Who's Using Fluorescent Markers and Tags?

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Sure Squintsalot
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Who's Using Fluorescent Markers and Tags?

#1 Post by Sure Squintsalot » Sun Jun 26, 2022 12:21 am

The whole fluorescent thing has lit a fire under under my butt. Before I empty my 401K on fluorescent markers ($300 per gram!!!), I figured I should learn at least the basics of how they're used, what they'll show, and if they'll help me win the Nikon Small Worlds Prize.

For example:
1) Can I dope the water used to feed a tomato plant, then section the tiny fruit to see fluoresent structures in just the seeds?
2) Can I feed fruit flies some marker that will make their eyes light up bright pink under UV? Or make only their digestive tract look like a neon blue sausage?
3) Where can I find the book called "Fluorescence Microscopy for Idiots"?

Hobbyst46
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Re: Who's Using Fluorescent Markers and Tags?

#2 Post by Hobbyst46 » Sun Jun 26, 2022 9:02 am

Sure Squintsalot wrote:
Sun Jun 26, 2022 12:21 am
The whole fluorescent thing has lit a fire under under my butt. Before I empty my 401K on fluorescent markers ($300 per gram!!!), I figured I should learn at least the basics of how they're used, what they'll show, and if they'll help me win the Nikon Small Worlds Prize.

For example:
1) Can I dope the water used to feed a tomato plant, then section the tiny fruit to see fluoresent structures in just the seeds?
2) Can I feed fruit flies some marker that will make their eyes light up bright pink under UV? Or make only their digestive tract look like a neon blue sausage?
3) Where can I find the book called "Fluorescence Microscopy for Idiots"?
Fluorescence microscopy is mostly science (IMHO - topped with some art).
The starting point is what you want to see. As the purpose is defined in your points no 1 and 2.
The rational approach IMHO would be to search the literature with appropriate keywords like :" tomato seed fluorescence" (for 1) or "fruit fly fluorescece" (for 2),
because the exact purpose dictates which marker to use; the marker dictates whcih filters and filter cube to have on the scope, and which excitation wavelength; which in turn dictate the illumination source; then there are potential difficulties which may or may not arise (for example bleaching) which again depend on the marker and light.
But, if one knows nothing about fluorescence microscopy, it would be wiser to read at least the Wikipedia entries about Fluorescence, Fluorescence microscopy; then read relevant texts in the Manufacturers supported sites, for example Microscopy4U (or similar). They explain the equipment.

There are many textbooks about fluorescence microscopy, but none of them targets "idiots". Yet the basic stuff is not reallt that difficult.
And to make some fun at the start, try watching autofluorescence, for which you need no stain. Illuminate green plant parts with intense violet light and watch red fluorescence.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

Microscopy_is_fun
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Re: Who's Using Fluorescent Markers and Tags?

#3 Post by Microscopy_is_fun » Sun Jun 26, 2022 11:38 am

Hobbyst46 wrote:
Sun Jun 26, 2022 9:02 am
There are many textbooks about fluorescence microscopy, but none of them targets "idiots".
This is certainly true, since fluorescence microscopy requires quite some technical skills, and also caution when dealing with the frequently toxic and/or carcinogenic fluorophores. The recently published book "Understanding light microscopy" (Sanderson) contains a decent and compact chapter about the optical side of fluorescence microscopy, and also an introductory chapter on fluorophores.

In my view, the easiest route is to start with samples which show autofluorescence (e.g. all botanical samples containing chlorophyl). Some of the cheaper and readily available fluorphores are e.g. Uvitex (a brightener used in detergents) or acridine orange. The fluorophores used in professional fluorescence microscopy are typically, as mentioned in the initial post, outrageously expensive. And this price level is probably one of the main reasons, why in recent years the gap between hobby microscopy and professional microscopy has become larger.

But when one wants to dig a little bit deeper in microscopy of biological samples, there is certainly no way around this technique. And some of the pictures obtained by fluorescence microscopy are definitely amazing!

Sure Squintsalot
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Re: Who's Using Fluorescent Markers and Tags?

#4 Post by Sure Squintsalot » Sun Jun 26, 2022 3:59 pm

I guess that by "Fluoresence Microscopy for Idiots" I really meant "Fluoresence Microscopy for the Science-Minded, Seriously Curious but Careful Bio-Noob".

After a lifetime of reading earth science articles, I'd only quickly review the occasional cell biology article where I'd often see fluorescence cited in concert with a singularly specific component of a cell, or even some part of a gene. It's finally dawned on me that somebody has to design a fluorophore that will be absorbed naturally and then only fit it those extremely specific cell locations. All of a sudden, a lot of that science you read about falls into place: the massive computer efforts to find molecular geometries that fit precisely into a specific slot with a cell, nevermind the resources to actually make these molecules. All those mysterious "shiny white electronic boxes" found in research labs. Makes sense for designing medicines; doubly so for designing markers that will let you see what you're doing. No wonder there are so many of these markers (4,000 at Fischer Scientific) and that they are so expensive ($500 per 100 μL!).

I guess I'll start by looking for undergraduate lesson plans for this sort of thing; hopefully there are basic, classroom level procedures that are easy to follow and yield cool results.

Alexander
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Re: Who's Using Fluorescent Markers and Tags?

#5 Post by Alexander » Sun Jun 26, 2022 6:08 pm

Your local university library is full of books on fluorescence microscopy.

The acridine orange mentioned above is cheap and a very versatile dye. It allows the separation of living from dead cells and of DNA from RNA.
While other dyes are expensive, you only need a small portion of it. A μg of DAPI for example may cost 40 USD (many times the price of gold) but will last for many years of a hobbyists use. The absolut expense is not that big.

Almost all fluorescence stains intercalate with DNA and are therefor potentially mutagen. Safe handling is a absolut must!

crb5
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Re: Who's Using Fluorescent Markers and Tags?

#6 Post by crb5 » Sun Jun 26, 2022 6:51 pm

Fluorescence dyes themselves usually have only limited specificity - they may preferentially stain certain classes of materials (e.g. Nile Red for fats, oils and plastics) or be just the right size to intercalate between the bases of DNA (e.g. DAPI). The specificity seen in many scientific micrographs is achieved by other means such as attaching a fluorophore (e.g. fluorescein or rhodamine) to an antibody where the antibody hones in on a protein only present in a limited location. The other trick to genetically engineer protein fusions with a fluorescent jellyfish protein (e.g. GFP) which are expressed in limited regions within a cell or organism. In general, these methods require extensive laboratory facilities and experience in molecular biology. The other problem is obtaining fluorophores, as chemical companies generally deal with organizations and not individuals. Collaboration with a local college is probably the best way forward. Otherwise, as mentioned above, chlorophyll autofluorescence is a good place to start if you want to explore the optics required. You may also find fluorophores around the home in some washing powders and highlighter marker pens (and molds/millipedes/scorpions!).

Leitzcycler
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Re: Who's Using Fluorescent Markers and Tags?

#7 Post by Leitzcycler » Sun Jun 26, 2022 7:55 pm

The methods mentioned above (antibodies, GFP) may be very expensive to use. It may also take months or even years to get a particular application to work even in a professional lab.

There are also some organelle-specific dyes which are rather easy to use. They may be rather expensive too and I am not convinced it is worth of buying them just for fun unless you are really doing research.

Here is an example of some staining from my lab: yeast mitochondria stain MitoView green from Biotium
https://biotium.com/product/mitoview-dyes/
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Wes
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Re: Who's Using Fluorescent Markers and Tags?

#8 Post by Wes » Sun Jun 26, 2022 10:30 pm

I can recommend acridine orange, as previously mentioned its very cheap and emits in multiple parts of spectrum depending on local conditions such as pH. I used to have a small collection of GFP-transgenic Arabidopsis seeds that gave rise to plants expressing GFP in different compartments (sadly I lost it with time). Below is an example of GFP-tagged mitochondria in a root hair cell (Z-stacked), but I did not produce this image with my hobbyist microscope (though GFP could easily be seen with it).

Image
Zeiss Photomicroscope III BF/DF/Pol/Ph/DIC/FL/Jamin-Lebedeff
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