Re: Overnight scan of human fibroblasts

Posted by Zoltan on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Overnight-scan-of-human-fibroblasts-tp2543999p2545481.html

Dear All,

Thank you all for the excellent ideas!!  To be more precise about our requirements:  we have to use either 405 or 458 (much preferably 405) because of the FRET pair that we are trying to image within the nucleus.  The 405 nm exc. is thus used not to label the nucleus but to excite the donor.   The time resolution is critical, we have to image every selected location every 3-4 minutes at least, otherwise we miss the time course of the process that we are trying to monitor.  The cells sometimes crawl over each other, hence the need to do a 6-um Z stack.
  Overall, we can right away try to lower the laser power and up the gain (we are currently using gains in the 750-900 range,  max. is 1250) to see if we can still resolve the events (the critical areas are only 1-3 um diameter), and we'll try the vitamins as well (C, E).  Our user is also trying to increase the expressed signal levels in the cells.
  Thanks very much everyone!!!

Zoltan

On Fri, Mar 27, 2009 at 4:10 PM, Sylvie Le Guyader <[hidden email]> wrote:

Hi Zoltan

 

I think using a short wavelength could be the problem. Not only will you induce more phototoxicity compared with longer wavelengths but you will also bleach your FRET acceptor and donor which presumably absorb blue light. One solution if you have a 633/640/647 laser is to label your nuclei with a far red fluorophore for live cells. You could use Draq5 http://www.biostatus.com/product/draq5/.

 

 

Med vänlig hälsning / Best regards

 

Sylvie

 

@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader

Dept of Biosciences and Nutrition

Karolinska Institutet

Novum

14157 Huddinge

Sweden

[phone]

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Zoltan Cseresnyes
Sent: 27 March 2009 13:19

To: [hidden email]
Subject: Overnight scan of human fibroblasts

 

Dear All,

One of our users would like to scan cultured human fibroblasts (focusing only on the nucleus) for about 15-17 hours at a time, 6 um Z stack (7 steps), 3-5 min time resolution, in 5% CO2 at 37C.  We have done a few very successful experiments this way already, using our SP5 inverted with an environmental chamber (The Box).  We used 458 nm or 488 nm excitation in those successful experiments at low laser power (<=20%).  Last night we tried 405 nm excitation (20% laser power) as well because we were attempting to do a live FRET scan; the scanned cells all died after about 2 hours.  Do you have any suggestions about how to protect these cells from photodamage for 15-17 hours?  We are trying to keep our lasers low, we were doing 20% power on both the 405 and 514 nm lines.  The temperature is fairly stable at 37C (+-0.8C perhaps) and the CO2 is okay as judged by the colour of the phenol red and the excellent survival of the cells in the non-scanned regions.    In one experiment last week we used 458 nm at 48% power (we had a poor fluorescence signal that time but we tried anyway) and then the scanned cells also died after about 6 hours.  These findings seem to point towards photodamage, hence my enquiry to this excellent forum of experts!
  Thanks very much,

Zoltan

--

Zoltan Cseresnyes
Facility manager, Imaging Suite
University of Cambridge, UK

--

Zoltan Cseresnyes
Facility manager, Imaging Suite