Incident fluorescence: Ficus leaf tip cross section

[MicroBob]

Hi together,
I just received my new Zeiss incident fluorescence condenser III RS for my 1960s Phomi.
Since I don't yet have the incident lighting field aperture I had to improvise with a little mirror and a flashlight.
This was green excitation and red long pass filter. There is a second filter set in the condenser but I couldn't bring it to a visible result with my white light flashlight.
For a first try with hand held flashlight I'm quite satified with the result.
I think about installing green, blue and white LEDs and a semi transparent mirror in one slot.
Having an incident lighting that is independent from the transmitted lighting might offer some nice opportunities. The Phomi normally uses the same lamp for both, it is switched mechanically by moving a periscope into the transmitted light path.

Bob

[Hobbyst46]

Very nice first image, Bob!
The optimal excitation wavelength for the red fluorescence in leaf images is 400/425nm. Shorter wavelengths will excite other chemicals like vitamin E, that might glow yellow but probably quite weak.
If possible, please show a photo of the fluorescence setup. I am experienced with epi-fluorescence but do not know the light path in the phomi. Many decades ago they were doing fluorescence with the transmitted beam, that is an inherently inferior setup, and I never saw such one in action.

[MicroBob]

Hi Doron,

if I remember right the leaf was stained in Etzold FCA, this is fuchsin, chryosidin, astra blau. So this could be the fluorescence of one of the stains.

Here is a manual of my condenser: https://www.google.com/url?sa=t&rct=j&q ... ZMeTPCsB5h

If it a hevy beast and magnificiently made. The filter changer block rides on small rollers. I just had a quick peek inside and have not yet checked what filters these are precisely.

Bob

[Wes]

Very interesting result and I'm also interested to see your setup. You have installed the Zeiss incident fluorescence condenser III RS on the first Phomi model?

[MicroBob]

Hi together,
here is a picture of the instrument with the III RS condenser.
With the Phomi 1 it is most important to know that there were two generally different versions: The first one (first black than hammerite/black) had a round nosepiece mount were the nosepiece fell right down when you loosened the screw. It had a white knob in the front top surface of the foot. From 1961 on this was still the "Phomi" without added number, but it had the same nosepiece mount as the later models. This was probably the most important change in the whole 30 years of production. And it occured within one model designation

Bob

[Hobbyst46]

if I remember right the leaf was stained in Etzold FCA, this is fuchsin, chryosidin, astra blau. So this could be the fluorescence of one of the stains.

Hi Bob,
1. I did not realize the Ficus leaf was stained, I thought it was just auto-fluorescence in the image.
2. Thanks for posting the condenser manual, now it is very clear and simple to understand.
3. It is a nice arrangement of epi-fluorescence. It differs from more modern setups in that there is no cube: the dichroic (also called dichromatic) mirror is fixed, whereas the two filter sets (excitation and emission) are carried on two separate slides. A cube has the benefit of speed and convenience, since it is preset combination for a given stain. But it is not a must.
4. Interference filters are sourced by aftermarket suppliers, e.g. Omega, Chroma, Edmunds, Thorlabs, Newport, Hammamatsu, and are farly expensive (~100 $ a piece). There are non-interference filters (e.g. the BG38, from Corning etc) that are much cheaper but are absorption and not interference. Of course, LEDs can simplify the choice and lower the overall cost of the optics. You might get along with Schott long-pass absorption filters for emission (and there is a rich choice of cutoff wavelength, every 10nm or so). They are inexpensive and last forever.
5. A question : have you got an HBO (or equivalent) mercury or xenon lamp ?
6. A question: where is the "periscope" you mentioned earlier ? within the arm of the microscope, to switch the ordinary illumination beam from the brightfield collector to the epi-condenser ?

[MicroBob]

Hi Doron,
the III RS actually has 4 complete filter sets including 4 dichroitic mirrors. The filters are not quick to change so it practically is limited to 4 fluorescence setups but can change quickly between them and nothing can get lost.
I have just tested an idea for getting illumination: An eyepiece is a fairly good and small LED condenser optic. If I let the LED extend into the opening for the incident lighting field aperture I would be able to find room for it and keep it as a part of the condenser to mount and unmount with it. Or I could use a 90° mirror or prism and have the LED sideways. This was a setup that was available anyway for the later Phomis. I plan to use a Cree XHP-50 LED that puts out a lot of white light at 450 and 500 nm but has a dent at 480nm. The green/red filter set will be fine with this. The other is BP 485, FT510, Emission 515-565. Here I may have to change the excitation and emission filters.
I don't have an HBO or xenon lamp and don't plan to get one. I have a strong LED in the foot of the microscope and probably will built a separate illuminator to be able to combine. The "periscope" in in the limb of the microscope. When moving it wownward into the transmitted light path this is directed upwards to be picked up by the mirror of the incident lighting field aperture. It is actuated by a lever on the left side of the stand.

Bob

[Hobbyst46]

The other is BP 485, FT510, Emission 515-565.

485 excitation, 510 cutoff(dichroic) and 515-565 emission should be good for fluorescein (FITC is a common stain).

I don't have an HBO or xenon lamp and don't plan to get one.

Applause.

I have a strong LED in the foot of the microscope and probably will built a separate illuminator to be able to combine. The "periscope" in in the limb of the microscope. When moving it wownward into the transmitted light path this is directed upwards to be picked up by the mirror of the incident lighting field aperture. It is actuated by a lever on the left side of the stand.

So, the brightfield light beam can be switched to enter the epi-condenser ? well, anyway, a dedicated monochromatic LED is evidently better. A monochromatic LED source might make the excitation filter redundant. The dichroic mirror will still be essential, the emission filter most probably as well.

[MicroBob]

The problem with most LEDs is that they have this gap at about blue-green 500nm. So my white LED will not be very powerful in this specific range. On the other hand side I have just bought some 500nm LEDs, originally meant for a room lighting project, to even out the spectrum of warm white LEDs. This leads me to give an angled design of the illuminator the preference as I can then change the LEDs more easily.
Apparently there are some types of observation that have only become possible with the less harmful LED light, like acrinidin orange stain of living plancton animals.

Most monochromatic LEDs have a spectrum like a wizzards hat, so still some intensity outside the core wave lenght. So it has to be tested in which cases the excitation filter can be omitted. It will be omitted if I can't get one for much less than 100€!
I might get access to some fluorescence filters and I have one type that I have not yet understood what to do with it.

Here:http://www.science-info.net/docs/zeiss/ ... cope-1.pdf on page 10 you can see the alternative light beams. In the upper image the periscope is lowered into the lightpath and reflects it upwards. At a time when a stong lamp was big this was probably no bad design. I would like to be able to combine transmitted and incident fluorescence. This is not too attractive for the green/red combination since only red and longer wave lengths of the transmitted image make it into the final image. But with blue excitation this can generate nice and unexpected images, I hope.

[MicroBob]

Hi together,
I forgot to report one thing: I usually just pop a Nikon 1j5 with eyepice adapter into a bino port and shoot my pictures. Usually this works well for my not too perfectionist approach.
With my improvised fluorescence setup I ran into faily long exposure times like 2 seconds. I saw a strange light effect in the image. Reson was the room light that shone into the other bino port!
This will have affected all my images, just not enough to become obvious. So better work in a more quality oriented way and use the 100% to the top port of the old Phomi1 - mode.

Bob

[Hobbyst46]

Bob,
The first principle in fluorescence microscopy has been - create a darkroom or at least a darkened area around the microscope. Fluorescence is weaker than other light sources by orders of magnitude. Fellows construct a "dark chamber" by means of a hanging U-form frame and a black cloth curtain around the scope. Like street photographers with their Linhof cameras on the boulvards ages ago!

[MicroBob]

Hi Doron,
thank you for this hint, it is obvious but I had overlooked it completely. I think I will go for a black cover for the microscope, with an opening for the camera.

Bob

[MicroBob]

Hi together,
I have built an LED lighting unit that shines from the side over a mirror and into the back of the reflected light fluorescence condenser III RS.
This was quite difficult as the gap is just 24mm wide and I wanted to attatch the lighting unit to the stand, not the condenser.
It works quite well wit hthe Cree XHP 50 18W LED but would probably work just as good with a Cree XM-L 10W LED with it's smaller emitter. The collector optic is just an old eyepiece that nearly gives a staight light bundle.

In my condenser III RS I've got two filter sets: BP 546, FT 510, LP 520 (blue-green excitation und BP 485/20, FT 510, BP 515-565. The second filter set is probably for a quite special application. I think I will change the blocking filter. The band pass filter BP 515-565 will give only monochromatic images. But I have to do more reading and thinking on this. I'm working on the mitosis-topic now and the condenser just came along for a very good price.
Attatched a picture of the side lighting unit (prototype) and some plant sections (I think W3A stained). To be honest: For these slides the fluorescence only offers a nice colourful variation.

Bob

[Hobbyst46]

Bob,

There are hundreds (at least) of fluorescent dyes.

The Toupview software, introduced to me by mrsonchus, is excellent and rich with features, not only image acquisition. Astonishingly, it contains a list of stains used in biology and cell structure. Look into the "Options" menu. Along with excitation (the poor guy who developed the interface named it "extraction"...) and emission wavelengths. Handy and convenient !

One should know, however, that not only peak exc and emi are important - complete separation between the exc and emi beams is even more important. So, one might prefer a lower exc peak wavelength than the nominal, and a higher emi wavelength than the nominal, to increase the separation (the latter is always higher than the former!). This moving away somewhat from the peak is OK since the peaks are fairly broad (relatively) so a shift of 20nm from the peak does not lower the absorption of excitation by the stain too much.
The dichroic mirror is crucial in this respect also.

Since I am pretty sure that you 485/515/ set is for fluorescein, I believe that the 546/ set is for rhodamine. They used to be a popular pair in the past. But please check out the wavelengths since I might be wrong. Also, a given exc - FT - emi combination usually fits more than one stains.

[MicroBob]

The big microscope makers offer nice listings of filters and dyes as they like to sell their expensive stuff.
For Rhodamin Zeiss has quite e few entries: https://www.micro-shop.zeiss.com/de/de/ ... tant/dyes/
If I haven't mixed this up in my short test this was a Wacker stain with Rhodamin B instead of Arinidine Red (wich is no longer available in cheap powder form).
I used the first filter set so this would have to match Zeiss data:
Rhodamine 551 nm 573 nm
Rhodamine 110 497 nm 520 nm
Rhodamine 110 pH 7.0 497 nm 520 nm
Rhodamine 123, MeOH 507 nm 529 nm
Rhodamine B 543 nm 565 nm
Rhodamine Green 497 nm 524 nm
Rhodamine phalloidin pH 7.0 558 nm 575 nm
Rhodamine Red-X antibody conjugate pH 8.0 573 nm 591 nm
Rhodaminen Green pH 7.0 497 nm 523 nm
/b]
In the case of Rhodamin B the excitation would be done with a lower wave lenght but also the emission would be blocked partly by the 546nm blocking filter.

Leitz states for Rhodamin B:
Excitation 540 and emission 625

They want to drive me mad!

[Hobbyst46]

The big microscope makers offer nice listings of filters and dyes as they like to sell their expensive stuff.
For Rhodamin Zeiss has quite e few entries: https://www.micro-shop.zeiss.com/de/de/ ... tant/dyes/
If I haven't mixed this up in my short test this was a Wacker stain with Rhodamin B instead of Arinidine Red (wich is no longer available in cheap powder form).
I used the first filter set so this would have to match Zeiss data:
Rhodamine 551 nm 573 nm
Rhodamine 110 497 nm 520 nm
Rhodamine 110 pH 7.0 497 nm 520 nm
Rhodamine 123, MeOH 507 nm 529 nm
Rhodamine B 543 nm 565 nm
Rhodamine Green 497 nm 524 nm
Rhodamine phalloidin pH 7.0 558 nm 575 nm
Rhodamine Red-X antibody conjugate pH 8.0 573 nm 591 nm
Rhodaminen Green pH 7.0 497 nm 523 nm

In the case of Rhodamin B the excitation would be done with a lower wave lenght but also the emission would be blocked partly by the 546nm blocking filter.

Leitz states for Rhodamin B:
Excitation 540 and emission 625

They want to drive me mad!

1. Here is one more tip about the LEDs: A 18W LED is better than a 10W. Do you use a dimmer or neutral density filters ? high intensity excitation might lead to photo damage and/or bleaching in sensitive cells and unstable dyes, but I would not worry about those at this initial stage of your project (and probably never).
2. Sorry that I did not comment on it, but a set of 546/510/520 does not make sense. The order of wavelengths must be: excitation<dichroic<emission
so 546/510/520 will not work at all. You may want to find the correct wavelength of each component of this set.
3. there are several Rhodamine variants (same molecular skeleton, slight side differences, which mean different opticl spectra). Also the pH and type of solvent sometime shift the wavelengths, because of interactions within the molecule or with other molecules (simplistic description). The popular Rhodamine was often B or 6G.

Now for example, Rhodamine B needs an excitation peak at 543nm (Zeiss table) or 540nm (Leitz). This difference is not important (side-note: excitation wavelengths have often been chosen according to the lamp; a mercury lamp has a strong line at 546nm and it will be fine for Rhodamine; a Xenon line allows more flexible choice of exc light). A laser light at 532nm can also excite it, since the light absorption line in the spectrum is fairly broad.
Now, about the emission of Rhodamine B: The emission peak is usually about 570-580nm, I do not know why the Leitz figure is 625nm. Anyway, if you choose a LP filter of 560nm, say, you catch all emission above 560nm. The problem is which dichroic to use. There are not so many of them, spaced at narrow wavelength intervals. Tentatively I would choose a dichroic of 550nm cutoff, if I excite at 546nm.
In critical fluorescence, they carefully study the spectra (or expected spectra) of the fluorophore to choose the filters. But, it might still be trial and error.

Please note, the choice of stain depends on other factors, not related to the optical spectra: You use the specific stain to make a biological feature or sub-unit or organelle stand out and become selectively visible, the rest of the specimen should remain invisible. So if you have a stain that specifically targets the nucleus, say, you consider it a candidate; then you look into its other features: excitation wavelength that is practical with your lamp, a large Stokes shift (so the emission wavelength will be as far from the excitation as practicable) and hopefully far from any auto-fluorescence of the specimen. So, it is a multi-parameter optimization process.

Hope this helps.

P.S. a little late to do it right now, I should find an illustrated guide based on the spectra. The excitation and emission spectra usually overlap each other; the tail (rightmost section) of the excitation overlaps the head (leftmost section) of the emission. So, there is a range of wavelengths around the crossing point, where excitation and emission occurs. But the challenge is to be able to visualize ONLY emission, because it is so much weaker than excitation. That is why it is so important to have a cutoff filter, that will block all wavelengths above the must-have minimum for excitation, so it will pass only emitted light at the longer wavelengths. Plant chlorophyll offers an advantage in this respect: the excitation and emission are far away from each other (400nm vs 600+ nm. respectively). Many artificial stains do not have such wide Stokes shifts.

Hope this clarifies.

[MicroBob]

Thank you for your explanations, Doron!
First: I got the filters of the first filter set mixed up (in writing). These are the specifications of my two filter sets:
Filtersatz 14 488014-0000-000 BP 510-560 FT 580 LP 590

Filtersatz 17 488017-9901-000 BP 485/20 FT 510 BP 515-565

So the order of excitation<dichroic<emission is intact.

The LEDs in question differ not only in power but also in the size of the emitter: The XHP 50 consists of four dies close to each other. The XM-L is just one die and the emitter is a lot smaller. The combination of XHP 50 and chosen eyepiece gives a wider beam than necessary so light is wasted. The XM-L would probably put out the same or even more useful light. Perhaps I find a more suitable collector optic instead to use the big XHP 50 or even a still bigger XHP 70. I left some space to be able to optimize it further. Unfortunately I mislayed all my LEDs when collecting the other bits for the LED illuminator. Cooling is active as there is no way to transfer the heat to the microscope stand and the 3D printed parts from PETG are not very heat resistant. Even with the wasted light everything stays cool enough now.

I got the illuminator light tight, capped the free eyepiece and darkened the room for the pictures.

Rhodamine emission: I found the 625 nm in this Leitz document: http://science-info.net/docs/leitz/1Fluor_Micro.pdf.
The 625nm are quite far off from what Zeiss states for the Rhodamine variants.

I have ordered a set of interference filters that will give me more options, new old stock from Jenoptik: https://www.ebay.de/itm/Farbfilter-set- ... 2749.l2649

There was an interesting thread in the german forum about fluorescence of pine needle sections: https://www.mikroskopie-forum.de/index. ... ic=35237.0
Rolf-Dieter was using Zeiss fliter set 9:
Filtersatz 09 488009-9901-000 BP 450-490 FT 510 LP 515
This differs mainly from my set 17 in that the barrier filter is a long pass, not a band filter as in my set. I think the yellow filter from the set I bought should act similar.
I think I will at first try to get attractive results from plant sections. This is not a very common application of fluorescence microscopy so I will have to look for stains that attatch to certain structures in the section and at the same time have a usable fluorescence. I have a couple of stains that work for plant sections, and John here in the forum is using a lot of other stains so I will make a table and look for suitable stains that could be combined.
I will keep on writing here when I make progress. The filter set will arrive today!

Bob

[Hobbyst46]

...These are the specifications of my two filter sets:
Filtersatz 14 488014-0000-000 BP 510-560 FT 580 LP 590
Filtersatz 17 488017-9901-000 BP 485/20 FT 510 BP 515-565
So the order of excitation<dichroic<emission is intact.

This is OK.

Rhodamine emission: I found the 625 nm in this Leitz document: http://science-info.net/docs/leitz/1Fluor_Micro.pdf.
The 625nm are quite far off from what Zeiss states for the Rhodamine variants.

And far off from the Rhodamine B wavelength that I have known and just verified it by a quick survey of articles. Either the 625nm is a typo error in the Leitz brochure (unlikely yet possible) or I do not know what. Fluorescence spectra depend somewhat on the excitation wavelength but not that much. I believe the range is 560-590nm and not above 600nm. The rightmost tail of the emission spectrum does cover as high as 650nm but the intensity is far lower than the peak.

The Leitz brochure has its flavor, although it is really very old and fluorescence microscopy has advanced tremendously, much more than other modalities of optical microscopy AFAIK.
There is a much broader choice of stains (those have been developed since the 1980's by an american named Haugland). What I like in the Leitz brochure is the first layout of trans-illumination fluorescence. It is highly outdated, but nice to see. Incident fluorescence (namely, epi-fluorescence) is the way they go for decades now.

I have ordered a set of interference filters that will give me more options, new old stock from Jenoptik: https://www.ebay.de/itm/Farbfilter-set- ... 2749.l2649

Excellent purchase.

There was an interesting thread in the german forum about fluorescence of pine needle sections: https://www.mikroskopie-forum.de/index. ... ic=35237.0
Rolf-Dieter was using Zeiss fliter set 9: Filtersatz 09 488009-9901-000 BP 450-490 FT 510 LP 515

the LP is what allowed Dieter to see the strong red auto-fluorescence. Your set #17 is not suitable for this red color. Both because the excitation wavelength is too high (485) and because the emission BP filter blocks all red and even orange light. It will pass green and yellow fluorescence though.

[MicroBob]

I got the filters and changed the setup:

Excitation: Jenmed Dichrolight M 47/63 plus B46 ( the B46 alone led to a green background!)
Dichroitic Mirror Zeiss FT 510
Blocking filter Jenmed Dichrolight Y 52

Object: Leaf of small (12cm) coniferous plant
Super quick hand section with cutter blade

Objective Neofluar 10/0,3
Free eyepiece blocked
Black fishing reel spool around nose of objective to block room light (can't get it dark in daytime)

I'm quite happy with the result!

Bob

[Hobbyst46]

Nice image!

Studying the spectrum of each filter will enable to estimate beforehand the wavelength of the excitation beam. Spectra are transmittance (percent) vs wavelength. In the seller info (link Jenoptic), one of the photos was the spectra, laid side by side. I caught them via print screen, but a better display would help. If the filters are from Schott, their spectra could be downloaded perhaps. The B46 for example has a cutoff at 470nm. So it transmits light (T=100%) below 470nm, namely blue light. It reflects all wavelengths>470nm. Since chlorophyll is excited at 400-430nm, this B46 is OK to serve as excitation filter for it.
The 47/63 IMHO does not contribute much to excitation of chlorophyll. It has a similar cutoff around 470nm as the BP. In addition, it transmits light >610nm. Such wavelengths (600+nm) are likely to excite very weak fluorescence IMHO. I think that the B46 could suffice, as excitation filter.

Then, to know the output from a combination of filters, you overlay the transmittance spectra. Since transmittance is a series combination. Two filters, each giving T=50% at a certain wavelength, and combined in series, will output T=25% at that wavelength, etc.
Some of your filters have a single cutoff wavelength, some have two.

The green fluorescence is possibly caused by excitation around 400-450nm, but I do not know which substances. Flavonoids perhaps? some of them are excited at 440nm and emit above 500nm, but are they present in this leaf ?

[MicroBob]

I repeated the test with the excitation filters: The B 46 filter alone gave a medium green background, so this light passes the dichroic mirror and the Y 52 emission filter. When I add the 47/63 as a second excitation filter, the background becomes nearly black. Not logical but repeatable. My idea is that the filters are not of perfect quality and leak a bit into longer wave lengths. I tried a second excitation filtration: While the B 51 gave a green background again it gave a good result in combination with the M49/65 filter. I attatach two pictures: One is the pine needle fresh in water. the second is a stained plant section in Euparal. Unfortunately Euparal has a green fluorescence itself, so is not really usable for fluorescent botanic section slides. Apparently natural resins have a tendency towards autofluorescence while some artificial resins don't. I was told Eukitt works as a mountant for fluorescence. As always in microscopy this is just one of the mountants I don't have.

Bob

[Wes]

Hi together,
I have built an LED lighting unit that shines from the side over a mirror and into the back of the reflected light fluorescence condenser III RS.
This was quite difficult as the gap is just 24mm wide and I wanted to attatch the lighting unit to the stand, not the condenser.
It works quite well wit hthe Cree XHP 50 18W LED but would probably work just as good with a Cree XM-L 10W LED with it's smaller emitter. The collector optic is just an old eyepiece that nearly gives a staight light bundle.

In my condenser III RS I've got two filter sets: BP 546, FT 510, LP 520 (blue-green excitation und BP 485/20, FT 510, BP 515-565. The second filter set is probably for a quite special application. I think I will change the blocking filter. The band pass filter BP 515-565 will give only monochromatic images. But I have to do more reading and thinking on this. I'm working on the mitosis-topic now and the condenser just came along for a very good price.
Attatched a picture of the side lighting unit (prototype) and some plant sections (I think W3A stained). To be honest: For these slides the fluorescence only offers a nice colourful variation.

Bob

Hi Bob,
I also ended up getting the III RS epifluorescence condenser but being a Phomi user its annoyingly more difficult to find the right accessories for epi illumination. Could you provide a bit more details on how you built your light reflection device (what materials, dimensions, maybe a few more pictures so I can get a better understanding of your DIY device).