Forum - About dual calcium imaging

Hi Jys000415,

This is still a work in progress in our lab but I am happy to share with you what we know so far.

• Search on [www.chroma.com Chroma.com] for the filter set that matches the fluorophore you want to image. We have been using filter set 49004 to image tdTomate and other red fluorophores.
• Find a Luxeon Rebel LED that has a peak wavelength inside the band of your excitation filter. There is a whole range of wavelengths of LEDs with the same footprint as our standard blue excitation LED.
• GRIN lenses have significant chromatic aberration which can shift your focal plane by as much as 100um. You will likely need to adjust the focal plane when changing from imaging green to red wavelengths. We are currently working on multiple solutions to this focal plane shift and will share them when ready.

Hi Daniel, have you tried using the new (or older) RCamp with the miniscope?

Do you think the quality improves over GCamp6f?

On a related topic: In the GUI there is the option of using a led for optogenetics. I understand this is not built-in at the current miniscope version but could you give me some info on how you are planning to do it?

As a possibility, I thought I could replace the blue LED with an RGB LED (eg. [this | http://www.digikey.com/product-detail/en/seoul-semiconductor-inc/F50360/897-1016-1-ND/2181964] one I think would fit).

Do you think this would work? In that case, how could the second color be powered? Is there any dedicated pin on the board?

Thanks in advance!

Hi Nikolas,

I think theoretically there are advantages to imaging at longer wavelengths but currently I don't think GCaMP6 out performs red calcium indicators. That being said, we have successfully imaged jRCaMP1a with very promising results.

Concerning optogenetics, the firmware, software, and electronics have all been design to hand an additional optogenetic light source along with the excitation LED. A new version of the scope body needs to be made (we have designed this but haven't machined it) to hold an additional optogenetic LED. We will put up a new page on our wiki once we find some time to pursue this further.

Your suggestion of a RGB LED is a good one but I would be very concerned with the broad spectrum produced by LEDs. I think the only way to both image a calcium indicator and control an opsin is by narrowly filtering the excitation wavelengths to make sure the excitation wavelength of your indicator does not overlap with your opsin. I am not sure how this would be possible on a single RGB LED.

Hi Daniel,

thanks for your response. I will give it a try with jRCaMP1b in the next few months and will report back here with updates.

As for the optogenetics, indeed the broad spectrum can be an issue. I have been thinking of combining such an approach with a the 59004 filter set from Chroma, and the following opsin varieties: a. jRCaMP1b + ChR2 b. GCaMP6f + BreaChes

Looking forward to checking the new page/body once you put it up.