Try to make this brief, currently trying to record action potentials across a neural network, using Di-3-ANEPPDHQ, a voltage sensitive dye which blue shifts during excitation of the cell. My current filter cube has a band pass of 485/20nm, a beam splitter of 510nm, and a long pass of 515nm (see Zeiss filter cube 16). As such, I would expect a reduction in intensity during depolarisation, as the blue shift will reduce the amount of exciting light hitting the sample. Due to limitations of my current camera, I am recording maximally at 500Hz.
I am trying to observe spontaneous action potentials in my neural cultures, however there seems to be no discernible difference between untreated and TTX treated (1uM) recordings (change of F/F0). I have also applied high concentrations of KCl to force depolarisation and that has only reduced signal intensity in a couple of experiments.
Does the problem stem from my current filter set up, or is it due to my low sampling frequency rate? I was planning on moving onto a faster camera next (>2KHz), however I'm anxious that I'm not seeing any change following the KCl. Any suggestions would be greatly appreciated, and if you need to know anything else, please ask.
Hi,
the S/N of this kind of dye is really small so ... recording spontaneous APs could be very difficult as you can't average ... On top of that I think that you'r right that you sampling freq. is too slow. I did this kind of measurements in the past ( spontaneous APs in cultured hippocampal neurons, recorded at 2KHz with a confocal microscope). You will find details here:
http://journal.frontiersin.org/article/10.3389/fncel.2011.00020/abstract
But in any case ... be careful on the toxicity of such dyes ... You'll have to find the correct imaging conditions to limit it.
Hope this helps,
Stephane
Hi,
I see different technical reasons, why you did not record spontaneous APs in your cell culture. Stephane already mentioned two:
1) the S/N of these dyes is small. If you can not correlate your recordings to a stimulus and average it, you will always have difficulties to detect APs.
2) your sampling frequency is too slow. Can you use calcium sensitive dyes? They show brighter and slower signals. If you just want to know, whether or not your neurons are firing, you might be more lucky with such dyes.
3) Most of the voltage sensors show a shift in the excitation spectrum during depolarization. In this case you have to use an excitation wavelenght, which is not at the maximum of the excitation spectrum but in the range of the maximum shift of the spectrum. This is usually at the steepest slope of the excitation spectrum. However, i am not familiar with this dye. Maybe you can get instructions on the right filter set from the vendor.
Hope i could help,
Stefan
Hi there, thanks for the answers guys!
With regards to recording spontaneous AP's, the only issue should be the acquisition rate. I've got a good signal because of where the excitation filter is, and due to recording background as a baseline, depolarisation would (over the course of ~10ms), result in the signal being on par with the background. Forcing the neurons to depolarise continuously using KCl though, regardless of acquisition rate, would surely result in a complete reduction of signal, when looked at over the course of say ten minutes, no?
I've carried out calcium experiments, now trying to move onto voltage sensitive dyes on a microfluidic platform as a novel design which is on par with electrophys.
Have emailed the venodr, so will hopefuly get back, thanks :)
Regards
Chris
I am not expert with dyes, but I suppose your problem is the acquisition rate. An AP has 1 ms durations. Sodium currents less than 1/2 of this. If your dye should change intensity for depolarizing currents you have high probability to loose depolarizing phase of the spike. Good probability can be bursting neuron but I guess would not be a precise method to analyze spike frequency in any case.
I have not used Di-3-ANEPDHQ, but for Di-4-ANEPPS, another commonly used dye, the excitation filter needs to be 520 nm with a width of 40-80 nm; emitting filter RG610. I am not sue 485 nm would work. Also, I would suggest you patch a cell then record from it so you know fore sure it has APS. You can also use spikes to trigger averaging. If you do average, then you should be able to see something even at low sampling rate.
Also, the dye signals can be small, e.g., 0.1-1% of the resting light. so you want the camera to be 12-16 bit, with a well size of 1million or up.
Questions about the EEG hardware may be posted to [email protected]
thanks
Francesco Ferro Milone
Using the ANEPP dyes to pick up spontaneous potentials in single neurons are just difficult due to its small S/N as mentioned before. If you look at the literature, it is often used to looked at population response or to look at APs when you know there is one (e.g., when you are patching a cell). There are 'tricks' to maximize your S/N but you are probably not going to spend that much time and money developing your hardware to do so. Molecular Probe is selling a new class of dye made by Evan Miller at UC Berkeley (VoltageFluor) that will give you better S/N and would give you a better chance at this.
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