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Messages - hsiao

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1
DAQ Software/Firmware / Event marker
« on: March 10, 2019, 03:18:24 PM »
Hi,

I am wondering if the elapsedTime in settings_and_notes.dat can be millisecond since I was using the note function as an event marker for stimuli. A second resolution is not enough for this.

Any suggestions?

Thank you!

hsiao 

2
General Discussion / Happy New Year 2019
« on: January 01, 2019, 04:26:03 AM »
Hello everyone,

Wish everybody has remarkable success in your experiments.

I'll keep working on Ca imaging this year, too.

I am curious about the difficulty of seeing Ca imaging in mice.
Does anyone like to talk about your percentage of successful seeing signal?

I am especially interested in the dorsal, ventral hippocampus and the amygdala.

I hope someone can give me some directions.


Thanks!

3
Surgery and Baseplating / Re: nothing but gray
« on: August 17, 2018, 02:38:43 AM »
I trimmed the brain today.

I believe the transparent part of the hole is the alveus/str. oriens ( Rat dCA1_5.JPG).
Is it a good place to implant? Or it is too far?

4
General Discussion / Virus injection and lens implantation
« on: August 16, 2018, 08:45:06 AM »
Hi,

I heard some researchers inject the virus and wait for ~2 weeks (or longer) then implant lens and wait for another week.

But the protocols from Shanna et. al. recommend combining them into one surgery.


Does anyone know the pros and cons of doing these protocols separately?

Thank you!

Hsiao

5
Surgery and Baseplating / Re: nothing but gray
« on: August 16, 2018, 05:24:32 AM »

But rat surgery is another story.
I feel like the 1st layer of CC of rats are tough and thick. (plz see the Rat dCA1_1.JPG and Rat dCA1_2.JPG)
After removing them, I saw some thin and transparent layers (plz see the Rat dCA1_2.JPG and Rat dCA1_3.JPG) so I think they are vertical CC and the alveus is below them.
Maybe this is a good place to implant lens?

6
Surgery and Baseplating / Re: nothing but gray
« on: August 15, 2018, 08:32:35 AM »
I did a practice surgery on mouse and rat targeting dCA1 and noticed some difference.

Thank you, Daniel, for reminding me of the tutorial videos.

For mice
Yes, everything just like the videos regarding the aspiration.
I saw two layers of the corpus callosum and I saw the fiber direction and blood vessels clearly under the stereoscope.
Using aspiration tools can easily remove the 1st layer of CC.
I am very excited about seeing this. I also took a picture of it (plz see the attached picture).

7
Surgery and Baseplating / Re: nothing but gray
« on: August 09, 2018, 03:22:13 PM »
Still have no luck on my second rat :'(,

3 weeks after virus injection, I check the image today.

This time, I did not use the objective lens.
The relay lens just sticks out the skull about 1mm.

I think I spotted a thin blood vessel (not sure since it is not clear either) but could not find any white neuron.
I cemented the baseplate anyway because I think maybe the cells will appear next week.


Maybe it is something wrong during the surgery...

I read the instruction again and found one step that I didn't do: Very slowly and carefully remove the horizontal white striations of the corpus callosum until you reach the striations that go vertical. 
Several questions I want to ask about it.

1. How do you remove the corpus callosum? by tweezer, needle or aspiration?
2. How to tell the horizontal and vertical corpus callosum?
3.
During the implantation surgery, will you see blood vessels through the relay lens?
I didn't see any blood vessel on the corpus callosum during the surgery. Is that normal?
Maybe I can't see the blood vessels during implantation and baseplating it is due to lack of removing corpus callosum?
I think it may be good to have a same blood vessel landmark during surgery and baseplating.

Thank you

8
Surgery and Baseplating / Re: nothing but gray
« on: August 01, 2018, 03:30:14 PM »
Hi Kristen,

Do you mind to tell me the information from Tad Blair's lab?

Thanks!

Hsiao

9
Surgery and Baseplating / Re: nothing but gray
« on: July 21, 2018, 07:33:55 AM »
Hi, kjlawlor

Would you mind to share your experience? Like where did you get your viruses and what kind of gene did they express?
How is the histology going?
Which lens did you use as objective and relay lens?

I had my second surgery done and would love to report the outcome 3~4 weeks later
This time I used addgene aav1 https://www.addgene.org/100843/

Thanks

10
Surgery and Baseplating / Re: nothing but gray
« on: July 20, 2018, 02:22:49 AM »
I am really appreciated your inputs.
 :D
Good luck on my second rat

11
Surgery and Baseplating / Re: nothing but gray
« on: July 16, 2018, 10:46:11 PM »
So I think the setup I was using is fine. However, I saw the histology slice yesterday.
It seems that my problem is the implantation.

The lens was above dCA1 cell body over ~500um. I should lower it further.

But here comes other questions.
1. The working distance of #64-519 is 0, so I should implant the lens as close to the cell bodies of dCA1 as possible (at least < 250 um)? If I choose a lens with a 0.2 mm WD, then it gives me more space so that I can implant the lens like 0.2mm or more above the cell bodies?

2. The medial hippocampus is higher than the lateral. Will you tilt the lens a little bit to fit the hippocampus?

3. In the dual lens system, the wiki page uses the title "Imaging With Thin GRIN Lenses". My question is that is it necessary to use a relay lens that is thinner than the objective lens? Will there be any problems if I use a relay lens with the same diameter with the objective lens?

Thank you!

12
Surgery and Baseplating / Re: nothing but gray
« on: July 14, 2018, 09:27:01 AM »
Hi Daniel,

Thank you for your fast reply

I am currently using a setup like attached file Fig1A.
What you mean is I need to use a setup like Fig2A or 2B?

13
Surgery and Baseplating / nothing but gray
« on: July 13, 2018, 06:28:56 AM »
Hi,

I was trying to do my first Gcamp experiment.

I planned to record dCA1 in rats. Since the grin lens (#64-519, Edmund optics) is not long enough, I stacked 2 lenses and screwed them in the bottom of Miniscope as an object lens. Then, I injected AAV into the dCA1 around the stratum radiatum and implanted 1 lens on the pyramidal layer (total 3 lenses, including 2 object and 1 relay lenses). After 3 weeks, I saw nothing. I even canít see the blood vessels. I could only see black and gray under the maximal power and gain. I waited another week but still had no luck. You can check the screenshot below. This is a screenshot after 4 weeks of incubation.

 
I just perfused this rat, no obvious blood clot is noticed. I am still waiting for the histology and want to make sure my Gcamp express well but canít wait to ask questions here for improving the protocol for my second rat.

 

I am not sure if my setup is correct (2 object lenses plus 1 relay lens) but I did test and see clear signals on a paper with green lines. I used some methods like this http://miniscope.org/index.php/Initial_Testing_of_Assembled_Miniscopes

 

Thank you!


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