Modular high power inverted scope

[ldflan]

Ok then well a protist will also have more height when crawling along the bottom of a dish. We're talking fractions of a micron when using immersion objectives

Fractions of a micron are my point. I'm not a pondwater guy, so this is outside my area of direct knowledge. But you're not going to squeeze the protists down that much. Most are too small. And at least from what little I have seen, there are pretty profound behavioral differences between nematodes and protists that seem like they could make all the difference. There is also an entire order of magnitude difference between the size of the very smallest nematode and the average protist, so the motion effect you probably get at high powers with a subject 10x larger is probably profound. Again that's just my guess, as I don't do much pondwater gazing. To give the idea a chance of working, I think I would certainly want to slow everything down as best I could with cold or a thickening agent.

Also, zzffnn, you may want to consider using a layer of mineral oil over your sample to reduce evaporation.

[zzffnn]

With respect to immersion. Have you considered mixing glycerin with water to get some viscosity?

Thank you, apochronaut. Did you mean mixing in glycerin at objective side of immersion? I am guessing that RI change may be too much for a NA 1.1 water objective to perform well? It depends on how much I mix in, I guess.

At condenser side of immersion, if I mix glycerin into pond water, wouldn’t protists be affected by its osmolarity?

[ldflan]

What about the stage micrometer controls? I would guess that trying to chase down live protists at 1000x is going to require great speed and precision in moving the sample. A lot of inverted scopes are just a blank table with no X-Y controls at all. That's the way most of the Nikon SEs are, though I have seen pictures of a neat-looking "joystick" control for the stage on some of them...

[ldflan]

Methyl cellulose is the usual thickening agent for protists, from what I have read. I understand it's birefringent, though.

[zzffnn]

Stage micrometer control is indeed a major problem. My hands are not smooth enough at 400x or above; I have tested them before.

I have made some methyl cellulose before but rarely use it, it does change protists’ natural behavior and is pretty messy to work with, in my hands.

[apochronaut]

With respect to immersion. Have you considered mixing glycerin with water to get some viscosity?

Thank you, apochronaut. Did you mean mixing in glycerin at objective side of immersion? I am guessing that RI change may be too much for a NA 1.1 water objective to perform well? It depends on how much I mix in, I guess.

At condenser side of immersion, if I mix glycerin into pond water, wouldn’t protists be affected by its osmolarity?

I wasn't considering an immersion condenser. I would think that you would normally be stuck with one of .95 or lower but now that you mention it, what would be the problems associated with using a dipping condenser, configured for use in water dipping?

Water/Glycerin. I was thinking in terms of having a correction collar. Might to some degree overcome gravity.
I believe glycerin can be used to slow down cilia and movement but it eventually, probably ends up mortifying the little things.

[apochronaut]

Stage micrometer control is indeed a major problem. My hands are not smooth enough at 400x or above; I have tested them before.

I have made some methyl cellulose before but rarely use it, it does change protists’ natural behavior and is pretty messy to work with, in my hands.

One way that works o.k.but you have to have one of the older above stage carriers with separate X and Y controls, is to remove the rack for the Y axis and lubricate it's slide with a very smooth slik lubricant with less tack than damping grease. i have used a moly grease emulsified with graphite on an old B & L research stand. The X is adjusted tight and smooth with damping grease. You turn the control for the X and use that knob as a handle to either push or pull the Y at the same time. With the right greasing and a dead true and tight guide it is much better than trying to manipulate two controls at once.
You might be able to do it with a dropdown coaxial control but I don't think it would be as easy : maybe once you got used to it.
I had to mess around with grease mixtures a bit to get the feel right but eventually got decent functioning.

I also cleaned up a Y axis rack and pinion on an older Spencer and lubed it with heavy oil. That worked about as well but the spinning knob drove me nuts and I got worried about wearing it out if I intended ever to return it to it's former self.

[PeteM]

With respect to stage controls - the early Nikons and several other models had sliding stages that could be either OEM or adapted to your inverted microscope. The old black Nikon version is very good.

These work wonderfully for chasing protists while, say, making a video. The movements are viscously damped -- you just grab the edges of the stage to follow the action. The microscopy equivalent of a fluid head tripod for making films.

Phil's approach, above, essentially turns a regular mechanical stage into somewhat a version of this.

There are also numerous top-mounted x-y controls that can be adapted, but most of the better ones I've seen want a fairly large stage to mount to. There are always the cheap Japanese, then Chinese, versions that mount with a thumbscrew with two pins either side of it. Not particularly smooth or ergonomic IMO.

[ldflan]

What about the stage micrometer controls? I would guess that trying to chase down live protists at 1000x is going to require great speed and precision in moving the sample. A lot of inverted scopes are just a blank table with no X-Y controls at all. That's the way most of the Nikon SEs are, though I have seen pictures of a neat-looking "joystick" control for the stage on some of them...

I took a look at the parts stand I have. The stage is square and has the same micrometer controls as the Nikon S, which are really quite good. Unfortunately, the fine focus gear is (somewhat predictably for these old Nikons) cracked, which is why it was in the basement...

[Phill Brown]

I would plan ahead for not popping the bottom out of a DIY container as there is no fine on the condenser.
I have an inverted scope, I'm not understanding the plan here.
Yes gravity pulls everything to the bottom but that means any debris will be between the objective and the specimen.
So clean water is essential anyway.
By the time the sample is clean and clear enough a compound is likely a better setup.
Definitely finding and isolating is good with inverted.
My inverted doesn't work with polarised analyser under the head, something birefringent in the prism/Camera port.
Good luck with the project.

[ldflan]

I would plan ahead for not popping the bottom out of a DIY container as there is no fine on the condenser.
I have an inverted scope, I'm not understanding the plan here.
Yes gravity pulls everything to the bottom but that means any debris will be between the objective and the specimen.
So clean water is essential anyway.
By the time the sample is clean and clear enough a compound is likely a better setup.
Definitely finding and isolating is good with inverted.
My inverted doesn't work with polarised analyser under the head, something birefringent in the prism/Camera port.
Good luck with the project.

All excellent points.

The inverted scope is great for raking through stuff and at many other tasks, but for the contemplated task I am kind of skeptical.

The old Nikon has a rack and pinion adjustment for the condenser in addition to being able to move it up and down the rod by hand, but it's not a fine control. It would surely be much safer to get a fairly high NA LWD condenser if you can, given that your sample is resting on a coverslip...

[ldflan]

Well, I am going to be interested to see what comes of this effort! I suspect it'll be as though a sports car suspension and steering were installed on a pickup truck. The resulting vehicle might run, and maybe even run well in some circumstances, but it probably won't really be much good as a sports car or as a pickup truck, per se...

[zzffnn]

I am building a sporty SUV (indeed a compromise in between)

Likewise goes for how clean the sample reservoir would be: I would clean it up a bit and remove all big pieces, but will keep it messy and dirty enough for the biology to be natural, yet hopefully clean enough for nice views.

If it doesn’t work, I can alway fall back and make it into a standard typical messy pickup truck.

For example, by keeping the pond reservoir at no more than 1-1.2mm deep, if condenser immersion is too shaky, I can simply lift the condenser top lens above water and get around NA 0.8, in which case is still better than the standard NA 0.5 from LWD inverted condensers. I don’t think I need to manipulate the sample that often, so I am happy to trade LWD condenser for a higher NA short WD one.

[apochronaut]

SUVs and pickup trucks use a lot of gas.

[ldflan]

SUVs and pickup trucks use a lot of gas.

Sports cars have no clearance.

[apochronaut]

Yea , terrible in snow. I never used one but I see them disabled quite frequently. You probably don't have that in "Morna Moruna"

[ldflan]

Yea , terrible in snow. I never used one but I see them disabled quite frequently. You probably don't have that in "Morna Moruna"

A snowy home, is it? I would have pegged yours for a place with a lot of hot air and wind. Funny, that.

[apochronaut]

Yes, terribly windy at times. Lots of blowing from afar, sometimes even blown steam comes this way..
Now . Fan is looking for some expert advice and you seem to have positioned your posterior to provide that. He might like to see some high resolution photos taken through a well appointed and set up inverted microscope, because that is his goal. I don't have any because it is a speculative possibility in my opinion only but I have some theories regarding how he can get there or at least partially. . You are the expert. Have you got any high resolution photos taken through an inverted microscope for him to assess? I believe the goal is to assist him, or have I got that wrong in your case.

[zzffnn]

Thank you, gents. Thank you for thinking of me, Apochronaut: Idflan did provide me a photo and descriptions of his available scope via private message and very generously offered me many parts; which helped me made decision to go with Nikon MS inverted. I am perfectly chilled if we want to poke some fun here in the thread. It is just a hobby…………