Excitation of fluorophores

[Aromis Storm]

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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  

[George McNamara]

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Hi Aro,

A quantum yield (Q.Y.) of 0.6 means that 60% of the time, an excited
fluorophore will emit a photon. The other 40% of the time, the energy is
lost non-radiatively. This is a different issue from having a
fluorophore molecule absorb a photon in the first place. Once in the
excited state (strictly speaking, once the molecule is in the S1 singlet
state), emission properties are independent of what wavelength was used
to excite it (Kasha's rule ... being a rule, there are a few exceptions).

When examining excitation wavelength vs absorption spectrum (or
excitation spectrum) if you are using  monochromatic light at the
excitation peak, then Q.Y. applies. for example, at

http://www.spectra.arizona.edu/

Alexa Fluor 488 H2O has an excitation maximum of ~492 nm. Using 488 nm
laser, you get 94% efficiency with respect to absorption of one photon.
476 nm light is 57.5% efficient. So, if you can only put one photon into
the specimen, ideally use 492 nm (488 nm close).

In practice, if you want to get the same fluorescence intensity out of a
specimen, you could use 488 nm or use higher power (more photons/second
or per um^3) at 476 or 458 nm.

***

At high laser power, non-linear effects happen enough to matter. In
particular,
1. a molecule in an excited state (typically S1 singlet state, lifetime
usually a few nanoseconds, that is ~1e-9 seconds) can absorb another
photon(s) to go to a higher excited state (S2, S3, etc), which is (are)
chemically highly reactive: often results in destruction of the molecule
("photobleaching"). And/or can go to a relatively long lived triplet
state, and stay there (more precisely, population in equilibrium) for
the duration of illumination. This results in loss of emission
intensity, since triplet state(s) do not fluoresce.
   Consequence: on a confocal microscope, it is better to scan fast
(8000 Hz * 100 exposures results in more photons than 1 exposure at 80 Hz).
2. multiphoton excitation can occur - sometimes useful (~980 nm can
excite Alexa Fluor 488, if two photons are absorbed "simultaneously"
[where simultaneous is around ~10e-12 seconds or less ... typicaly MP
laser has ~200 femtosecond duration pulses]. Can also result in greater
photobleaching (not usually an issue, but worth mentioning).

***

Excel file (xlsx inside zip) of most of the "spectra.arizona.edu" data
is available for download at
http://works.bepress.com/gmcnamara/9/

***

Another way to increase brightness in cells is to localize the signal,
as in my Tattletales concept -- also nice because it greatly facilitates
multiplexing reporters:
http://home.earthlink.net/~pubspectra/McNamara_20121023Tue_Tattletales_GFP_Public_Domain.jpg 

For a presentation about Tattletales, I calculated

1 uM GFP, 0.6 molecules/um^3, is approximate detection limit vs
autofluorescence (Niswender et al 1995 /J Microscopy/) (autofluorescence
can be lowered by using riboflavin free media for a day or so; also
increases in cells stability of blue-yellow FPs - see DMEMgfp at
www.evrogen.com).

1 *molecule *in diffraction limited volume (200x200x600 nm) is ~40 *uM**
*(0.2x0.2x0.6 um = 0.024 um^3).

1 *molecule *in OMX (or other 3D-SIM) nanoscope volume (8x higher
resolution) is~320 *uM** *(0.1x0.1x0.3 um = 0.003 um^3).

Loci will be essentially stationary (for short acquisition time), can be
imaged longer time(s) to improve signal to noise ratio.

    With respect to 3D-SIM, I am especially looking forward to doing
    this on Andrew York and Hari Shroff's MSIM2, which reduces original
    MSIM (York et al 2012 Nature Methods) 225 exposures 9of say 40 ms),
    down to a single exposure of 40 ms (25 fps), while reatining MSIM's
    other benefits. I think MSIM2 will kill the spinning disk confocal
    market - and hopefully take a big dent out of the laser scanning
    confocal market.
    I showed the poster to Ann Plant of NIST - she immediately "got it"
    and suggested that several loci with defined numbers of operator
    sites (256, 128, 64, 32, etc) would make for nice in cell intensity
    standards. Would also be a way to test the on:off ratio of
    photoswitchable etc FPs.

As mentioned in the Tattletales poster, not necessary to have just one
fluorescent protein per DNA binding protein. Steve Vogel and colleagues
have published Venus6 (V6) - and made it available on addgene.org (I
suggested he make V8 and V12 versions ... other colors and color
combinations would be nice).

Enjoy,

George




another way to get brighter signal per pixel is to rethink the biology.
I recently placed online a poster on how to multiplex fluorescence
reporters.
http://home.earthlink.net/~pubspectra/McNamara_20121023Tue_Tattletales_GFP_Public_Domain.jpg



On 11/18/2012 3:38 PM, Aromis wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>
>    

[Guy Cox-2]

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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:[hidden email]] On Behalf Of Aromis
Sent: Monday, 19 November 2012 7:38 AM
To: [hidden email]
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  

[Sudipta Maiti]

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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

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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:[hidden email]] On Behalf Of Aromis Sent:
> Monday, 19 November 2012 7:38 AM To:
> [hidden email] Subject: Excitation of fluorophores
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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: [phone]
alternate e-mail: [hidden email]
url: biophotonics.weebly.com