Posted by
Guy Cox-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/xyzt-image-tp7580102p7580125.html
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This is a REALLY bad idea. Exciting FITC at a 2P wavelength that will also excite DAPI will either give you ultra-rapid fading of your FITC or pathetic signal from your DAPI or (in my experience) both. If you look a the fluorescein spectra from the Webb lab, you'll see that there is a dual peak, one at about 910-920 the other at around 760. This means that the shorter one is clearly accessing a higher excited state. If you excite FITC at the 910 peak you'll get good fluorescence that does not fade.
Guy
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Arvydas Matiukas
Sent: Friday, 5 April 2013 6:26 AM
To:
[hidden email]
Subject: Re: colocalization between confocal and two-photon: voxel matching
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Hi Pascal,
Thanks for the suggestion to acquire both DAPI and FITC signals
in 2P mode. Actually I typically use double DAPI/FITC
configuration with FITC channel disabled.
I agree that by exciting both DAPI and FITC at the same 2P
wavelength provides a perfect colocalization. I will tweak
the settings to get less noisy images.
I did not initially use FITC acquisition in 2P mode because this mode
yields more noisy signal compared with confocal. Additionally,
LSM software was giving incorrect optical slice for 2P mode
which confused me.
Best regards,
Arvydas
>>> Pascal Weber <
[hidden email]> 4/4/2013 2:10 AM >>>
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In fact for colocalization signals, DAPI and FITC, just use the laser 2P. You'll
always have a perfect colocalization. The fluorescence comes as a single dot.
Why would you want to use two different lasers?
For the LSM510 the only way to adjust the filling of the rear lens you have to
ajust the beam size with a lens. I did it and i can modulate the beam size and the
uniformity of the field.
Regards, Pascal