How does the flow of calcium ions through neurons cause the dyes to activate? The voltage is extremely small, so the dyes have to be extremely close to the neurons, which would disrupt the cells, so is there any additional process?

Fluorescent protein dyes are used, however this article prompted me to ask this question, which might be different:


  • $\begingroup$ What type of dye are you referring to? Can you name one? $\endgroup$
    – Bryan Krause
    Jul 12 '21 at 15:34
  • $\begingroup$ There are entire families of fluorescent protein dyes used, so it not a clear cut answer, which is why I am asking this question. $\endgroup$ Jul 12 '21 at 20:13
  • $\begingroup$ And I'm asking to narrow it down. Are you talking about calcium dyes? Voltage-sensitive dyes? Bath-applied dyes or genetically expressed? SE Q&A should be specific questions with specific answers, not for questions that beg an entire tutorial for an answer. $\endgroup$
    – Bryan Krause
    Jul 12 '21 at 20:14
  • $\begingroup$ @C-Consciousness Small correction in your description. Fluorescent protein or fluorescent dye. Not Fluorescent protein dye. $\endgroup$
    – Science123
    Jul 13 '21 at 4:07
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    $\begingroup$ I agree with @BryanKrause here. The question is a bit vague and there are multiple questions in a single question. The title says about membrane depolarization based dyes. Description talks about calcium dyes. Also lack of source validating "dyes close to neurons disrupting the cells". Finally as mentioned above either fluorescent protein or fluorescent dye. Fluorescent protein dye is technically confusing. $\endgroup$
    – Science123
    Jul 13 '21 at 4:21

They are using a GCaMP, specifically GCaMP6s and GCaMP6f.

This is a genetically encoded sensor. Cells expressing the gene make a protein. The protein is inside the cell, in the cytoplasm. This protein includes a domain that can bind calcium ions. There are lots of other proteins in a cell that bind calcium, too, so this is just one of them.

When bound to calcium, the protein is in a different conformation than when not bound to calcium. When a particular wavelength of light shines on the calcium-bound protein, it emits a photon of a different color. Two-photon imaging takes advantage of a summation trick in that two longer-wavelength (reddish) photons of light combine to provide enough energy to release a single shorter-wavelength photon (greenish).

No voltage or energy from the cell is needed to operate the fluorophore, except the energy used to synthesize the protein originally and the small voltage changes that cause calcium channels to open and allow calcium into the cell. It only fluoresces because of the excitation light used in the microscope.

It would be possible that if you had too much calcium binding protein around it would actually bind enough calcium to interfere with cellular signaling. However, people use these proteins quite a bit and it doesn't seem to affect function all that much. Lab mice who have these proteins expressed throughout their brains all their life aren't any behaviorally different than wild type animals.

  • $\begingroup$ Thank you for your answer. This is more of a question related to the fluorophore. As I understand, green fluorescent protein transform ultraviolet-blue light to green light, however I am unclear how the protein do the non-linear photon conversion of infrared light. Could you expand on that? Thank you $\endgroup$ Jul 14 '21 at 3:21
  • $\begingroup$ @C-Consciousness The wikipedia page I linked on two photon microscopy explains it fairly well. It's not really anything special about biology, it's a chemistry/physics thing about excited states. Two long wavelength photons can have just as much energy as one shorter wavelength photon so it excites the molecule to the same state from which it then emits a photon is the short story. $\endgroup$
    – Bryan Krause
    Jul 14 '21 at 3:56

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