LED

[Dechuan Sun]

Hello!

I'm looking for a proper LED to stimulate mCherry and GFP at the same time, and I plan to do the work on miniscope V3. The general RGB LED is not within the excitation range. Is there a secret way to get the LED I needed?

Many thanks,

Dechuan

[Daniel Aharoni]

Hi Dechuan,

We have done this once before (though using tdTomato and not mCherry) using the same standard LED in the V3 Miniscope. This will only weakly excite mCherry but it should be enough to see, especially if you are using it in combination with GCaMP (and not just GFP) since GCaMP is generally a lot dimmer than mCherry to begin with. You need to check to make sure all the optical filters you are using can pass mCherry along with GFP.

[Dechuan Sun]

Hi Daniel.

Thanks for the reply. I want to mark the hippocampal neurons with GCaMP(mark all the neurons) and mCherry(mark only a specific neuron type) At the beginning of the recording, switch on the LED for mCherry to find the position of the specific neuron type and then switch on the LED for GCaMP to record the activities of all the neurons. So I need a special LED that can emit two different colors of light. Because the CMOS sensor is a mono-color one, I'm a little confused about how can you distinguish the neuron type in your experiment? Just set a threshold like the bright is the mCherry and the dim is Gcamp? 

Thanks,

Dechuan

[Daniel Aharoni]

I see. So it sounds like you want to use 2 separate LEDs to excite one or the other fluorophore. So this isn't really possible with the V3 Miniscope but we are actively working on a dual color V4 Miniscope.

With the V3, I would suggest using a color CMOS image sensor and actually excite both fluorophores simultaneously with a single LED. You can then try to use the color filtering of the CMOS sensor to separate the 2. We have tried this in the past with mixed success as the Bayer color filters on image sensors have a lot of spectral overlap with each other and require some tricks in changing color spaces to make things work.

The other major issue here is GRIN lenses cause a large amount of chromatic aberration. This results in a possibly large focal plane shift between green and red fluorescence. The V4 Miniscope uses all achromatic optics which helps here a lot but the V3 requires a rather large diameter GRIN lens which is an issue.

[Dechuan Sun]

Hi Daniel,

Thanks for the suggestion. But I'm a little bit confused about the difference between V3 and V4.  I think the aberration in V3 is caused by the grin lens. If I want to observe the neurons in the hippocampus, I must implant a grin lens into the brain. So even using V4, the grin lens should still incur aberration. Is my understanding right?

[Daniel Aharoni]

Hi,

Yes this is correct but there are a few important points. Chromatic aberration of GRIN lens scales with the lens diameter. The V3 uses a 2mm diameter Objective GRIN lens where the V4 does not. If you were to image cortex through a cranial window then there shouldn't be any chromatic aberration from the V4. If however, you are imaging deeper and are using a relay GRIN lens, then yes there will still be some aberration causing the focal plane to shift between green and red fluorescence. This shift will be pretty small if using a 0.5mm diameter GRIN lens and a bit larger using a 1mm lens. But the V4 also has an electro-tunable lens inside which possibly would allow you to correct for this focal plane shift when imaging red vs green.

[Dechuan Sun]

Thanks Daniel, I will have a try and see how it goes :)

[adrienne kinman]

To follow up on V4 dual color optics - is it know what the ~chromatic aberration is with a v4 scope and/or what the depth of field is generally? I.e. if I image two cell types that are spatially separated and have different flurophores(RFP in one and GFP in another), how far could they be and still be in focus?

Also very interested in how could we could use the electro-tunable lens to correct for the focal plane shift between red and green channels? 

Thanks for all the great info and open source resources! :) 

[Daniel Aharoni]

The V4 optics are chromatic ally corrected and shouldn't have any noticeable focal shift due to wavelength within the visible range. That said, if you image through a relay grin lens, that grin lens will introduce moderate to large chromatic aberration and shift the focal plane between green and red anywhere from between 20um to 80um (depending on the diameter of the grin lens).

For depth fo field, I don't know that number off the top of my head but I think it should be around 30um or so.

Daniel Aharoni
Assistant Professor, Department of Neurology, UCLA
Dbah...@gmail.com
daha...@mednet.ucla.edu

   

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