Update: Fluorescent stained radiolaria
Moderators: rjlittlefield, ChrisR, Chris S., Pau
Update: Fluorescent stained radiolaria
Years ago, shortly after acquiring my fluorescence microscope, I bought a fluorescent dye kit on Ebay. A pity it has never been used, I thought last weekend and started to figure out what it was about.
Six different dyes were found inside (did not test them all yet), and the idea was to add some of it to unmounted radiolaria. I already noted that radiolaria as they are, do not show a lot additional detail under the fluorescence microscope.
After half an hour drying time the slide was observed with a 40x/0.65 Na objective, with the green filter block (accomodating the yellow barrier filter) and ultraviolet excitation with the YOC6-3 filter.
The result I found rather three dimensional with specific detail in the surface structure. The staining itself resulted in "lighter" yellow-red images.
It will be interesting to stain different kinds of items that are not auto-fluorescent by nature, to find out how the will show under UV excitation light created by the mercury lamp. For example parts of insects or diatoms.
But first some more radiolaria...
Six different dyes were found inside (did not test them all yet), and the idea was to add some of it to unmounted radiolaria. I already noted that radiolaria as they are, do not show a lot additional detail under the fluorescence microscope.
After half an hour drying time the slide was observed with a 40x/0.65 Na objective, with the green filter block (accomodating the yellow barrier filter) and ultraviolet excitation with the YOC6-3 filter.
The result I found rather three dimensional with specific detail in the surface structure. The staining itself resulted in "lighter" yellow-red images.
It will be interesting to stain different kinds of items that are not auto-fluorescent by nature, to find out how the will show under UV excitation light created by the mercury lamp. For example parts of insects or diatoms.
But first some more radiolaria...
Last edited by WalterD on Thu Feb 07, 2019 7:54 am, edited 1 time in total.
- iconoclastica
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- Robert Berdan
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Very interesting
Nice photos and interesting application of fluorescent dyes.
Very nice!
I have probably the same kit from ebay, of half a dozen dyes. I have noticed how the different dyes appear to concentrate in different parts of plants if you put a little in the water. (Much to the amusement of schoolchildren equipped with a nichia-led torch.)
I intend to try tiny quantities with bryophytes, etc
I have probably the same kit from ebay, of half a dozen dyes. I have noticed how the different dyes appear to concentrate in different parts of plants if you put a little in the water. (Much to the amusement of schoolchildren equipped with a nichia-led torch.)
I intend to try tiny quantities with bryophytes, etc
Chris R
Sometimes they doMASZEK wrote:... From your post I conclude, that radiolaria without dye doesn't give you FL effect.
http://www.photomacrography.net/forum/v ... 725#222725
Pau
Thanks everybody!
@Jacek: I think everything made from Silica (and many other materials as well) can be stained, all you need is unmounted radiolarian shells
e.g. from Krantz, see topic https://www.photomacrography.net/forum/ ... hp?t=38688
@Maszek: I've been using Rhodamine 6G for this set, available in different Ebay kits. Five more colours to go!
@Chris: Sounds good, have fun! Looking forward to see the results.
Now three more pictures, same dye but blue filter block (and again ultraviolet excitation)
All 40x/0.65 magnification.
@Jacek: I think everything made from Silica (and many other materials as well) can be stained, all you need is unmounted radiolarian shells
e.g. from Krantz, see topic https://www.photomacrography.net/forum/ ... hp?t=38688
@Maszek: I've been using Rhodamine 6G for this set, available in different Ebay kits. Five more colours to go!
@Chris: Sounds good, have fun! Looking forward to see the results.
Now three more pictures, same dye but blue filter block (and again ultraviolet excitation)
All 40x/0.65 magnification.
Last edited by WalterD on Sat Feb 02, 2019 12:46 pm, edited 2 times in total.
- carlos.uruguay
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Thanks for your kind words, Carlos.
@ Pau: when comparing "pale blue autofluorescence" with rhodamine, the results are interesting.
In my opinion more than just irregular deposits of dye on the surface. The staining is more consistent than you suggest, and shows the item lighter with increased contrast.
When we look e.g. at the first 2 photos, the red as well as the yellow colour are both result of the staining, albeit in a different way.
I noticed rhodamine is a very powerful stain, in below (not processed) image applied on an Amaryllis pollen. Stained on the right, unstained on the left within the same slide.
The image shows much stronger emission of the stained pollen and better definition of surface structure. (@95x). What it did with the pollen was more or less what it did with the radiolaria.
@ Pau: when comparing "pale blue autofluorescence" with rhodamine, the results are interesting.
In my opinion more than just irregular deposits of dye on the surface. The staining is more consistent than you suggest, and shows the item lighter with increased contrast.
When we look e.g. at the first 2 photos, the red as well as the yellow colour are both result of the staining, albeit in a different way.
I noticed rhodamine is a very powerful stain, in below (not processed) image applied on an Amaryllis pollen. Stained on the right, unstained on the left within the same slide.
The image shows much stronger emission of the stained pollen and better definition of surface structure. (@95x). What it did with the pollen was more or less what it did with the radiolaria.
Last edited by WalterD on Thu Feb 07, 2019 7:58 am, edited 1 time in total.
To be a bit more specific about my attempts to stain radiolaria with fluorescent dyes, let's jump back to the kit I have got from Ebay.
It contains 6 different fluorescent dyes (in powder); these are Rhodamine B, Rhodamine 6G, Fluorescein, Bromofluor, Acridine Orange and something called "Fluorescent Green"
From my end I would like to know from each of these: the excitation and emission peaks, solubility in water and/or alcohol, known application and how to process (how long should the item be exposed to the stain, (how long) should it be rinsed?). To start with the last mentioned item: I did not find it easy to recover that info, the out of print reference book on this subject sells for $400,- second hand. About solubility: generally a very small amount of powder is enough to create a saturated solution. The 50 gram in the set would altogether be enough for a couple of buckets.
The first 3 questions are easier to answer, I made below overview:
Rhodamine B ex=546 nm em=568 nm (green)
Rhodamine 6G, ex=524 nm em=547 nm (green)
Fluorescein, ex=501 nm em=516 nm (cyan-green) Note: I've got the yellow emission version
Bromofluorescein no data
Acridine Orange ex=490 nm (?) em=520 nm (cyan-green)
Fluorescent Green no data, though maybe green fluorescein.
The info on excitation and emission peaks and bandwiths I found was contradictory. E.g. nowhere I found stated that 6G works with UV excitation, which appeared to work in my setup. The amount of specialized applications and dye-families is overwhelming. Rhodamine 6G alone can be found in several different product names, each with their own application and specs.
About the staining and to reply on Steve's observation:
Previously got the impression that the stain was actually highlighting surface structure and cavities.True or not, this is not how these dyes are designed to work. (This is actually how dye-penetrant works for industrial non destructive testing). To be sure, the over-stained slide was rinsed in a cup of water and the radiolarian shells were collected from the bottom and put on slides, that were slowly warmed. When the water evaporates too fast, the radiolaria present will remain together in small heaps.
Observation showed a diferent picture, as can be seen in 3 below images. Furthermore the staining appearded to have had a random effect: some showed a strong emission, while other radiolaria went back to their natural colour or dark-red after rinsing. No irregular staining noted within each individual item though. Colours as can be seen within the green spectrum range, after correcting the camera white balance setting. Objective was again Lomo epi 40x/0.65.
Zerene as well as Photoshop had difficulties to perform satisfactory stacking. The 3rd image was stacked "manually" from a small selection of pics.
As mentioned I did not find too many reference articles or posts on this subject, I mean on amateur level. https://www.leica-microsystems.com/scie ... cent-dyes/ you may find an interesting link.
Below site has very interesting spectrum viewers for different dyes.
https://www.aatbio.com/spectrum/Rhodamine_6G
https://www.aatbio.com/products/rhodami ... -9?unit=69
https://www.aatbio.com/products/fluores ... 7-5?unit=1
https://www.aatbio.com/products/acridin ... unit=17502
It contains 6 different fluorescent dyes (in powder); these are Rhodamine B, Rhodamine 6G, Fluorescein, Bromofluor, Acridine Orange and something called "Fluorescent Green"
From my end I would like to know from each of these: the excitation and emission peaks, solubility in water and/or alcohol, known application and how to process (how long should the item be exposed to the stain, (how long) should it be rinsed?). To start with the last mentioned item: I did not find it easy to recover that info, the out of print reference book on this subject sells for $400,- second hand. About solubility: generally a very small amount of powder is enough to create a saturated solution. The 50 gram in the set would altogether be enough for a couple of buckets.
The first 3 questions are easier to answer, I made below overview:
Rhodamine B ex=546 nm em=568 nm (green)
Rhodamine 6G, ex=524 nm em=547 nm (green)
Fluorescein, ex=501 nm em=516 nm (cyan-green) Note: I've got the yellow emission version
Bromofluorescein no data
Acridine Orange ex=490 nm (?) em=520 nm (cyan-green)
Fluorescent Green no data, though maybe green fluorescein.
The info on excitation and emission peaks and bandwiths I found was contradictory. E.g. nowhere I found stated that 6G works with UV excitation, which appeared to work in my setup. The amount of specialized applications and dye-families is overwhelming. Rhodamine 6G alone can be found in several different product names, each with their own application and specs.
About the staining and to reply on Steve's observation:
Certainly picks out different detail. Nice shots. But are they really "stained"? I think it's just surface residue really - isn't it?
Previously got the impression that the stain was actually highlighting surface structure and cavities.True or not, this is not how these dyes are designed to work. (This is actually how dye-penetrant works for industrial non destructive testing). To be sure, the over-stained slide was rinsed in a cup of water and the radiolarian shells were collected from the bottom and put on slides, that were slowly warmed. When the water evaporates too fast, the radiolaria present will remain together in small heaps.
Observation showed a diferent picture, as can be seen in 3 below images. Furthermore the staining appearded to have had a random effect: some showed a strong emission, while other radiolaria went back to their natural colour or dark-red after rinsing. No irregular staining noted within each individual item though. Colours as can be seen within the green spectrum range, after correcting the camera white balance setting. Objective was again Lomo epi 40x/0.65.
Zerene as well as Photoshop had difficulties to perform satisfactory stacking. The 3rd image was stacked "manually" from a small selection of pics.
As mentioned I did not find too many reference articles or posts on this subject, I mean on amateur level. https://www.leica-microsystems.com/scie ... cent-dyes/ you may find an interesting link.
Below site has very interesting spectrum viewers for different dyes.
https://www.aatbio.com/spectrum/Rhodamine_6G
https://www.aatbio.com/products/rhodami ... -9?unit=69
https://www.aatbio.com/products/fluores ... 7-5?unit=1
https://www.aatbio.com/products/acridin ... unit=17502