Fluorescent differences between photosynthetic prokaryotes and eukaryotes

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Wes
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Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#1 Post by Wes » Sat Nov 09, 2019 12:27 am

I sampled a small drop of green slime from the bottom of one of my cultures and observed it under epifluorescent conditions. When I illuminate the sample with a bandpass filter of 450-490 nm combined with a longpass filter of 515 nm it seems like the eukaryotic algae light up in red. When I use green interference filter (~546 nm) and 590 nm longpass filter and image the exact same spot its the cyanobacteria that light up in an intense red color while the eukaryotic algae don't emit much.

If anyone has a better idea of what the responsible pigments are I'd be interested in learning more from you.

The light source is a 12 V 100 W tungsten halogen lamp and the objective is 40/0.75 neofluar.

Image
BP 450-490 LP 515

Image
BP 546 LP 590

Image
I converted the above images to 8 bit gray scale and merged them as individual channels where the BP 450-490 LP 515 image is in cyan and the BP 546 LP 590 is in magenta.

MicroBob
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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#2 Post by MicroBob » Sat Nov 09, 2019 9:24 am

Hi Wes,
you make interesting use of your epi-fluorescence kit!
Are you happy with the filter sets in you condenser?
I have no answer for your question, but probably you will be able to find the excitation and emission ranges for these type of objects.
The mixed image is an attractive way to show the different image contents that are acheived with different fluorescence settings.

Bob, who is experimenting with coconut-water-turbocharged mitosis since yesterday

Wes
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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#3 Post by Wes » Sat Nov 09, 2019 11:02 am

MicroBob wrote:
Sat Nov 09, 2019 9:24 am
Are you happy with the filter sets in you condenser?
Hi Bob,

The condenser came with only two filter sets, one for FITC and one for rhodamine. The dichroic mirror of the FITC set was really fried so I had to replace it with a FT 510 beam splitter and it seems to work ok now. I quite like the use of longpass barrier filters because I can see more than one color at the same time. I also tried epi-brightfield but there is a lot of flare from the non-epi objectives I used.

The mixed image I did in ImageJ (FIJI). Its especially convenient for channel mixing, splitting, composite production, 32-bit floater ratiometric images and many, many more. I highly recommend it to anyone who has in interest in photomicrography.

Let us know how your cytokine experiments are working out, I think its super interesting :D

Wes

MicroBob
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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#4 Post by MicroBob » Sat Nov 09, 2019 12:29 pm

Hi Wes,
my III RS was originally equipped with a filter set with narrow band excitation and narrow band emission and a 510nm dichroitic mirror. I changed both emission and excitation filter against some out of this affordable kit: https://www.ebay.de/itm/133223733959?Vi ... 3223733959
I'm quite happy with blue excitation and emission green and above which I was able to assemble mit the kit.
Epi brightfield is always lacking in contrast, even with the right equipment. It can be much enhanced when using polarizers. Dark field is not available with the III RS but I'm working on the completion of a set.

I have used Fiji in the past but then forgot about it for a while. I really have to start using it again. Another interesting scientific image editing software is Fitswork, it comes from the astronomy corner.

I added 30% coconut water to the rain water I grow my onion roots in. I get a good amount of core divisions, but have no real idea how much of this comes from the coconut water. In theory it should work and the mitosis test is probably the most staightforward way to test this property. Problem is that this would need real scientific slave work, counting cells and counting divisions, comparing.... And no slave in sight! :lol:

Bob

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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#5 Post by Hobbyst46 » Sat Nov 09, 2019 1:46 pm

wes wrote:...
I am not quite clear on your setup. The first one seems OK. A BP 450-490nm, 510nm dichroic mirror (beam splitter) and 515nm LP is OK to excite chlorophylls A and B and some similar molecules, although their excitation peaks are actually lower (400-425nm). 450nm is adequate.

For the second one, 546BP and 590LP, a dichroic mirror of 510nm (or 515nm) is inappropriate! the purpose of the dichroic mirror (DM) is to separate the excitation from the fluorescence. When aligned at 45 degrees towards the lamp and specimen, it should reflect the exc towards the specimen (and pass the rest of light to an opaque wall). At the same time, it passes the fluor from the specimen upwards the eyes/camera, and reflects the rest (namely, the exc) back towards the lamp. So the exc does not mix with the fluor. Hence, The cutoff (step) wavelength of the DM must be higher than that of the exc and lower than that of the fluor. This does not hold for your 510/515nm DM.

If, for the 546/590, setup, I assume:
The optical components are OK;
The focus of the second image IS EXACTLY THE SAME as with the first (450-490...) image, no Z-axis difference at all!

Your 546nm BP filter passes light to the 510nm DM, which in turn passes all of it to the opaque wall. That is, the DM reflects a tiny or none amount of light to the specimen. Hence, no significant exc. Then no red fluorescence is expected.

Since the image shows strong fluorescence, which of the assumptions is true ?
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

Wes
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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#6 Post by Wes » Sat Nov 09, 2019 2:24 pm

Hobbyst46 wrote:
Sat Nov 09, 2019 1:46 pm
For the second one, 546BP and 590LP, a dichroic mirror of 510nm (or 515nm) is inappropriate!
You are correct, this would be an inappropriate combination. In this case I used the original DM that came with the condenser which is a FT 580 splitter.

I will try a BP 430 exciter for chlorophyll next time.

MicroBob
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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#7 Post by MicroBob » Sat Nov 09, 2019 2:53 pm

My impression was that it is the most universal setup to have a short pass excitation filter and a long pass emission filter. So that all light below a certain wave length is used for excitation and everything above is passed to the eyepiece. This is how my two setups work now. With my 18W LED I get a normal visual image in the eyepieces.
In one of the remaining slots I want to install a half-silvered mirror to use the III RS for epi polarized light.

Wes
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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#8 Post by Wes » Sun Nov 10, 2019 9:38 am

MicroBob wrote:
Sat Nov 09, 2019 2:53 pm
My impression was that it is the most universal setup to have a short pass excitation filter and a long pass emission filter. So that all light below a certain wave length is used for excitation and everything above is passed to the eyepiece. This is how my two setups work now. With my 18W LED I get a normal visual image in the eyepieces.
In one of the remaining slots I want to install a half-silvered mirror to use the III RS for epi polarized light.
One minor issue I have is that the replacement dichroic mirror I use does not fit in the round slots and is 1-2 mm above its intended position. Since only the 580 DM fits in the round slot there is a small lateral shift in where the lamp field stop is seen when switching back and forth between the two sets.

I thought of using a circular drill bit to cut out a 22 mm piece from the larger rectangle DM but I'm not entirely sure if this would work out as intended

Image

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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#9 Post by Hobbyst46 » Sun Nov 10, 2019 10:32 am

Wes wrote:
Sun Nov 10, 2019 9:38 am
One minor issue I have is that the replacement dichroic mirror I use does not fit in the round slots and is 1-2 mm above its intended position. Since only the 580 DM fits in the round slot there is a small lateral shift in where the lamp field stop is seen when switching back and forth between the two sets.

I thought of using a circular drill bit to cut out a 22 mm piece from the larger rectangle DM but I'm not entirely sure if this would work out as intended

Image
What you need is a diamond coated drill bit, the one in the photo appears to be a used one. They last between 10 and 20 drillings, depending on the glass. Drilling should be done under a layer of water. Place the DM on a piece on scrap glass, and both should lie in a flat tray (of water) on the drill press stage.

I have cut DM's made of 2-3mm thick glass. But it goes much better when the drill bit is not post heavy use.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

Wes
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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#10 Post by Wes » Sun Nov 10, 2019 12:09 pm

Hobbyst46 wrote:
Sun Nov 10, 2019 10:32 am
Wes wrote:
Sun Nov 10, 2019 9:38 am
One minor issue I have is that the replacement dichroic mirror I use does not fit in the round slots and is 1-2 mm above its intended position. Since only the 580 DM fits in the round slot there is a small lateral shift in where the lamp field stop is seen when switching back and forth between the two sets.

I thought of using a circular drill bit to cut out a 22 mm piece from the larger rectangle DM but I'm not entirely sure if this would work out as intended

Image
What you need is a diamond coated drill bit, the one in the photo appears to be a used one. They last between 10 and 20 drillings, depending on the glass. Drilling should be done under a layer of water. Place the DM on a piece on scrap glass, and both should lie in a flat tray (of water) on the drill press stage.

I have cut DM's made of 2-3mm thick glass. But it goes much better when the drill bit is not post heavy use.
Excellent! Very useful info, thanks!

MicroBob
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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#11 Post by MicroBob » Sun Nov 10, 2019 12:17 pm

Hi Doron,
thank you for sharing your experience with glass hole cutting. I have a rectangular half silvered mirror on the way and have the same job before me. The mirror is not extremely expensive and easy to get so it's not that big of a problem when it doesn't work.
My new excitation filters are a bit smaller than the originals and I use an O-ring to secure them. One new emission filter is quite a bit smaller and I 3D-printed a black spacer to hold it.

Bob

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mrsonchus
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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#12 Post by mrsonchus » Sun Nov 10, 2019 12:44 pm

Hey, that drill-cutter looks to be just what I need to reduce some large filters down to a 45mm for my Oly!

Thanks for the post.
John B

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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#13 Post by Hobbyst46 » Sun Nov 10, 2019 4:56 pm

mrsonchus wrote:
Sun Nov 10, 2019 12:44 pm
Hey, that drill-cutter looks to be just what I need to reduce some large filters down to a 45mm for my Oly!

Thanks for the post.
Wow, 45mm diameter ? the largest I ever cut was 25mm.
BTW - I operated the drill at low speed. Cannot remember how low, but lower than I would with 8-10mm drilling into aluminum, etc.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#14 Post by mrsonchus » Sun Nov 10, 2019 5:01 pm

Yes, they're quite a size! The BX40 has a neat little filter changer that fits onto the field-port and takes 3 in-out filters, unframed 45mm type. A very handy device but the 45mm filters are hard to find....
John B

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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#15 Post by MicroBob » Sun Nov 10, 2019 5:23 pm

When using the hole saw the wobble has to be considered as it changes the final size of hole and remaining disc.
@John: In my experience the filter diameter is not fully used to the border so a smaller filter in a spacer ring could work just fine.
This is something that is easily designed and 3D-printed so you can contact me if you need help.
What I don't like in filters close to the field aperture: Every bit of dirt tends to show in the image!

Bob

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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#16 Post by mrsonchus » Sun Nov 10, 2019 5:43 pm

A kind offer Bob. Yes, avoidance of the dreaded conjugate image planes! The Oly's are well-away from the FI, no problems there.
Your idea of a set of holders is a good one I think, as the 3D-printer is probably easily capable of producing a holder with a thin lip, into which a glass filter of <45mm may be fitted, then loaded into the cassette...
The filter cassette requires filters that are no thicker than about 2.8mm I think - plenty large enough for a filter and 3D printed adapter...

I may get back to you re this Bob, thanks. I'm going to buy a 44mm cutter tomorrow and give it a go with some glass ND filters I have that are oversized, fingers crossed....
John B

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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#17 Post by MicroBob » Sun Nov 10, 2019 5:58 pm

For my III RS i printed a holder to adapt from 13,3 to 17,8mm. Since there is not much force to control a single print layer is enough for the recess, ca. 0,25mm.
3D-printig is not extremely precise as the filament diameter and other thing influence the part dimensions, but for such an adapter it is easily good enough. Turning something like this on the lathe is not much fun as one ends with a light part and a big heap of chips. At work I developed a bigger tool that mainly consisted of a bowl like shape with a stub at the bottom, 80% material removal on the lathe - the guy who made these deserved the good money he got!

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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#18 Post by Hobbyst46 » Sun Nov 10, 2019 6:53 pm

MicroBob wrote:
Sun Nov 10, 2019 5:23 pm
...What I don't like in filters close to the field aperture: Every bit of dirt tends to show in the image!
Yes, I find that if the filter is raised above the field aperture by several millimeters the dust is visible, but if the filter is laid flat, directly on the FA, the problem disappears. Hence, those filters that are used on top of the FA I take out from their metal sleeve or frame.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

MicroBob
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Re: Fluorescent differences between photosynthetic prokaryotes and eukaryotes

#19 Post by MicroBob » Sun Nov 10, 2019 8:32 pm

Hi Doron,
when setting up Köhler illumination the condenser id focussed upon the edge of the field aperture. So for me it would be more logical if the dust would leave focus when lifting the filter away from the field aperture. I will have a closer look at the Phomi, this should be more or less the same as with the GFL.
What I find interesting: Dust becomes more of a problem with high powered objectives, also Coolpix rings show more. I have no explanation for this.

Bob

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