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April 2009

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Subject:
From:
Cameron Nowell <[log in to unmask]>
Reply To:
Confocal Microscopy List <[log in to unmask]>
Date:
Sat, 25 Apr 2009 10:09:01 +1000
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Hi Shiva,
 
Thanks for the references, this is exactly what i was looking for. We even have the same microscope system:)
 
 
Cam
 
 

________________________________

From: Confocal Microscopy List on behalf of Shivaprasad Bhuvanendran
Sent: Sat 25/04/2009 2:42 AM
To: [log in to unmask]
Subject: Re: Multiple Fluorophores and Two Photon Imaging


Hi Cameron,

We have used ECFP, EGFP, EYFP and DsRed at the same time for intravital multiphoton imaging of Lymph nodes using the Olympus Fluoview 1000MPE with a SpectraPhysics MaiTai laser set to 910nm.   
Dichroic Mirrors 505 and 570 were used along with bandpass filters 460-510, 515-560 and 570-625 for CFP, YFP and DsRed respectively. The GFP was collected in both the CFP and YFP channels and could be distinguished both by the morphology and the distinct color. 

If you need further details, you can look up the publication cited here or contact me offline. 
 
Science April 2009 Liu et al., pp. 392 - 397 
http://www.sciencemag.org/cgi/rapidpdf/1170540.pdf
http://www.sciencemag.org/cgi/data/1170540/DC1/1


Regards,
Shiva



Shivaprasad Bhuvanendran
Research Support Specialist 
Bio-Imaging Resource Center
The Rockefeller University

1230 York Avenue
Box 209 (DWB 201)
New York NY 10065

tel +1 212 327 7487
fax +1 212 327 7489


On Thu, Apr 23, 2009 at 7:20 PM, Cameron Nowell <[log in to unmask]> wrote:


	Howdy List,
	
	Has anyone out there had any experience with imaging multiple
	fluorescent constructs in the one sample using two photon microscopy? I
	have a user that already has a zebrafish with GFP and DsRed in it and
	would like to add a third fluorophore if possible.
	
	Does anyone know if this is possible to image, or have any ideas on a
	good choice for the third fluorophore?
	
	A blue protein like CFP would be ideal as its 2PE is ~800nm, leaving GFP
	at 920nm and DsRed at ~980nm. But my experience in the past with CFP
	under single photon and widefiled fluorescence has been that it is quite
	dim. Has anyone used CFP under 2PE before?
	
	
	Any advice would be greatly appreciated.
	
	
	Thanks
	
	
	Cam
	
	
	
	Cameron J. Nowell
	Microscopy Manager
	Centre for Advanced Microscopy
	Ludwig Institute for Cancer Research
	PO Box 2008
	Royal Melbourne Hospital
	Victoria, 3050
	AUSTRALIA
	Office: +61 3 9341 3155
	Mobile: +61422882700
	Fax: +61 3 9341 3104
	Facility Website
	
	
	
	
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