my workflow out knowing what to expect going in.

That would be super interesting. I was wondering, and I don't know if this makes sense, how all this relates to the transfer function of the filter. A pinhole is supposed to be a low pass filter but when I close the iris, there is some "edge detection" happening. In a previous post, I was wondering what would happen if you had a hole, a black annulus, then open transparency again. That should do something to the spectral response?

[josmann]

Can you take a pic of that?

Yeah I'll have it up soon.

That would be super interesting. I was wondering, and I don't know if this makes sense, how all this relates to the transfer function of the filter. A pinhole is supposed to be a low pass filter but when I close the iris, there is some "edge detection" happening. In a previous post, I was wondering what would happen if you had a hole, a black annulus, then open transparency again. That should do something to the spectral response?

Okay this one I know. So you're right that a pinhole is a lowpass spatial filter, but that only applies if you're focusing imaging data through the pinhole (in a Fourier plane). What you're seeing when you stop down the iris is the effect of coherent light. You're creating a point source at the focus of the condenser which results in a highly collimated beam of light exiting the condenser. You can check this by holding a little tissue paper about the condenser and moving it up and down at different iris sizes. When light emerges from a point source, it will have a high degree of spatial coherence. You can see this sometimes when the sun reflects brightly off a car windshield and travels a long way into a building (think of this the next time you catch some harsh sun glare off a parked car). The reflected light casts very hard shadows and if you hold a hair up and look at its shadow on the wall, you'll be able to see interference fringing around the main shadow. Textured surfaces catching such light will often have a strangely chromatic graininess as well. The "edge detection" you speak of is an interference effect. Coherent light creates very high contrast outlines, but you pay for that with lots of ringing artifacts which often makes non/semi-coherent light the better choice for quality imaging.

Also, going back to the main topic - I found another Closterium and did a super rough 12 image manual focus stack thinking I'd try out Picolay for the first time. I had precisely 0 confidence that I'd achieve anything that looked like a decent image. Glad to see I was proven wrong!

[Rossf]

Nice vids josmann-I’ve got tadpoles in a big bowel with water plants and found these a couple of weeks ago-and noticed the moving crystals as well-I’ve been more interested in micro critters but algae are starting to really catch my eye more now-ditto on Davinci Resolve-been a using it for a while now-I would say it’s pretty easy to use as long as you keep away from the Fusion page-node based compositing does my head in!
Keep it up
Ross

[josmann]

Nice vids josmann-I’ve got tadpoles in a big bowel with water plants and found these a couple of weeks ago-and noticed the moving crystals as well-I’ve been more interested in micro critters but algae are starting to really catch my eye more now-ditto on Davinci Resolve-been a using it for a while now-I would say it’s pretty easy to use as long as you keep away from the Fusion page-node based compositing does my head in!
Keep it up
Ross

Yeah fusion is one of those things where you learn a little about it, think "hey this is stupid easy" and then realize that you actually don't know squat .

Here's another video I put together today pushing my Varimag II, 100x objective, and camera to its limits: https://youtu.be/xGJz9KU7DiM

[josmann]

Can you take a pic of that?

Here's an album with the filters I'm using, what they look like stacked, and the image they can create: https://ibb.co/album/RzkdY3

[Rossf]

josmann that last video was really rad!-like a little space city with flying vehicles racing all over-was that time lapse? Also what were the really long strands wriggling around the algae? I need to put some effort into 1000x oil immersion again! Lazy me…

[smollerthings]

Okay this one I know. So you're right that a pinhole is a lowpass spatial filter, but that only applies if you're focusing imaging data through the pinhole (in a Fourier plane). What you're seeing when you stop down the iris is the effect of coherent light. You're creating a point source at the focus of the condenser which results in a highly collimated beam of light exiting the condenser. You can check this by holding a little tissue paper about the condenser and moving it up and down at different iris sizes. When light emerges from a point source, it will have a high degree of spatial coherence. You can see this sometimes when the sun reflects brightly off a car windshield and travels a long way into a building (think of this the next time you catch some harsh sun glare off a parked car). The reflected light casts very hard shadows and if you hold a hair up and look at its shadow on the wall, you'll be able to see interference fringing around the main shadow. Textured surfaces catching such light will often have a strangely chromatic graininess as well. The "edge detection" you speak of is an interference effect. Coherent light creates very high contrast outlines, but you pay for that with lots of ringing artifacts which often makes non/semi-coherent light the better choice for quality imaging.

That is awesome. Now I can sleep at night. Reminds me of my Michelson days.
Nice pic and video. It is amazing what one can do with a relatively inexpensive microscope and elbow grease!

[smollerthings]

I'm using a Journey to the Microcosmos kickstarter microscope (Modified with a 7W LED source)

Wanted to ask, could you please share how you did it?

Also, what do you guys think those closterium's cristals' role?