Questions about my new CX40 w. epifluorescence

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erikwetterskog
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Questions about my new CX40 w. epifluorescence

#1 Post by erikwetterskog » Thu Sep 29, 2022 3:57 pm

Hello forum,

This week I managed to buy my 3rd microscope, a CX40 in pristine condition w. 5 plans, phase-contrast and a fluorescence illuminator(!). The epi-fluorescence was really the reason why I pulled the trigger (and the price) since I have very similar option in terms of features (Nikon E400).

Testing the epi-fluorescence I managed to get everything working, and I am super-happy and over the moon with my new toy. The microscope was supplied with the manual for the scope itself, the vertical illuminator (CX-RFA or RFL, the manual says two different things). It came with a new Hg-burner, but no ND-filters and a single CX-DMG fluorescence cube. I'm really surprised regarding how difficult it is to find manuals and general info for microscopes of this vintage, given how common they are. I have a couple of questions, but I will limit myself to inquires about the fluoro-part here (I'll save some others for another post).

1. It seems impossible to find ND-filters for the illuminator on eBay or anywhere for that matter, they are named 20ND25-W and 20ND6-W in the manual. Basically a circular filter in a plastic frame (drop-in). I was thinking to perhaps buy a ND-filter of similar diameter and 3D-print a frame. Does anyone know the dimensions of the original part or at least the diameter of the filters in use? My eyes are really strained from yesterdays session (aligning the burner etc), so I really want to have the option to work with ND-filters in the future.

Thorlabs seem to have a good selection with some different diameters.

2. Is it possible to use this illuminator for brightfield observation? I found a "OLYMPUS U-MBF3 BRIGHTFIELD CUBE" (on eBay), which I guess is essentially a conventional half-reflecting mirror instead of a dichroic mirror. But I think the cube does not work with my basic illuminator, since it lacks the dovetail of the CX-DMG/DMB cubes. (Are U-cubes for the more advanced illuminator?). A dead link says should work with a 0.5% transmission ND filter in BF-mode.

I have a "dummy" cube in the other position, which is simply just a piece of metal blocking the light. As far as I can tell, the only two options mentioned in the manual are CX-DMG and CX-DMB. I was at least lucky to have one of them, as they seem difficult to find.

I would be interested to work with brightfield occasionally: imaging of semiconductors components, butterfly-wings and the like (probably also for focus stacking).

I always get fantastic help here, so I hope you will not let me down :).
Nikon S-Ke - Old goodie
Olympus CX40 - Fluorescence [Selling]
Olympus BX61 - Phase contrast, DIC, fluorescence [Work-in-progress]

Hobbyst46
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Re: Questions about my new CX40 w. epifluorescence

#2 Post by Hobbyst46 » Thu Sep 29, 2022 4:45 pm

Although I am not familiar with that specific illuminator and filters, here are my thoughts.

1. The Hg burner is not really appropriate for brightfield, except for very short sessions. The spectrum of the mercury lamp differs very much from natural daylight or halogen or LED white light. Moreover, it contains harmful UV. Furthermore, the life time of that lamp is relatively short.
For the BX microscope series, there exists a "T" adapter piece (from Olympus), that allows the simultaneous connection of two light sources for epi-illumination. A 45 degrees flip-out mirror switches between the two. One would be halogen and the other Hg (say). Cost might be high though.

2. To utilize the Hg lamp for BF, one would need to a cube with a dichroic mirror (cutoff 400nm or higher) and an emission filter (longpass, say >420nm). These need to be high quality to block all UV. I would add an excitation filter with similar features to the DM, as extra blocking (in series).
Personally, I consider this option as unsafe.

3. On the other hand, for fluorescence, I am not sure that the ND filters are so important. Fluorescence is very weak and intense excitation is wanted. There are two common problems with intense excitation that I know of: biological damage to the tissue and photo bleaching. The latter can be eliminated by an electromechanical shutter (or filter wheel) that lowers the duty cycle. Sometimes the shutter can be installed between the lamp and the optical train.
Anyway, it seems that a makeshift plastic or metal frame for the ND frame as in the 20ND25-W can be made.

4. A LED illuminator will avoid all of the above problems, but again, depends on budget.

erikwetterskog
Posts: 68
Joined: Mon Apr 08, 2019 9:18 pm

Re: Questions about my new CX40 w. epifluorescence

#3 Post by erikwetterskog » Thu Sep 29, 2022 5:18 pm

According to the manual, there is a UV filter in the lamphouse itself, so there should be no risk whatsoever in exposing yourself to UV. It is safe to pop up the hatch to insert ND-filters and the like. But I agree that the Hg-burner is perhaps that well suited for the task. I was excited when I found the "brightfield-cube" though, since I've always wanted to do regular epi-microscopy at home. Even with a regular light source, I would not be able to do that. I would still need a suitable "cube".
Nikon S-Ke - Old goodie
Olympus CX40 - Fluorescence [Selling]
Olympus BX61 - Phase contrast, DIC, fluorescence [Work-in-progress]

Hobbyst46
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Re: Questions about my new CX40 w. epifluorescence

#4 Post by Hobbyst46 » Thu Sep 29, 2022 5:33 pm

OK, that is good, your illuminator was configured for specific visible light excitation as shown in the spectra. So the safety issue has been eliminated.

Indeed, a cube is needed for BF, and a 50/50 beamsplitter installed in place of the DM.

PeteM
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Re: Questions about my new CX40 w. epifluorescence

#5 Post by PeteM » Thu Sep 29, 2022 7:09 pm

I'm pretty sure the Olympus BF and DF cubes were meant to be used with their halogen lamps and don't screen UV. As you've noted, they are also configured differently to avoid being used in fluorescence heads.

Personally, I would never trust a lamp-mounted UV filter as my sole eye protection from a mercury burner. First, they are subject to heat and can crack. Second, in an old scope who knows if they were replaced by a more common IR or even plain glass filter? Third, a barrier filter on the cube's exit to the eyepieces is spec'd and required by Olympus (far as I know).

Most labs these days don't view UV visually. Instead, viewing-composing-imaging are done through a camera and display. That would be a safer and generally affordable option for hobbyists as well.

erikwetterskog
Posts: 68
Joined: Mon Apr 08, 2019 9:18 pm

Re: Questions about my new CX40 w. epifluorescence

#6 Post by erikwetterskog » Thu Sep 29, 2022 9:29 pm

Good thoughts and points! I should have checked the instrument more carefully. Perhaps I will bring a spectrometer from work to pick up an emmission spectrum through the binoculars. I'm usually a safety guy, that's why I read the manual so carefully. But things do get lost and compromised, even if in this case get the feeling that the whole scope itself is probably put together like it was bought (except for the phase condenser which is of BH-2 vintage).

And I do have a camera to go with it, but no trinocular head so I have to shove it down front. Fluorescence microscopy through the eyepieces is spectacular though.
Nikon S-Ke - Old goodie
Olympus CX40 - Fluorescence [Selling]
Olympus BX61 - Phase contrast, DIC, fluorescence [Work-in-progress]

PeteM
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Re: Questions about my new CX40 w. epifluorescence

#7 Post by PeteM » Thu Sep 29, 2022 11:35 pm

Another option for your camera (avoiding the cost of a trinocular head) is the Olympus U-TLU tube lens. These sometimes show up used at a fraction of the cost of a CX/BX trinocular head. Simply remove the binocular head and swap it for a U-TLU with your camera and any needed adapters mounted.

It sounds like you'll have a great setup.

Sure Squintsalot
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Re: Questions about my new CX40 w. epifluorescence

#8 Post by Sure Squintsalot » Fri Sep 30, 2022 12:54 am

I'd thought that fluoresence microscopy was a little more complicated than getting a mercury lamp source and a filter cube.

Don't you need UV transparent objectives? Never mind the camera modifications that have to be made....

PeteM
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Re: Questions about my new CX40 w. epifluorescence

#9 Post by PeteM » Fri Sep 30, 2022 1:37 am

Fluorite glass is relatively transparent to UV and widely used (and widely available) for fluorescence microscopy.

In general, the specimen is excited at a UV wavelength specific to the fluorophor in use - with the fluorescence showing up higher in the visible range. So a normal camera can work, if it's sensitive enough to relatively low light levels.

Alexander
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Re: Questions about my new CX40 w. epifluorescence

#10 Post by Alexander » Fri Sep 30, 2022 6:40 am

In most cases a UV wavelength of about 360 nm is used for fluorescence microscopy. This wavelength passes any typ of glass without problems. The UV filtering problem hits shorter wavelength below 300 nm. Under 250 nm one needs very special glass often made from quartz crystals.

Since the objective is the condenser as well, the most important factor is its NA. In fluorescence microscopy immersion objectives with moderate magnification and high NA are widely used. 10/0.45, 25/0.75 and 40/1.30 are what I use most for fluorescence work.

As PeteM explained, emission wavelength is always higher than excitation wavelength. A typical UV fluorescence dye like DAPI has its absorption maximum at 360 nm and emits light at 460 nm which is in the blue range of visible light. The cell cores glow in a bright blue. No need to modify the camera.

viktor j nilsson
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Re: Questions about my new CX40 w. epifluorescence

#11 Post by viktor j nilsson » Fri Sep 30, 2022 7:07 am

Sure Squintsalot wrote:
Fri Sep 30, 2022 12:54 am
I'd thought that fluoresence microscopy was a little more complicated than getting a mercury lamp source and a filter cube.

Don't you need UV transparent objectives? Never mind the camera modifications that have to be made....
Epi fluorescence can actually be done in an even simpler way if you illuminate the sample from the side with a UV flashlight, then you only need a single filter after the objective to block off UV and let visible light through. Here's an example:

http://www.photomacrography.net/forum/v ... hp?t=33123
http://www.photomacrography.net/forum/v ... hp?t=33112

Requires objectives with a bit of free working distance, so not great for high magnification, high NA. But great images can be made at 10x and 20x with this technique, for sure.

Hobbyst46
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Re: Questions about my new CX40 w. epifluorescence

#12 Post by Hobbyst46 » Fri Sep 30, 2022 8:44 am

This old thread could be somewhat relevant
viewtopic.php?f=24&t=7104&p=62889&hilit=365nm#p62889
But I believe that in some other post (which of course I failed to find) there were transmission spectra of compare "fluorescence" vs "general purpose" objectives.

erikwetterskog
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Joined: Mon Apr 08, 2019 9:18 pm

Re: Questions about my new CX40 w. epifluorescence

#13 Post by erikwetterskog » Sun Oct 09, 2022 8:35 pm

Ok, so I thought I should give you guys the result of my findings.

I measured the UV-Vis spectrum from the lamp, both the "unfiltered" excitation-light, as well as the filtered light (green) that passed through the cube. There is absolutely nil, zero of an UV-component in the excitation light. This probably means that the excitation filter (UV-filter) in my illuminator is still functioning as it should.

I can post the spectra later if anyone finds it interesting, but the software for this particular unit is giving me more than enough headaches.

The light from the illuminator is still extremely intense, I guess that is the purpose of the screen. It's probably not a great idea to stare into a intense green beam nonetheless. In the binos (after the 2nd filtering), the light is very dim but comfortable to view. Now I'm at least convinced that I will not destroy my eyes
Nikon S-Ke - Old goodie
Olympus CX40 - Fluorescence [Selling]
Olympus BX61 - Phase contrast, DIC, fluorescence [Work-in-progress]

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