Several Alexa Fluor dyes extinction coefficients and quantum yields tabulated // new deconvolution papers

[George McNamara]

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Dear Confocal listserv

http://www.abcam.com/index.html?pageconfig=resource&rid=15527 
<http://www.abcam.com/index.html?pageconfig=resource&rid=15527>

Dye Absorption Emission Extinction Quantum Brightness

max (nm) max (nm) coefficient yield (Ec*QY/1000)

Alexa Fluor 405 402 421 35,000 - -

Alexa Fluor 488 495 519 73,000 0.92 67

Alexa Fluor 555 555 565 155,000 0.1 15

Alexa Fluor 568 578 603 88,000 0.69 61

Alexa Fluor 594 590 617 92,000 0.66 61

Alexa Fluor 647 650 668 270,000 0.33 89

Alexa Fluor 750 749 775 290,000 0.12 35


No E.c. or quantum yield, but an interesting new fluorophore from BD
Sirigen is Brilliant Ultraviolet 395 (BUV395),
http://www.bdbiosciences.com/documents/BD_Brilliant_Violet395_Datasheet.pdf
excitation max 348 nm, emission max 395 nm, more usefully with an
emission tail into the blue. I spoke with one of the BD Sirigen chemists
recently, who hinted that the performance would be similar (and maybe
better than) BV421:

http://www.biolegend.com/brilliantviolet

Dye Absorption Emission Extinction Quantum Brightness

max (nm) max (nm) coefficient yield (Ec*QY/1000)
BV421                     405 nm                              421
nm                             2,400,000                          0.6
(in DPBS)     1,560

Sirigen lists the E.c. of BV421 as 407 nm,
http://www.sirigen.com/sirigen_products.html

When I tried BV421 (anti-human CD4 on human PBMCs in PBS), it was very
photostable with an Hg arc lamp, standard Zeiss DAPI filter cube, and
Zeiss widefield microscope, 63x/1.4 NA oil immersion lens. Photobleached
quickly (did acquire nice single focus image) on a Leica SP5 confocal
microscope, 63x/1.4 NA oil immersion lens, with brand new 405 nm laser -
my thanks to BioLegend for the specimen.

**

A couple of papers (open access) that caught my attention this weekend:

High-resolution restoration of 3D structures from widefield images with
extreme low signal-to-noise-ratio.
<http://www.ncbi.nlm.nih.gov/pubmed/24106307>

Arigovindan M, Fung JC, Elnatan D, Mennella V, Chan YH, Pollard M,
Branlund E, *Sedat* JW,*Agard* DA.

Proc Natl Acad Sci U S A. 2013 Oct 22;110(43):17344-9. doi:
10.1073/pnas.1315675110. Epub 2013 Oct 8.

PMID:
    24106307


Towards real-time image *deconvolution*: application to confocal and
STED microscopy. <http://www.ncbi.nlm.nih.gov/pubmed/23982127>

Zanella R, Zanghirati G, Cavicchioli R, Zanni L, Boccacci P, Bertero M,
Vicidomini G.

Sci Rep. 2013;3:2523. doi: 10.1038/srep02523.

PMID:
    23982127

In particular, Figure 4j, right panel,
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3755287/figure/f4/
shows -- for that specimen -- confocal + SGP deconvolution performs
equally well (better than the raw data in 4i, right panel) as CW-STED +
SGP (I realize that their STED may not be performing as well as it could).


http://www.nature.com/srep/2013/130204/srep01208/full/srep01208.html


  Simple super-resolution live-cell imaging based on diffusion-assisted
  Förster resonance energy transfer

    * Sangyeon Cho
      <http://www.nature.com/srep/2013/130204/srep01208/full/srep01208.html#auth-1>,

    * Jaeduck Jang
      <http://www.nature.com/srep/2013/130204/srep01208/full/srep01208.html#auth-2>,

    * Chaeyeon Song
      <http://www.nature.com/srep/2013/130204/srep01208/full/srep01208.html#auth-3>,

    * Heeyoung Lee
      <http://www.nature.com/srep/2013/130204/srep01208/full/srep01208.html#auth-4>,

    * Prabhakar Ganesan
      <http://www.nature.com/srep/2013/130204/srep01208/full/srep01208.html#auth-5>,

    * Tae-Young Yoon
      <http://www.nature.com/srep/2013/130204/srep01208/full/srep01208.html#auth-6>,

    * Mahn Won Kim
      <http://www.nature.com/srep/2013/130204/srep01208/full/srep01208.html#auth-7>,

    * Myung Chul Choi
      <http://www.nature.com/srep/2013/130204/srep01208/full/srep01208.html#auth-8>,

    * Hyotcherl Ihee
      <http://www.nature.com/srep/2013/130204/srep01208/full/srep01208.html#auth-9>,

    * Won Do Heo
      <http://www.nature.com/srep/2013/130204/srep01208/full/srep01208.html#auth-10>

    * & YongKeun Park
      <http://www.nature.com/srep/2013/130204/srep01208/full/srep01208.html#auth-11>

Can someone enlighten me how Cho et al's figure 2 obtained a 3x
improvement in resolution, to 33 nm, by deconvolving their dSOFI output?
http://www.nature.com/srep/2013/130204/srep01208/fig_tab/srep01208_F2.html



Enjoy,

George

[Csúcs Gábor-2]

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Dear All,

I made a quick search but found nothing really useful. My question is the
following: does anyone have some data/literature how efficient microscopes
are in detecting the photons of a fluorescent sample? Obviously the NA and
the QE of the camera (plus the performance filters/dichroics) are
important factors but taken e.g. A single molecule experiment with a 1.49
NA objective and a back-illuminated EM-CCD what ration of the emitted
photons really reach the detector? Of course the transmission of the
objectives and other lenses in the microscopes can vary but is there still
an estimate?

Thanks     Gabor

[Alessandro Esposito]

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Dear Gabor,
    you may find some information in Qiaole Zhang et al  published in J Biomed
Opt "Photon budget analysis for fluorescence lifetime imaging microscopy"

As you said, a major impact is the NA of the objective. For single molecule
detection using top end EM-CCD, you will not lose much at the camera.

However, you will lose quite a bit into the filters and relay optics. I've written
a short description about this in Suppoprting Text S1 here:
<a href="http://www.plosone.org/article/info%3Adoi%2F10.1371%">http://www.plosone.org/article/info%3Adoi%2F10.1371%
2Fjournal.pone.0077392

Cheers,

Alessandro

>*****
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>*****
>
>Dear All,
>
>I made a quick search but found nothing really useful. My question is the
>following: does anyone have some data/literature how efficient microscopes
>are in detecting the photons of a fluorescent sample? Obviously the NA and
>the QE of the camera (plus the performance filters/dichroics) are
>important factors but taken e.g. A single molecule experiment with a 1.49
>NA objective and a back-illuminated EM-CCD what ration of the emitted
>photons really reach the detector? Of course the transmission of the
>objectives and other lenses in the microscopes can vary but is there still
>an estimate?
>
>Thanks     Gabor

[John Oreopoulos]

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Gabor,

I think the reality is that a fluorescence microscope (especially operating in the confocal mode) is actually a very inefficient light filtering device. There is a passage from a book chapter written by Kennith Spring in the book Digital Microscopy (3rd Edition) that states:

"The geometric constraints also imposed by the numerical aperture of the objective lens as well as the inevitable losses in the optical train of the microscope lead to a loss of 80% or more of the original fluorescence signal. Depending on the properties of the detector, the resultant electronic signal may represent as little as 3% or as much as 16% of the total fluorescence emission.”

As you say, the exact value depends on the detector, the optics of the system, etc.

Cheers,

John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2013-11-07, at 7:33 AM, Csúcs Gábor wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All,
>
> I made a quick search but found nothing really useful. My question is the
> following: does anyone have some data/literature how efficient microscopes
> are in detecting the photons of a fluorescent sample? Obviously the NA and
> the QE of the camera (plus the performance filters/dichroics) are
> important factors but taken e.g. A single molecule experiment with a 1.49
> NA objective and a back-illuminated EM-CCD what ration of the emitted
> photons really reach the detector? Of course the transmission of the
> objectives and other lenses in the microscopes can vary but is there still
> an estimate?
>
> Thanks     Gabor

[Arne Seitz]

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Dear Gabor,

in the Handbook of Biological Confocal Microscopy in Chapter 16 ("Fluorophores for Confocal Microscopy: Photophysics and Photochemistry" by R. Tsien, L. Ernst, A. Waggoner) you can find a nice calculation of the efficiency of confocal systems. In essence: Per fluorophore you obtain photoelectron per sweep. This might serve a s a good starting point to make calculations for WF systems.
For the single molecule experiment you should be able to measure the photons which arrive at the camera (as done in many point localization papers). Knowing the fluorophore and the excitation intensity (at least roughly) you should be able to calculate the efficiency of the microscope pathway.

Have a nice day.

Cheers
Arne

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Csúcs
> Gábor
> Sent: jeudi 7 novembre 2013 13:34
> To: [hidden email]
> Subject: Photon detection efficiency of microscopes
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All,
>
> I made a quick search but found nothing really useful. My question is the
> following: does anyone have some data/literature how efficient microscopes
> are in detecting the photons of a fluorescent sample? Obviously the NA and
> the QE of the camera (plus the performance filters/dichroics) are important
> factors but taken e.g. A single molecule experiment with a 1.49 NA objective
> and a back-illuminated EM-CCD what ration of the emitted photons really
> reach the detector? Of course the transmission of the objectives and other
> lenses in the microscopes can vary but is there still an estimate?
>
> Thanks     Gabor

[phil laissue-2]

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These papers might be helpful.

Comparing systems:
Evaluating performance in three-dimensional fluorescence microscopy
JOHN M MURRAY, PAUL L APPLETON, JASON R SWEDLOW, JENNIFER C WATERS
J Microsc. 2007 December; 228(3): 390–405. doi:
10.1111/j.1365-2818.2007.01861.x
PMCID:     PMC2438600

Calibration, theory etc.:
Precise nanometer localization analysis for individual fluorescent probes.
Russell E Thompson, Daniel R Larson, Watt W Webb
Biophys J. 2002 May; 82(5): 2775–2783.
PMCID:     PMC1302065

_____________________________________
Philippe Laissue, PhD, Bioimaging Manager
School of Biological Sciences, Room 4.17
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / (0044) 07842 676 456
[hidden email]
privatewww.essex.ac.uk/~plaissue


On 7 November 2013 12:33, Csúcs Gábor <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All,
>
> I made a quick search but found nothing really useful. My question is the
> following: does anyone have some data/literature how efficient microscopes
> are in detecting the photons of a fluorescent sample? Obviously the NA and
> the QE of the camera (plus the performance filters/dichroics) are
> important factors but taken e.g. A single molecule experiment with a 1.49
> NA objective and a back-illuminated EM-CCD what ration of the emitted
> photons really reach the detector? Of course the transmission of the
> objectives and other lenses in the microscopes can vary but is there still
> an estimate?
>
> Thanks     Gabor
>

[James Pawley]

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>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear All,
>
>I made a quick search but found nothing really useful. My question is the
>following: does anyone have some data/literature how efficient microscopes
>are in detecting the photons of a fluorescent sample? Obviously the NA and
>the QE of the camera (plus the performance filters/dichroics) are
>important factors but taken e.g. A single molecule experiment with a 1.49
>NA objective and a back-illuminated EM-CCD what ration of the emitted
>photons really reach the detector? Of course the transmission of the
>objectives and other lenses in the microscopes can vary but is there still
>an estimate?
>
>Thanks     Gabor

Dear Gabor,

Please excuse the plug but you will find many useful references by
looking at the page numbers called out by the entry "Photon
Efficiency" in the Index of the Handbook, Third edition.

It was an item of major interest since the First Edition.

Regards,

Jim Pawley
--
James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt,
BC, Canada, V0N3A0,
Phone [phone], email <[hidden email]>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146