Virus coated lens

[Md. Mamun Al-Amin]

Hi Folks,

I would like to record calcium activity from pyramidal CA1 neuron using virus coated lens to reduce surgery step. Inscopix supply virus coated lens with baseplate. Is there any major limitation of this type of virus coated lens?

Kind regards

Md. Mamun Al Amin

Postdoctoral fellow

Stark Neuroscience Research Institute

Indiana University

[Matthias Klumpp]

Am Do., 29. Okt. 2020 um 17:12 Uhr schrieb Md. Mamun Al-Amin
<mdalam...@gmail.com>:

>
> Hi Folks,
> I would like to record calcium activity from pyramidal CA1 neuron using virus coated lens to reduce surgery step. Inscopix supply virus coated lens with baseplate. Is there any major limitation of this type of virus coated lens?

[I am coating my own lenses, so I have no experience with the
Inscopix-coated ones, but I expected them to be the same or better
than the manually coated ones]
Other than you potentially needing a bit more virus, and the coating
step taking quite a while (but you can coat multiple lenses at once) I
so far have found no issues. The viral expression is better localized
for my use case, and the tissue (CA1) appears normal as well (I am
still looking for any issues though, just in case).
Having coated lenses greatly simplifies the surgery, so it is
definitely worth trying!

Cheers,
Matthias Klumpp

[Md. Mamun Al-Amin]

Hi Matthias Klumpp,

Thanks for your quick reply.

I am happy to use more virus if I could coat the lens by myself. Is there any specific protocol to prepare virus coated lens?

Thanks.

[Matthias Klumpp]

Am Do., 29. Okt. 2020 um 19:10 Uhr schrieb Md. Mamun Al-Amin
<mdalam...@gmail.com>:
>

> Hi Matthias Klumpp,
> Thanks for your quick reply.
> I am happy to use more virus if I could coat the lens by myself. Is there any specific protocol to prepare virus coated lens?

There is one in Jackman SL, Chen CH, Chettih SN, et al. Silk Fibroin
Films Facilitate Single-Step Targeted Expression of Optogenetic
Proteins. Cell Rep. 2018;22(12):3351-3361.
doi:10.1016/j.celrep.2018.02.081 =>
https://pubmed.ncbi.nlm.nih.gov/29562189/
But the basic idea is to simply slowly mix your virus mix (possibly
pre-diluted) 1:1 with the fibroin (slowly in order to reduce bubbles)
and then apply that in small amount to the lens surface, waiting for
the first drop to dry before applying the second one, so you get
multiple layers of fibroin. The biggest challenge is to get a somewhat
homogenous layer on the lens surface.
Then in theory the lenses could be stored in the fridge for a week (so
I was told by others), but personally I never kept them in the fridge
for more than two days.

Cheers,
Matthias

> On Thursday, October 29, 2020 at 12:21:17 PM UTC-4 Matthias Klumpp wrote:
>>
>> Am Do., 29. Okt. 2020 um 17:12 Uhr schrieb Md. Mamun Al-Amin
>> <mdalam...@gmail.com>:
>> >
>> > Hi Folks,
>> > I would like to record calcium activity from pyramidal CA1 neuron using virus coated lens to reduce surgery step. Inscopix supply virus coated lens with baseplate. Is there any major limitation of this type of virus coated lens?
>>
>> [I am coating my own lenses, so I have no experience with the
>> Inscopix-coated ones, but I expected them to be the same or better
>> than the manually coated ones]
>> Other than you potentially needing a bit more virus, and the coating
>> step taking quite a while (but you can coat multiple lenses at once) I
>> so far have found no issues. The viral expression is better localized
>> for my use case, and the tissue (CA1) appears normal as well (I am
>> still looking for any issues though, just in case).
>> Having coated lenses greatly simplifies the surgery, so it is
>> definitely worth trying!
>>
>> Cheers,
>> Matthias Klumpp
>

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[cke...@gmail.com]

Hey Matthias,

Thanks much for sharing that protocol. I've often wondered how hard this coating process was. Do you typically just do it in a biosafety cabinet and then implant the lens in the next day or two? And has your experience been that this approach is better than trying to do an injection prior to aspiration? I had imagined the main advantage was that they could be coated far in advance in bulk, but presumably in terms of time, coating is just as time consuming as injection...

PS Thanks again for your work with pomidaq!

thanks!

Caleb

[earthw...@gmail.com]

Hey  Matthias 

Thanks so much for sharing such detailed protocol. I would like to know the titer of virus that you mixed with fibroin? I also want to know the concentration of fibroin that you used to mix with virus. Thank you! 

Gaeun Park 

2020년 10월 30일 금요일 오전 4시 1분 19초 UTC+9에 cke...@gmail.com님이 작성:

[Matthias Klumpp]

Am Do., 29. Okt. 2020 um 20:01 Uhr schrieb cke...@gmail.com <cke...@gmail.com>:
>
> Hey Matthias,
>
> Thanks much for sharing that protocol. I've often wondered how hard this coating process was. Do you typically just do it in a biosafety cabinet and then implant the lens in the next day or two?

Yes, that is exactly what I do :-)

> And has your experience been that this approach is better than trying to do an injection prior to aspiration? I had imagined the main advantage was that they could be coated far in advance in bulk, but presumably in terms of time, coating is just as time consuming as injection...

So, for my application I heavily dilute my virus because I need very
sparse labelling of neurons (which usually really isn't what you
want). Using the coating method ensures the diluted viruses are
exactly where I want them. I had troubles with that in the past when
doing injections, with cells not being labeled in the FOV of the lens
- that problem disappeared completely when using coated lenses
(granted, I could also have improved my injection method which
apparently wasn't that great).
If you coat more than one lens, you are likely faster than doing
injections - if you coat just one, the coating method will - in my
experience - be slower for sure.
Another thing to consider though is that coating the lenses isn't time
when an animal is anaesthetized, so you can relax while coating the
lenses and then have a faster surgery the next day. And that's a
pretty neat thing for the experimenter, but also nicer for the animal
:-)

Usually I coat two lenses and then do two surgeries on the subsequent
days. (More is possible any may be even better, but I am admittedly a
bit paranoid about leaving the lenses in the fridge for too long).

> PS Thanks again for your work with pomidaq!

Thank you :-) Hopefully soon with head orientation sensor support for
the V4 scope as well ;-)

Cheers,
Matthias

[Alessandro Di Filippo]

Hi Matthias,

I would like to try your method to reduce the number of surgeries and increase the rate of success.

I ordered the fibroin and I already diluted the virus.

Do you have any suggestion on how practically prepare the mix and put it on the lens?

I checked the paper you suggested and it seems the final result is a sort of bag under the lens.

Is there any risk of it breaking during the insertion?

Thanks!

Ale

[Matthias Klumpp]

Am Mo., 16. Nov. 2020 um 12:35 Uhr schrieb Alessandro Di Filippo
<alessandr...@gmail.com>:

>
> Hi Matthias,
>
> I would like to try your method to reduce the number of surgeries and increase the rate of success.
> I ordered the fibroin and I already diluted the virus.
>
> Do you have any suggestion on how practically prepare the mix and put it on the lens?

I carefully mix it when pipetting the ingredients together. The most
important thing is that you do not get any bubbles, as having those
will result in uneven distribution of the virus.
If you do get bubbles, sometimes centrifuging the Eppendorf tube used
for the preparation works to get rid of them.

When coating, I do stick the lens into Rodico putty, a kind of
cleaning putty usually used by people repairing watches (it's great
stuff, I use it all the time for work on small parts, and when using
it with lenses I get way less dirt on them compared to regular putty -
regular one works too for holding the lens though).

> I checked the paper you suggested and it seems the final result is a sort of bag under the lens.
> Is there any risk of it breaking during the insertion?

I've never had a "bag" under the lens - it's more like a thin film or
ultra-flat doughnut shape (very much dependent on the lens size). The
fibroin film also dries out completely, and I never actually needed to
apply much of it. At the moment, I am using about 8µl (4µl Fibroin +
4µl virus dilution) for two 1mm lenses, which is probably *way* too
much - it feels like I could likely coat 3-4 lenses with that amount.
It does help to experiment with these parameters a bit though.

Good luck with your experiment!
Cheers,
Matthias

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[Alessandro Di Filippo]

Thanks Matthias!

Finger crossed for my try then!

Cheers,

Ale

[Sylar Grayson]

Hi all!

I am recently trying to coat lens by myself.

Do you use fibroin powder, or you just order fibroin solution?

Could you pls tell me the Stock Number of the fibroin?..so that I could order some:)

Thanks,

Kaii

[Raphaël Lavoie]

Sigma:  5154-20ML

https://www.sigmaaldrich.com/catalog/product/sial/5154?lang=en&region=CA&cm_sp=Insite-_-caSrpResults_srpRecs_srpModel_fibroin-_-srpRecs3-1

[Zachary Zeidler]

Similar to a virus coated lens, has anyone tried simply doing a gcamp "injection" onto the surface of the aspirated brain, and then immediately implanting a lens? Functionally, the idea is the same as using a virus coated lens, in that you deliver the gcamp directly to the imaging site, but it would save the step of coating the lens. I have a suspicion that minor bleeding or CSF might wash away the virus and prevent good infection, but in theory it seems like it could work. 

To view this discussion on the web visit https://groups.google.com/d/msgid/miniscope/1b8f792f-dc9d-44f8-8b76-f1da1f607df4n%40googlegroups.com.