Problems with flourescence picture on ZEISS Axiovert 25

[highbeta]

All,

I have problems with a fluorescence microscope (Zeiss Axiovert 25 CFL) looking at GFP fluorescing cells. I see a lot of background light and can even see the mercury lamp arcs. Please see attached pictures, unfortunately the background noise isn't captured good by the camera, but it is there when looking with eyes.

I disassembled the fluorescence filter cube to look at the used filters. The excitation filter has the correct bandpass (CWL= 470 nm / 40 nm) but the surface that is oriented towards the mercury lamp does not not look great.

For the dichroic mirror and barrier/emission filter I can’t really determine the wavelength but since I see the GFP I think it’s not too far off.

Question:
What can be the reason for seeing so much background light and even the arcs from the lamp? Is this a sign of a burnt out excitation filter?
Can there be something wrong with the mirror or emission filter? Since both have no marks or bevels to verify the correct orientation, I can’t really proof the correct assembly.

Is there anything that I missed?

Any help is appreciated.

Thanks,
Highbeta

[Hobbyst46]

Never worked with GFP but from common tables, the wavelengths for the cube are 470-480/510/516 (excitation, mirror, emission respectively). Possibly somewhat different, like 470/490/510.

The image of the lamp as shown is definitely a problem.

Indeed the surface of the excitation filter looks as if it has been damaged by the heat from the lamp. But I doubt that incorrect orientation of any of the components is the cause of trouble. It is not that important. But the filter itself is possibly damaged. This is one possible cause of the problem, but I would look for others as well.

The components themselves: When looking through the exc filter, you should see blue-green or green. The mirror should reflect green and pass through yellow and red. The emission should likewise pass yellow and red.

Note also, that interference filters (and the mirror of course) do have a limited shelf life even if securely wrapped and tucked away in the depth of a drawer. Several years, not decades. The coating deteriorates.

If in doubt, try and run the transmittance spectrum of each of the three optical parts. On any spectrophotometer. Often easy to do by just placing the filter vertically, adjacent to the empty cuvette port.

BTW, your emission filter looks like an absorption (colored glass) rather than interference filter. Possibly inadequate, but what counts is its transmittance spectrum.

[highbeta]

Thanks a lot for that information. I think have to bite the bullet and invest into a new filter or even a set to be sure that everything fits together. The current filters came together with the microscope. I do not know, if this is still the original setup from 2002 or was replaced at some point.

The idea with the spectrometer is great, but I have to look if I can get access to one.

Thanks,
Highbeta

[Leitzcycler]

interference filters (and the mirror of course) do have a limited shelf life even if securely wrapped and tucked away in the depth of a drawer. Several years, not decades.

I am curious: what is the physical nature of the process of deterioration? What is the structure and composition of the mirror and filter coating?

I have cubes which seems to be in good condition and they must be decades old rather than years... Sadly, non-institutional researchers are not able to buy new cubes every other year.

[viktor j nilsson]

interference filters (and the mirror of course) do have a limited shelf life even if securely wrapped and tucked away in the depth of a drawer. Several years, not decades.

I am curious: what is the physical nature of the process of deterioration? What is the structure and composition of the mirror and filter coating?

I have cubes which seems to be in good condition and they must be decades old rather than years... Sadly, non-institutional researchers are not able to buy new cubes every other year.

Is this really true? I regularly see filter cubes from Nikon and Olympus' finite era that must be several decades old that looks to be in excellent condition (of course, some may have been replaced). Filters obviously degrade with use, and some will surely degrade even in a drawer. But I doubt that all interference filters have such a short lifespan when stored in a drawer. If not, I probably shouldn't have bought so many filters for my DIY fluorescence setup that I expect will take 'several years' to complete...

[Hobbyst46]

interference filters (and the mirror of course) do have a limited shelf life even if securely wrapped and tucked away in the depth of a drawer. Several years, not decades.

I am curious: what is the physical nature of the process of deterioration? What is the structure and composition of the mirror and filter coating?

I have cubes which seems to be in good condition and they must be decades old rather than years... Sadly, non-institutional researchers are not able to buy new cubes every other year.

Is this really true? I regularly see filter cubes from Nikon and Olympus' finite era that must be several decades old that looks to be in excellent condition (of course, some may have been replaced). Filters obviously degrade with use, and some will surely degrade even in a drawer. But I doubt that all interference filters have such a short lifespan when stored in a drawer. If not, I probably shouldn't have bought so many filters for my DIY fluorescence setup that I expect will take 'several years' to complete...

Well, I should have added "sometimes". True - according to my personal experience with 6-7 high quality interference filters. Not a representative sample of all filters in the world... the coating was visibly degraded, although I could not trace the reason. They were stored in various storage-rooms, and temperature and humidity were highly variable.
BTW in very old microscopes, fluorescence filters were often absorption filters, not interference, and (at least if not too close to the excitation lamp) might have lasted decades. I still have some from Corning and Schott that are just fine.

[lightshape]

I know this shop as a good source for filters if you don't want to spend too much.

To start with excitation replacement:
https://www.ebay.de/itm/Ottico-Filtro-4 ... 23a9bd7ba6

Also your beamsplitter could be worn-down.

Stefan

[viktor j nilsson]

I can vouch for Bjomejag being excellent. It's Omega optical's official surplus store, and it's basically being run by Robert Johnson - the president of the company - in his spare time. Or as he put it when we exchanged some email: "I can't stand to see good and helpful materials thrown away". Stellar company!

[highbeta]

I was able to find a Chroma ET485/20x FITC Excitation Filter Ring Mounted CWL 485 nm BW 20 nm D 25mm for a decent price. As soon as I get it and test it, I will post the results.

Thanks for the the hint with the Omega shop.

highbeta

[highbeta]

I received the EX485/20X excitation filter. By looking without any fluorescent specimen the image of the lamp disappeared but I still saw blue background.
So I ordered a new beam splitter 500 DCLP and an emission filter 515 EFLP. Will keep you posted hopefully with nice pictures soon.

thanks,
highbeta

[lightshape]

Hi,
good news, hope you'll get it sorted with all the new filters.
Curious to see your results