I would like to know if it is possible to use the the calcium indicator dyes to stain tissues in a live animal.
I am not talking about genetically encoded calcium indicators of course. My main interest for this question is common calcium dyes such as Flu 4, etc.
Dear Hanie Kavand,
You may want to look over the data below:
Walter T. Piper
I'm a little confused by the purpose of the question. Fixed tissue is fixed and unchanging. Fluorescent tools like GCaMP are there to detect changes. It seems like a mismatch of methods. If you want to quantify ions or other chemicals in fixed tissue, you'd be better off with analytical chemistry/biochemistry methods. Even with chemistry methods on fixed tissue, you won't be able to establish absolute firing rates of the neurons before death.
https://www.researchgate.net/post/Is_it_possible_to_quantify_calcium_GCaMP6_reporter_from_fixed_tissue
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Vick Gardner
Former Nuclear Reactor Operator at United States Navy (USN) (1966–1973) Author has 2.1K answers and 764.4K answer views11mo
The staining is intended to increase the resolution of areas of the cell which may be of low contrast, making them stand out more and be more easily visible.
https://www.quora.com/Why-do-we-have-to-stain-cells-under-microscopes
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Genetically Encoded Calcium Indicators: A New Tool in Renal Hypertension Research
https://www.frontiersin.org/articles/10.3389/fmed.2019.00128/full
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Haha, thanks Len Leonid Mizrah . Two third of the suggested topics were almost relevant. I am not talking about transgenic calcium indicators, I am mainly looking for calcium dyes.
One big challenge would be the opacity of the tissue. To measure fluorescence, excitation light must reach the fluorescent substance, and the emitted light must reach the detector. In both cases, the tissue would absorb and scatter the light. You would only be able to measure fluorescence from the surface of the exposed tissue. The depth of light penetration increases with the wavelength, so the longer the excitation wavelength of the indicator the better.
A second big challenge would be the background fluorescence from the tissue.
A third challenge would be delivering the calcium-sensitive dye to the tissue in a living animal. I think microinjection has been used for nerve cells.
There is a high concentration of calcium in the extracellular fluid and a low concentration of calcium in the cytoplasm of cells, so any intravenously or intraperitoneally delivered indicator is probably just going to show you the extracellular calcium concentration.
Thank you Adam B Shapiro for the the great reply. I don't have much problem with challenges 1 and 2 as I will be visualizing the tissue through the cornea, which is quite transparent.
I do think the only way is microinjection but due to the clearing of the aqueous humor, playing with the concentration and adjusting it would be challenging.
And the forth challenge is actually the major one. However, I am looking to measure oscillations, but again I suppose each tissue or residing cell would have a certain oscillatory regimen and picking the one which is of interest would be very hard. Except that we also dye the desires cell group and only include the oscillation of the overlapping dyes. That's the only strategy I can think of :)
The problem of clearance of the dye might be reduced somewhat by using a ratiometric dye, which reports the calcium concentration by the ratio of the intensities measured at two wavelengths. This ratio will remain the same for a given calcium concentration even as the dye concentration decreases due to clearance, until the dye concentration gets too low to measure.
Since the dye works by binding calcium, it is important not to allow the dye concentration to get too high relative to the calcium concentration, because then the free calcium concentration will be perturbed.
Another consideration is matching the calcium affinity of the dye to the calcium concentration being measured, so that changes in the concentration are reflected in the signal. Ideally, the Kd for calcium would be approximately equal to the calcium concentration.
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