I use the red dye, Di-4-ANEPPS for my ex vivo rat slices. RH-1691 washes off pretty easily in aCSF whereas DI-4-ANEPPS is more lipophilic and is able to stay embedded in the cell membranes for longer. Plus, it was specifically made to to be used in vivo as it is less affected by the brain pulsation artefact and so there is no real need to use it in vitro.

For incubation time, I've found roughly 20-25 minutes of incubation time in the dye solution works extremely well with just a quick rinse in aCSF to wash it off. 

As for excitation, we use a  filter to emit green light (530 nm) and then the emitted fluorescence is then filtered >590 nm high pass filter.

I've attached a nature protocol that goes over the technique in pretty good detail, although they seem to favour RH-1691 as well. From our lab's experience, Di-4-ANEPPS is much better for in vitro VSDI experiments and have found that RH-1691 might actually give an enhanced signal to noise ratio in in vivo preparations (in the 2nd paper I linked).

Article In vitro functional imaging in brain slices using fast volta...

Article An evaluation of in vivo voltage-sensitive dyes: Pharmacolog...

Hi Alba, answers to your questions. 1. we use a different dye, RH482, (NK3630), which is an absorption dye. The good thing about absorption dye is that it is not excited by the light so is not phtotoxic. Another good thing about NK3630 is that it works in near infrared, 705-720nm.  In contrast,  Di-4-ANEPPS is a fluorescent dye excitation 520nm emission 610 and longer. It has larger signals but the recording time is shorter due to bleaching and phototoxicity. 2. We incubate 2 hrs in diluted dye solution. In order to incubate slice in a long time you will need to circulate the ACSF to keep the slice viable. 3. all dyes work on exact wavelength. RH482 works on 705 and longer, RH1691 works on excitation 630 and emission >690; Di-4-ANEPPS works on excitation 520 and emission > 610. you can get LED light to work. Laser sometimes are noisier then LED. We had a method paper for RH482 :

Jin, W., Zhang, R.J., and Wu, J.Y. (2003). Voltage-sensitive dye imaging of population neuronal activity in cortical tissue. J Neurosci Methods 115, 13-27.

A method paper for in vivo RH1691:

Lippert, M.T., Takagaki, K., Xu, W., Huang, X., and Wu, J.Y. (2007). Methods for voltage-sensitive dye imaging of rat cortical activity with high signal-to-noise ratio. J Neurophysiol 98, 502-512.    

Hi,

Thank you both for answering, this will be very helpful. I have a few more questions regarding the dyes you use:

Alastair, from what company do you buy Di-4-ANEPPS? How do you dilute it and storage it? Do you use Neuroplex to record and analyze the VSD signals? What frame interval do you typically use and how many frames per ms?

Thank you very much again fror your help.

Cheers

Alba

Hi Alba, we buy ours from Sigma and dissolve it in a solution of 7% cremophore EL and DMSO. It's approximately 1.5 ml of that solution to dissolve 5 mg of the dye. In powder form, we store it at 2-8 degrees Celsius with the vial wrapped in foil so as to minimise the amount of light exposure, however once dissolved we keep it at room temperature, with the vial of the solution kept inside another opaque container that itself contains desiccant in it. 

I'm afraid I can't help with you Neuroplex, as we use the BrainVision Ultima and MiCam02 cameras that come with their own software. In regards to frames, with the MiCam02, I record at 2.2 frames per ms for a length of 256 ms for a total of 30 repeats.

I hope that helps.

Alastair

Hi,

Thanks for your answers! I just ordered DI-4-ANEPPS, we'll see if it works ;) 

Is the dilution that you typically apply to incubate the slices in aCSF around 1 uM at room temperature? What dischoic mirror and amplifier gain do you use?

Thank you very much!

Alba

Hi Alba,

The solution we apply to the dye is 0.75 ml FBS, 0.75 ml aCSF and 80 uL of the di-4-ANEPPS solution, which should bring the total concentration of di-4-ANEPPS to 0.2 mM

As for the dichroic mirror, we use one that emits green (530 nm) light and filters the emitted fluorescence through a >590 nm high pass filter. As for amplification gain, I'm not sure if we use any sort of amplification in the hardware for VSDI, only for e-phys experiments (which I'm not too familiar with).

Hope that helps

Alastair

Hi again Alastair,

Do you think I could use 20% Pluronic F-127 in DMSO to prepare the DI-4-ANEPPS stock solution instead of 7% cremophore EL and DMSO? Will it work fine in rat slices?

Thank you,

Alba

Hi Alba, I would think so, although I haven't had much experience with it myself. From a quick search and given it's use in calcium imaging, it should be ok.

Hi,

Do you typically record movies exciting all the time or you it's better to use frame intervals?

Thanks!

Alba

Hi again Alba, We recorded in intervals, typically we aligned our stimulation protocol to the imaging software to make discrete recording periods. We used a stimulation of 30V every 28s for 30 repeats as we found it gave a nice compromise between getting a clean signal and allowing us to carry out multiple experiments in a day. But you can use as many repeats as you want.

This paper introducing the MiCam system has a nice figure that explains the need for averaging multiple images. (Figure 3C)

Article Quantification of optical signals with electrophysiological ...

Hi!

Do you know why is it necessary to use Fetal Bovine Serum to prepare Di-4-ANEPPS staining solution? Could I use just ACSF?

Thank you,

Alba

Hi Alba, as far as I'm aware, the FBS just acts a medium for the dye. I personally don't know if just using aCSF would result in less staining and therefore a reduced response.

I'm not sure if the answer still matters, I think 1691 is mainly an in-vivo dye, where its red absorption doesn't interfere with blood. For slices, I'd recommend one of the slice dyes people are using.

Of course, the signal is tiny. It needs sufficient excitation light to come out of shot noise and a camera with a sufficient well depth to go with that (typical CCD or CMOS microscope cameras are not suitable due to optimization for low dark noise which is irrelevant for VSDI).

Laser excitation is generally a very bad idea for VSDI due to the inherently large noise in a laser. It might be necessary for specific applications but should be substituted whenever possible with LED - or better - filtered filament bulb light, which are both incoherent and much more easily stabilized.