Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Sorry, no TIRF experience, but we recently purchased a 561 nm DPSS (diode
pumped solid state) laser for widefield imaging. The most powerful I could
find about 9 months ago was the 75mW Jive laser from Cobolt (www.cobolt.se).
We also have ordered a 100mW 491nm DPSS laser from the same manufacturer.
Good pricing, very small laser housing, very easy to use, basically just
plug and play.

We are using an AOTF (AA optoelectronics, www.a-a.fr) to control the
individual laser lines and intensities. (same AOTF as used in most confocals
I believe)

The 561nm DPSS laser on our Nikon confocal is from Coherent I believe, but
is not as powerful (10mW).

Best regards,

Kevin Braeckmans, Ph.D.
Lab. General Biochemistry & Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Ghent
Belgium
Tel: +32 (0)9 264.80.78
Fax: +32 (0)9 264.81.89
E-mail: [log in to unmask]
 

> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List 
> [mailto:[log in to unmask]] Namens Christophe Leterrier
> Verzonden: maandag 11 september 2006 10:43
> Aan: [log in to unmask]
> Onderwerp: Lasers for TIRFM
> 
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> 
> Hi everybody
> 
> First, thanks to the pepole who replied to me concerning the 
> live imaging microscope I'm about to set up. I will try to 
> add TIRF (Total Internal Reflexion Fluorescence) to this 
> setup, and I think it's easier to do TIRFM with lasers (I 
> know Olympus has a widefiled TIRF solution from a regular 
> illumination lamp and ring-shaped patterns...). The thing 
> here is to choose the right lasers (I'm prefering solid-state 
> lasers for their ease of use and low heating):
> 
> - which wavelenght ? I want to do 2-color imaging, assuming 
> my fluorophores will be eGFP (beacause we already have tons 
> of constructs with that) and a red fluorophore (I've tested a lot,
> DsRed2/HcRed/mRFP/mCherry) and mCherry seems to be the best. 
> So one 488nm line, but the other laser can be 532 nm (cheap 
> doubled YAG) or 591 nm diode, or maybe something inbetween ? 
> Any advice or feedback about the laser wavelenght for 
> red-fluorescence TIRF would be appreciated.
> 
> - which power ? Diode lasers start at 25 mW, but can be found 
> at 50 or 100 mW. What are the power requirements for 
> GFP-based probes TIRF, assuming it will image constructs 
> expressed in hippocampal neurons (not so bright...). It woud 
> be a waste to have a laser that is too weak to image my 
> cells, but also to have a powerfull (and expensive) laser 
> that will instantly bleach (or worst, kill) the cells and 
> will always be behind a neutral density filter...
> 
> Thank you all for your time and consideration,
> 
> Christophe Leterrier
> 
> Equipe Canaux Ioniques et Polarité Neuronale :: Bénédicte 
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> Ioniques :: Mike Seagar Institut Jean Roche :: Université de 
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> Marseille Cedex 20 Téléphone 04 91 69 89 30 :: Fax 04 91 09 
> 05 06 site web : http://ifrjr.nord.univ-mrs.fr email : 
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