Re: Overnight scan of human fibroblasts

Posted by Leigh Silvester on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Overnight-scan-of-human-fibroblasts-tp2543999p2544180.html

One answer, although not helpful in the short run is to use a tuneable infra red laser with multiphoton capability.

This can actually excite a wide range of fluorochromes. The excitation wavelength is approximately half that of the set wavelength.

Hence an IR laser pulsing nicely at 750 ish nm will excite DAPI beautifully.

 

As a result of the wavelength you don't get any DNA damage - just slightly warmed cells.

 

However, these lasers do not come cheap, and they are hard work/expensive to keep in running condition - although newer ones may be better.

 

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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Zoltan Cseresnyes
Sent: 27 March 2009 12:19
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

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

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