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While Guy is right, one must remember that you may have enough laser power to 
saturate  fluorophores even while exciting them off the peak wavelength, so 
the "60% efficiency" may not matter at all.  Depending on the filter 
characteristics, such off-peak excitation may even help in tackling bleed-
through.
Sudipta
  On Sun, 18 Nov 2012 21:43:12 +0000, Guy Cox wrote
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> 
> If a given laser line is said to be 60% efficient at exciting a 
> fluorochrome, it means that you will get 60% of the number of 
> photons you would get if you excited at the optimum wavelength.  It 
> does NOT mean that you will get 60% quantum yield.
> 
> The two factors that determine how much fluorescence you get are 
> 1 Extinction coefficient (how well the fluorochrome captures photons)
> 2 Quantum efficiency (what fraction of photons absorbed give rise to 
> fluorescence).
> 
> These two factors (which vary widely between fluorochromes)
>  determine much fluorescence you will get at the optimum excitation. 
>  At a 60% efficient wavelength you will get 60% of this.
> 
> I hope this helps,
> 
> 				Guy
> 
> -----Original Message-----
> From: Confocal Microscopy List 
> [mailto:[log in to unmask]] On Behalf Of Aromis Sent: 
> Monday, 19 November 2012 7:38 AM To: 
> [log in to unmask] Subject: Excitation of fluorophores
> 
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> 
> Hello Everyone:
> 
> I'm recently analyzing the bleed-through effects in my multi-color imaging
> analysis and have met a conceptual problem:
> 
> When a certain laser beam excites a fluorophore with 60% efficiency 
> does it mean 60% of the fluorophores are excited and  will emit 
properly/normally?
> 
> Thanks a lot for your feedback in advance.
> 
> Best regards,
> 
> Aro


Dr. Sudipta Maiti
Dept. of Chemical Sciences
Tata Institute of Fundamental Research
Homi Bhabha Raod, Colaba, Mumbai 400005
Ph. 91-22-2278-2716 / 2539
Fax: 91-22-2280-4610
alternate e-mail: [log in to unmask]
url: biophotonics.weebly.com