<p>When using a genetically encoded fluorescent dye like <a href="https://en.wikipedia.org/wiki/GCaMP" rel="nofollow noreferrer">GCaMPs</a>, the calcium-sensitive proteins are <strong>produced in and located inside the cell</strong>. That's where they have to be because they are measuring the calcium concentration inside the cell.</p>
<p>When using an injected dye like Oregon Green BAPTA, it's common to use an AM ester form of the dye (for example see this product from ThermoFisher: <a href="https://www.thermofisher.com/order/catalog/product/O6807#/O6807" rel="nofollow noreferrer">https://www.thermofisher.com/order/catalog/product/O6807#/O6807</a> - also not meaning to promote any particular merchant, you can search &quot;oregon green BAPTA AM&quot; and get other results). The esterified version of these dyes are cell-permeant and then are cleaved by an intrinsic esterase to become non-cell-permeant. This means that again, the <strong>fluorescent calcium sensor molecules are within the cells where you want them to be so that they measure internal calcium concentrations</strong>. Any AM dye that remains in the extracellular space will not fluoresce without the esterase, which is good because that would otherwise contribute to unwanted background signal.</p>

CC BY-SA 4.0
2021-07-12T22:58:48.470Z
2021-07-12T22:58:48.470Z
Bryan Krause
<p>Considering the minute electrical excitation produced by neural cells needed to excite fluorescent dyes, how close are they to these cells, considering that being too close will disrupt cell processes. Is there any ratio of how many fluorescent proteins are excited by each cell?</p>

CC BY-SA 4.0
2021-07-12T17:16:25.203Z
This seems highly overlapping with https://biology.stackexchange.com/questions/101632/what-is-the-process-by-which-fluorescent-dyes-in-two-photon-microscopy-are-optic What sort of fluorescent dyes are you talking about? Often dyes used to measure neural activity are intracellular, so I suppose you can't get any closer to a cell than being literally inside it...
Bryan Krause
CC BY-SA 4.0
2021-07-12T20:40:38.743Z
Well, two photon microscopy can only image inside tissue up to one millimetre is size, of which neural cells being almost one order of magnitude smaller than that, so I do not think they go inside the cell
C-Consciousness
CC BY-SA 4.0
2021-07-12T20:43:26.150Z
See my answer on your question on biology. The fluorescent proteins they are using are definitely inside of cells. I do not understand the logic in your comment: yes, you can image a millimeter deep, so any fluorescent protein has to be within that millimeter. A lot of that space is inside of cells.
Bryan Krause
<neuroscience><neurophysiology><neuroimaging>
How close are fluorescent dyes to individual neurons in two photon microscopy?
C-Consciousness
# How close are fluorescent dyes to individual neurons in two photon microscopy?

(Asked by: username_0 on 2021-07-12)

Considering the minute electrical excitation produced by neural cells needed to excite fluorescent dyes, how close are they to these cells, considering that being too close will disrupt cell processes. Is there any ratio of how many fluorescent proteins are excited by each cell?

**Tags:** neuroscience, neurophysiology, neuroimaging

> **Comments:**
> **username_1**: This seems highly overlapping with https://biology.stackexchange.com/questions/101632/what-is-the-process-by-which-fluorescent-dyes-in-two-photon-microscopy-are-optic What sort of fluorescent dyes are you talking about? Often dyes used to measure neural activity are intracellular, so I suppose you can't get any closer to a cell than being literally inside it...  
> **username_0**: Well, two photon microscopy can only image inside tissue up to one millimetre is size, of which neural cells being almost one order of magnitude smaller than that, so I do not think they go inside the cell  
> **username_1**: See my answer on your question on biology. The fluorescent proteins they are using are definitely inside of cells. I do not understand the logic in your comment: yes, you can image a millimeter deep, so any fluorescent protein has to be within that millimeter. A lot of that space is inside of cells.  

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## 1 answers

### Answer by username_1 on 2021-07-12. Score: 1

When using a genetically encoded fluorescent dye like [GCaMPs](https://en.wikipedia.org/wiki/GCaMP), the calcium-sensitive proteins are **produced in and located inside the cell**. That's where they have to be because they are measuring the calcium concentration inside the cell.

When using an injected dye like Oregon Green BAPTA, it's common to use an AM ester form of the dye (for example see this product from ThermoFisher: <https://www.thermofisher.com/order/catalog/product/O6807#/O6807> - also not meaning to promote any particular merchant, you can search "oregon green BAPTA AM" and get other results). The esterified version of these dyes are cell-permeant and then are cleaved by an intrinsic esterase to become non-cell-permeant. This means that again, the **fluorescent calcium sensor molecules are within the cells where you want them to be so that they measure internal calcium concentrations**. Any AM dye that remains in the extracellular space will not fluoresce without the esterase, which is good because that would otherwise contribute to unwanted background signal.


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