Zeiss III RS - Getting started in Fluorescence

[Adam Long]

I recently picked up a box of Zeiss bits which included a Zeiss III RS head which I've been playing around with on my Universal III.

The filters are mostly in good condition thankfully. One beam splitter has a bubbly look but still gives a viewable image. A few of the dye filters had hazy surfaces which improved with polishing (all on the illumination side). I have no dyes or UV light so looking to start with halogen and autofluorescence. I tried a few slides and slider positions but saw little through low power Neofluars. I don't have the 100W lamp so possibly underpowered. A toy uv flashlight didn't show much either.

Can anyone suggest some good subjects to start with?

Acridine Orange seems to be the commonest dye - but almost as hazardous as a UV illuminator. Is it a big deal?

I have downloaded the III RS manual and have been trying to compare the fitted filter sets with those listed. Position one is empty but Position II looks like the 'Violet excitation for autofluorescence and conventional fluorchromes', due to two dark blue and violet dyed exciter filters. The other positions have the metallic-looking yellowish interference filters that are harder to identify. Is it possible to photograph them on a lightbox and examine the spectra/ histograms?

Any other advice welcome.

[Alexander]

Acridine Orange seems to be the commonest dye - but almost as hazardous as a UV illuminator. Is it a big deal?

Yes, acridine orange is about as hazardous an UV-illuminator which is much less hazardous than a walk on the beach under a blue sky.

Today people fear their shit into the pants for thing nobody cared at all when I was young.
Today people believe they might die the same day, if they go out of the house without sun-protection. When I was a kid, we did't use any sun-protection at all being all day out. For some strange reason I am still alive.

[Hobbyst46]

Acridine Orange seems to be the commonest dye - but almost as hazardous as a UV illuminator. Is it a big deal?

Yes, acridine orange is about as hazardous an UV-illuminator which is much less hazardous than a walk on the beach under a blue sky.

IMHO this is overlooking the danger of viewing UV directly. Damage to the eye can occur. When using an UV illuminator on the microscope, I would double check that no radiation is directly emitted into the viewing optical path. This mostly depends on the quality of the dichroic mirror and excitation filter.

The commonest and easiest fluorophore is chlorophyll, either in green plants or in briophytes or algae. I would suggest the latter. Moss "leaves" are excellent. The best excitation is violet light of wavelength 405-425nm, NOT UV. A peace of moss in water will fluoresce red.

Interference filters are easily identifiable : the color of reflected light differs from that of light that passes through. For example, violet light is reflected and yellow is transmitted. Dichroic mirrors are interference filters as well, and for fluorescence microscopy, they reflect a short wave and transmit a long wave. Like, reflect blue, transmit red. About it.

Interference filters do have a relatively short life span - the coating deteriorates over the years, depending on the use or storage conditions.

[Scarodactyl]

Today people fear their shit into the pants for thing nobody cared at all when I was young.

Like leaded gas?

High pressure lamps have to be treated with a lot of respect, but a lot of things can be stimulated with visible light instead of UV, and LEDs can make very affordable and much safer light sources for these.

[Alexander]

IMHO this is overlooking the danger of viewing UV directly. [/b]Damage to the eye can occur. When using an UV illuminator on the microscope, I would double check that no radiation is directly emitted into the viewing optical path. This mostly depends on the quality of the dichroic mirror and excitation filter.

Looking directly into UV concentrated through an optical system is like looking into the sun using binoculars. Strongly not recommended!
A contemporary microscope fitted with an epi-fluorescence illuminator is safe to a very high degree.

[Hobbyst46]

IMHO this is overlooking the danger of viewing UV directly. [/b]Damage to the eye can occur. When using an UV illuminator on the microscope, I would double check that no radiation is directly emitted into the viewing optical path. This mostly depends on the quality of the dichroic mirror and excitation filter.

Looking directly into UV concentrated through an optical system is like looking into the sun using binoculars. Strongly not recommended!
A contemporary microscope fitted with an epi-fluorescence illuminator is safe to a very high degree.

IMHO the epi-fluorescence illuminator, whether contemporary or old, is only safe for UV if the dichroic mirror and emission filter are safe. If those are interference filters, and if their coatings have been worn out, some of the reflected UV from the slide might be transmitted back vertically into the eyepieces. And that is dangerous. Plain glass transmits UV, although not 100%.

[Adam Long]

The commonest and easiest fluorophore is chlorophyll, either in green plants or in briophytes or algae. I would suggest the latter. Moss "leaves" are excellent. The best excitation is violet light of wavelength 405-425nm, NOT UV. A peace of moss in water will fluoresce red.

Thanks, I'll give that a go. Pretty sure position II will provide the correct violet light. I guess the fluorescence may be pretty dim?

Interference filters are easily identifiable : the color of reflected light differs from that of light that passes through. For example, violet light is reflected and yellow is transmitted. Dichroic mirrors are interference filters as well, and for fluorescence microscopy, they reflect a short wave and transmit a long wave. Like, reflect blue, transmit red. About it.

Thanks, I'll have another look and make some notes. Position three has a stack of three of these so presumably a fairly narrow band making it through. I'll try taking some photos and looking at the histograms too.

Interference filters do have a relatively short life span - the coating deteriorates over the years, depending on the use or storage conditions.

IMHO the epi-fluorescence illuminator, whether contemporary or old, is only safe for UV if the dichroic mirror and emission filter are safe.

Yeah that's my concern. The head is likely over thirty years old and at least one dichroic mirror is obviously shot. Until I'm sure they are 100% working I'll not be using UV. I note contemporary scopes typically have a UV shield for looking at the stage too. I'm not risk averse at all but I do value my eyesight highly (and my ten year-old son's who likes to take a look too).

[Hobbyst46]

...I guess the fluorescence may be pretty dim?

Yes. Fluorescence in general is dimmer by orders of magnitude than the excitation light. Often, fluorescence microscopy is done in a half-darkened room.

[Adam Long]

Yes. Fluorescence in general is dimmer by orders of magnitude than the excitation light. Often, fluorescence microscopy is done in a half-darkened room.

Hmm. had a 'look' at some moss last night. With my 30W halogen lamp at full power (same luminance as the more common finned tube 60w lamphouse), it was so dark I couldn't really focus in any of the three positions. The room was fairly dim, and I swapped my normal 12.5x/20 eyepieces for the 10x/18s to try to get a bigger exit pupil and get my eyes a bit closer in, but basically the view was darkness. I took some long exposure (30s @ ISO 800) photos in each position and there didn't seem to be any red fluorescence visible there either, although with the violet light the moss was a sort of ghostly white.

I''m now wondering if the filters have been mixed up. There are no sets listed in the manual with three exciter filters for starters, unless the 'filter set' means a stack of more than two. Does anyone else have this unit to compare?

I'll order some Acridine Orange too...

[Hobbyst46]

In principle, any dye or other fluorophore is characterized by its excitation and fluorescence spectra, which in turn determine the set of filters. Usually a "set" consists of an excitation filter, dichroic mirror and emission filter. They are arranged within a frame called "cube".
Not all older microscopes had cubes. Some had a different mechanical arrangement of the filters (although their order in the optical train was the same).
So it is best to know the specific identity of each of the three components of the set.
Some filters, especially interference filters, bear ID marks or code numbers along the rim. Or the wavelengths are printed there.
It is possible to acquire the transmittance spectrum of a filter by means of an ordinary spectrophotometer. Or find it in the manufacturer's site or other net-available catalogue
(like Schott, Chroma, Omega and other makers of optical components).

A very rough estimate of the filter features can sometimes achieved from the color of the reflected and transmitted light. Old corning UV excitation filters for example appear nearly black and transmit extremely faint violet color.

In practice, there are thousands (or more) or synthetic and natural fluorophores, so on the market one sees filter sets that accomodate groups of fluorophores. That is possible because the excitation and emission peaks are relatively wide, so usually a deviation of even 10-s of nanometers from the emission peak still allows some fluorescence. Moreover, the optimal excitation and emission wavelengths can depend on many factors: bonding of the fluorophore to another molecule; pH of the environment; solvent; and more.

[Adam Long]

Thanks. The III RS doesn't have cubes, it has two sliders. One holds the exciter filters, these are push fit and can be easily changed, the other slider has the mirrors and emission filters held more securely. So in use it's possible to experiment by swapping the exciter set with the mirror/barrier set and vice versa. I'll have a look at the filter edges but haven't noticed any marks so far.

I don't have a spectrophotometer but this paper suggests it might be possible to cobble something together.

The sets are listed here:

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[Hobbyst46]

Yes, some of those, at least the non-interference ones, are familiar Schott filters. Zeiss collaborated with Schott.
BP means band pass, LP mean long pass. Bandpass of the colored glass filter is not the same as that of an interference filter. But for non critical work it can work OK.
Filter thickness of a colored glass filter also affects the results. Typical thickness is 2-3mm.
Anyway, the set for violet excitation, for example can serve for moss.
Perhaps photos of your filters (such that the transmitted light is visible) might help to identify.
A DIY spectrophotometer from cardboard seems a fun project but perhaps the ID can be achieved without it.

[Alexander]

Zeiss collaborated with Schott.

Both Zeiss and Schott are owned by Carl-Zeiss-Stiftung, a non-profit-foundation founded in 1889.

[Adam Long]

Thanks both, useful info. There are no markings on the filter rims. Here are some pictures with afternoon sunshine as the illuminator:
Exciters, hopefully showing both reflection and transmission:

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Slot I is empty, slot 2 is dye filters only so only 3 and 4 the reflections from the interference filters. These are mostly sandwiches of two thin glass layers with metal(?) in between. Note the violet/ blue dye filter combo in slot 2 transmits almost no visible light.
I just noticed Zeiss states 'interference filter first, with coated surface towards light source, then color glass or sandwich filters) so I need to double check these filters are all correctly oriented and stacked.

Beam splitter/ barriers transmission:

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Reflection:

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To me they look to be in good condition, and I guess the fact that the view with all three sets is completely dark suggests they are working as intended, to block all light except fluorescence. The nice thing about the Zeiss III RS head is you can swap the exciters vs the rest just with the sliders, so I'm fairly confident if the filters have been moved about I could still get a usable combination..

If the violet/ blue set in slot 2 is the standard set for autofluorescence I suspect my illumination just isn't anywhere near bright enough. I did try adding a bright LED headtorch to my flash port but no improvement. I'm using the Neofluar 10/0.30 as recommended by Zeiss (plus NF 6.3x, 16x, 40x). Other possibilities are I'm using an unsuitable subject (Zeiss suggests a strongly fluorescing to start with), or the slide is unsuitably prepared or all three.

[Hobbyst46]

Sorry for the delayed response.
I would guess that the filters in port 2 (of the four ports of the carrier) are the combination 467968 + 466302 + 467861.

[Alexander]

If the violet/ blue set in slot 2 is the standard set for autofluorescence I suspect my illumination just isn't anywhere near bright enough. I did try adding a bright LED headtorch to my flash port but no improvement. I'm using the Neofluar 10/0.30 as recommended by Zeiss (plus NF 6.3x, 16x, 40x). Other possibilities are I'm using an unsuitable subject (Zeiss suggests a strongly fluorescing to start with), or the slide is unsuitably prepared or all three.

There is a number of possible causes for this.
- Is a pol-filter present? Remove it.
- Is there a trinocular tube? Is it switched to 100 % eye-pieces?
- Does the head have some switch to block the excitation light?

Some simple test. Remove one objective, put a piece of plain white paper on the stage and switch your UV-source on. Does the paper emit a patch of blue light?

A good test slide is some thin cut from a plant stem. Most of the stains used on those show strong fluorescence.

[Hobbyst46]

I would guess that the filters in port 4 (of the four ports of the carrier) are the Rhodamine combination 467901 + 466305 + 467869.

And in port 3, the Fluorescein combination 467966 + 466304 + 467865.

[Adam Long]

Thanks again for the advice.

Actually had some success last night after going through my box of prepared slides - a couple of stem sections showed a fairly strong result in Position 4. The top one is a sunflower stem. There's no info on stains used.

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The illumination reaching the slide in Position 4 is bright green. The bright areas were fairly dim at low mags but got brighter as I stepped up the mag and NA. This was in a dark room except for the light escaping from the lamphouse. Exposures were about 10 secs @ ISO 800. In the other positions I got a slight effect but much weaker. Most of my slides appeared completely dark in all three though.

Here is the illumination as it appears on the stage (the top highlight is a reflection off the objective barrel) L-R 4, 3, 2.

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I have bought this set of dyes from ebay so hopefully will be able get a better idea of what filter sets I have. It didn't come with any instructions so any suggestions of good dye/ sample combos would be welcomed. Anyone know if the optical brightener is used as a dye in it's own right or as an enhancer?

[Hobbyst46]

Congratulations on first success.
The starter kit you bought indeed consists of strongly fluorescent chemicals. Fluorescein is moderate but still OK.
A tiny drop of a solution of the dye in a liquid on a slide (and covered with a coverslip) will make an appropriate sample. The fluorescence shines only from the top surface if the solution is very concentrated.

Problem with the dyes is the terrible mess if the dye solution (or powder) is accidentally spilled - on furniture, clothing, floor, walls...
So, opening the package and handling should be done with nitrile rubber gloves on, over a flat container to catch unwanted spills.
Once they stain anything, sometimes the only remedy is cleaning with hypochlorite-based home cleaners, if the stained surface is compatible.

These dyes can stain plant tissues, if used according to known protocols. For microscopy.

Note that the intensity of fluorescence is directly proportional to the intensity of excitation.

Also note, that fluorescence brightness benefits from high NA, since the amount of light gathered by the objective strongly grows with the NA. A 40X1.4 objective is considered excellent for fluorescence microscopy.
Since on your microscope there is no UV excitation (as far as I guess), special objectives that transmit UV are not required.

Moss/alga autofluorescence should be achieved with the filters in position 2 or 3, I guess position 2 will be better.

[Adam Long]

Problem with the dyes is the terrible mess if the dye solution (or powder) is accidentally spilled

Thanks for the warning!

Also note, that fluorescence brightness benefits from high NA, since the amount of light gathered by the objective strongly grows with the NA.

Yes, I was aware of this and took the observation as confirmation that I wasn't just seeing a darkfield type effect.

A 40X1.4 objective is considered excellent for fluorescence microscopy.

Ahh, just when I thought I had enough objectives...