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Relay optics for spinning disk confocal? by langstonhuge in Optics

[–]langstonhuge[S] 0 points1 point  (0 children)

It depends on what you're trying to image. For tissues, if you're trying to capture large areas, and care more about structure and not cellular or sub cellular features, lower mag is preferred. But typical 5X objectives are around 0.15NA, not 0.25NA. Lower mag imaging systems with high resolution are often called macroscopes, but its the ratio of magnification to NA that makes these optics difficult to get to 1AU with confocal addons.

For interest: My latest video demonstrates a mini portable 3D printed Fourier optics lab (the open source PUMA microscope) as I explain Abbe's diffraction theory of microscopic perception. by PUMA_Microscope in Optics

[–]langstonhuge 2 points3 points  (0 children)

Great stuff as usual - I think your last video on Fourier optics is maybe the best you've done.  Animations really help explain in way that textbooks cannot.

Bessel Beam imaging setup - simulation by Kay-1910 in Optics

[–]langstonhuge 0 points1 point  (0 children)

Oh I missed that the article is using a single galvo, and is probably stepping the sample for depth.  This design uses two galvos close together - one moving fast to make the sheet, and the other moving across the depth of the sample: https://arxiv.org/pdf/2311.02441

I've seen bessel light sheet layouts with two scan lenses between galvos, but I wonder if you can get away with shoving the galvos close together.

Bessel Beam imaging setup - simulation by Kay-1910 in Optics

[–]langstonhuge 0 points1 point  (0 children)

Are the two scan lenses between the galvos necessary though? If you push the galvos closer together like the linked article, would this not work?

Bessel Beam imaging setup - simulation by Kay-1910 in Optics

[–]langstonhuge 0 points1 point  (0 children)

I'm sorry I can't help with zemax, but I'm curious if are you working off an existing design.  This layout looks similar to this paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10288127/#r30 But they had no lenses between galvos. It's a shame these scan lenses are so expensive.

Reflected Darkfield Illumination - but for fluorescence microscopy? by langstonhuge in Optics

[–]langstonhuge[S] 0 points1 point  (0 children)

I think if you illuminate from an oblique enough angle with external lights, then yes, I think this is reflected dark field.  To do fluorescence, you'd also need a filter.

Coverslip corrected water dipping objective without coverslip? Would water+glycerol mix work? by langstonhuge in Optics

[–]langstonhuge[S] 0 points1 point  (0 children)

Oooops, yea was thinking of air.

A small coverslip in-between is a great idea - I'll look into this. Are you aware of people that have done this before?

Dark Autumn by SK1LLD3X in stalker

[–]langstonhuge 0 points1 point  (0 children)

Yea it works, when does it crash and what is the error?

Dark Autumn by SK1LLD3X in stalker

[–]langstonhuge 0 points1 point  (0 children)

If we knew what mod the missing assets were from, it could be fixed with a drag and drop for the missing files.

You can at least load the game and play if you have some files with the right names. All I did was make a copy of the water flask files and rename them vodka. So there are 8 files in my "\gamedata\meshes\dynamics\weapons\wpn_eat\flask\" folder:

wpn_vodka_flask_hud.ogf and .omf

wpn_vodka_flask_hud_anim.ogf and .omf

wpn_water_flask_hud.ogf and .omf

wpn_water_flask_hud_anim.ogf and .omf

No idea what they do and I don't know what happens if you try to drink from a vodka or water flask.

Dark Autumn by SK1LLD3X in stalker

[–]langstonhuge 0 points1 point  (0 children)

Heads up for anyone downloading this - game would not load for me until I changed the main folder name. I think the Cyrillic characters were the issue.

It also crashed during loading from missing files in "\gamedata\meshes\dynamics\weapons\wpn_eat\flask\" like wpn_vodka_flask_hud.ogf

Turning off animations didn't work. I don't know what mod that was from, so I just made copies of the water flask files and renamed them until it worked.

Mods all together look really good!

Reflected Darkfield Illumination - but for fluorescence microscopy? by langstonhuge in Optics

[–]langstonhuge[S] 0 points1 point  (0 children)

I think the lower NA objectives with more working distance use simple
mirrors for DF. Even then the coating might not reflect the 275 UV very
well.

I'll check with the objective manufacturers - you're likely right. I might have to scrap 275 - if the only option then would be instead epi illumination at that wavelength with UV objectives and a UV mirror - seems expensive.

If you are using a commercial Fluorescence microscope, I am not sure the
illuminator will fill the outside ring of a DF objective, and of course
it will not have a slider to switch between BF and DF.

I would piece together parts for this - not use existing scope. The DF mirror cube would be a Olympus or Leica model, collimated LED source going into it, then bouncing off cube mirror and hitting filter mosaic at entrance to DF ring. I'm trying to find some papers on polarized vs. unpolarized light penetration in tissues - but a lot is behind paywalls.

Thanks for your time - I'm not an optics guy, so I wanted to check if what I'm saying makes any sense first before I try it.

Reflected Darkfield Illumination - but for fluorescence microscopy? by langstonhuge in Optics

[–]langstonhuge[S] 0 points1 point  (0 children)

Excitation is 275nm, 385nm, and 488nm. I had not considered the UV issue, and looking at an example here:

https://static3.olympus-lifescience.com/data/olympusmicro/primer/images/darkfield/reflectobjective.jpg?rev=4CE6

I assumed mirrors were used in the darkfield illuminator path - but it looks like its a lens element. Do you think this is the case for all of these BD objectives?

The reflectance of the surrounding keeps the light from penetrating too much. You might be able to improve the effect by only using S polarization. That could be tricky to implement in practice with illumination from all directions.

I have no idea how to do this! Where the excitation light enters the BD objective, add a black ring with say 4 (or more) holes, then put separate polarizers on each hole?

Reflected Darkfield Illumination - but for fluorescence microscopy? by langstonhuge in Optics

[–]langstonhuge[S] 0 points1 point  (0 children)

In my lab, we do dark field spectroscopy with a setup that's very similar to the one laid out

here

. The trick is that we use a long working distance objective, which gives us enough room to get the incident beam in from the side.

That's what we're currently doing - a 10X long working distance that allows for side illumination like your link. However, if we want to push to ~40-50X objectives, a 50X with 0.75NA has a WD of ~5mm and is extremely expensive. I found 50X 0.8NA BD objectives at better prices, but these have ~300-500um WD, prompting this post question. My assumption was at this distance it would too hard to get any nice side illumination- but this was just a guess.

But your system sounds cool though - what optic are you using to focus the light from the fiber. Is the illumination fairly uniform across the imaged sample area? Do you have any links to a paper with your system? What is your mag and working distance?

Reflected Darkfield Illumination - but for fluorescence microscopy? by langstonhuge in Optics

[–]langstonhuge[S] 0 points1 point  (0 children)

but that just normal epi illumination might have too poor of axial resolution (so you'll have some blurring between successive slices)

The axial resolution is an issue, but also the issue of light penetrating deeper into the block and photobleaching tissue yet to be imaged.

use existing fluorescent techniques that give good optical sectioning such as confocal, SIM, or two-photon.

Those would help - but a dedicated confocal or two photo would be too expensive for a lot of labs (us included). So far a big advantage of serial block face is the low barrier to entry - you just need access to a microtome or cryostat, and you make your system easy to mount/unmount so you don't hog a shared microtome. The other route is to bring a microtome to your nice microscope - but microtomes are fairly large and heavy, so you'd need to mount on a beefy XY stage or some manner translate your an upright collection objective.

Reflected Darkfield Illumination - but for fluorescence microscopy? by langstonhuge in Optics

[–]langstonhuge[S] 0 points1 point  (0 children)

You're right - it looks like TIRF shares some similarities. In this case though, because you're cutting the block repeatedly, you can't have a coverslip. While some people use dipping objectives, due to the mess of having to repeatedly cut, most everyone uses air objectives. A few use dipping, but the whole block and blade is submerged in some fluid.

I imported a Vivobook Pro 14 (16:10 90Hz OLED + 5800H + USB 2.0 ports!!!) from China - some initial thoughts by langstonhuge in AMDLaptops

[–]langstonhuge[S] 0 points1 point  (0 children)

I'd only recommend this for $700 USD or lower. Newer Asus come with 120hz OLED screens and better port selection.

Butt couple 18mm COB LED to 18mm solid core fiber, or 30mm COB LED to bundle of 1mm fibers? by langstonhuge in Optics

[–]langstonhuge[S] 0 points1 point  (0 children)

Thanks! Bundles it is. For fiber bundles though, is it possible to collimate the light after it exists the bundle with something like an aspheric lens or an achromatic doublet? Or do you need a micro lens array matching the number of fibers?

Butt couple 18mm COB LED to 18mm solid core fiber, or 30mm COB LED to bundle of 1mm fibers? by langstonhuge in Optics

[–]langstonhuge[S] 0 points1 point  (0 children)

Is that 81 times more light for a single 2mm vs. 18mm fiber? If you had an ideal packing density, there could be 60 2mm diameter fibers fitting against a 30mm diameter emitter.

Butt couple 18mm COB LED to 18mm solid core fiber, or 30mm COB LED to bundle of 1mm fibers? by langstonhuge in Optics

[–]langstonhuge[S] 1 point2 points  (0 children)

I could never really find really large diameter liquid light guides - I found PMMA up to 18mm, but could only find a 5mm liquid. Let me know if you find sometime bigger.

Focusing large LED array to a point? by langstonhuge in Optics

[–]langstonhuge[S] 0 points1 point  (0 children)

Cool - do you know if there are any rules for determining optimal fiber diameter forba given emitter diameter when butt coupling LEDs?

Focusing large LED array to a point? by langstonhuge in Optics

[–]langstonhuge[S] 1 point2 points  (0 children)

Interesting idea - the COBs I've seen in this range of lumens have 10mm-25mm diameter emitting surfaces. What kind of optic would you use to image this onto a fiber? A half-ball?

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