Working distance of V4

[Ji hun Kim]

Hi, I recently started setting up Miniscope V4 and have a question on calculating working distance.

In the Inscopix spec sheet, the GRIN lens has 0 to 200um working distance.

I believe that mean GCaMP positive cells should be between 0~200um from the surface of GRIN lens.

Then how does the electrowetting focusing apply?

The homepages says +/- 200um, then can I image GCaMP cells upto 400 um from the lens surface? 

Thanks.

[Daniel Aharoni]

Hi,

The working distance range of the standard V4 Miniscope is approximately 600um to 900um below the bottom surface of the Miniscope. Relay GRIN lenses (from Inscopix or else where) generally are designed such that a plane roughly 200um below the bottom surface of the GRIN lens is reformed roughly 200um above the top surface of the GRIN lens. You want this reformed top image to sit at around the working distance of the Miniscope. In other words, the gap between the bottom of the Miniscope and the top of the GRIN lens should be around (200um + 750um). Then when you change the focus of the Miniscope using the electrowetting lens, you will be focusing up and down on different image planes reformed by the GRIN lens. You can also adjust the gap between the Miniscope and GRIN lens to push the range of focal planes deeper (by decreasing this gap). 

With all that said, in most cases you will be limited to imaging no deeper than about 250um of tissue due to fluorescence scattering through the tissue. In some cases this can be pushed to 300um or 350um but it will depend on the brain region and the sparsity of expression.

[Ji hun Kim]

Thank you for your explanation!

Would you mind if I ask what happens to the cells closet than the working distance (between 0~200)?

Will they become blurry as they get close to the GRIN lens surface?

Thanks.

2021년 6월 23일 수요일 오전 4시 51분 2초 UTC+9에 dbah...@gmail.com님이 작성:

[Daniel Aharoni]

You should be able to adjust the focal plain of the system (using the EWL and also adjusting the gap between the Miniscope and GRIN if necessary) to image all the way up to ~0um tissue depth. Cells very close to the bottom of the GRIN lens will look really good once in focus because their fluorescence will be going through very little tissue and therefor not scatter much. As you shift the focus away from cells they will become blurry.

[Ji hun Kim]

Thank you!

2021년 6월 26일 토요일 오전 5시 5분 47초 UTC+9에 dbah...@gmail.com님이 작성: