why does high NA excitation illumination give better resolution in fluorescence microscopy?

[Daniel White-2]

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Can can anyone describe, as if to an idiot, in simple terms:

Why does high NA excitation illumination give better resolution in
fluorescence microscopy?

Simplistically, it seems that only the NA of detection/emission matters....
since the fluorescence absorption/excitation and emission processes are
separated in time, and the excited fluorophore emits light in all
directions...?

Is it something to do with the fact that there is anisotropy in the
emission process direction, and that the fluorophore can move between
excitation and emission?

Is it explained in Pawley's confocal handbook? If so, what page?

cheers

Dan

[Guy Cox-2]

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Geometrically, obviously (as you say) it doesn't.  But practically attainable resolution depend on the signal to nose ratio, and that is typically a limiting factor in fluorescence.  High NA illumination will improve the S/N ratio, and therefore the real world resolution.

                                        Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Daniel White
Sent: Sunday, 2 February 2014 11:57 PM
To: [hidden email]
Subject: why does high NA excitation illumination give better resolution in fluorescence microscopy?

*****
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*****

Can can anyone describe, as if to an idiot, in simple terms:

Why does high NA excitation illumination give better resolution in fluorescence microscopy?

Simplistically, it seems that only the NA of detection/emission matters....
since the fluorescence absorption/excitation and emission processes are separated in time, and the excited fluorophore emits light in all directions...?

Is it something to do with the fact that there is anisotropy in the emission process direction, and that the fluorophore can move between excitation and emission?

Is it explained in Pawley's confocal handbook? If so, what page?

cheers

Dan

[Mark Adelman (Work)]

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Some follow-on questions.  Asked by a relative novice, so please be  
gentle.

1.  Assuming we are talking about epifluorescence, isn't there just  
one NA in play - i.e. that of the objective?  Or are we also  
considering the aperture of the illumination path by which the bfp of  
the objective is 'filled'?

2.  Why should high NA illumination give a higher S/N ratio?  It seems  
to me (recall - very naive) that a lower NA illumination will excite  
fluorophores adequately (so long as one is not using a REALLY small  
cone of illumination) and that anything beyond that would just give  
more 'stray' light (unless the dichroics are 'perfect') and hence a  
lower S/N ratio.

3.  More to the point that Dan originally raised.  How 'certain' is it  
that high NA illumination gives better resolution in fluorescence  
microscopy?  This is perhaps bridging Dan's question and Guy's reply,  
but are we talking about theoretical resolution or 'practical'  
resolution.  Can someone provide a reference to this?

Mark Adelman

On Feb 2, 2014, at 9:09 AM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Geometrically, obviously (as you say) it doesn't.  But practically  
> attainable resolution depend on the signal to nose ratio, and that  
> is typically a limiting factor in fluorescence.  High NA  
> illumination will improve the S/N ratio, and therefore the real  
> world resolution.
>
>                                        Guy
>
> Guy Cox, Honorary Associate Professor
> School of Medical Sciences
>
> Australian Centre for Microscopy and Microanalysis,
> Madsen, F09, University of Sydney, NSW 2006
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ] On Behalf Of Daniel White
> Sent: Sunday, 2 February 2014 11:57 PM
> To: [hidden email]
> Subject: why does high NA excitation illumination give better  
> resolution in fluorescence microscopy?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Can can anyone describe, as if to an idiot, in simple terms:
>
> Why does high NA excitation illumination give better resolution in  
> fluorescence microscopy?
>
> Simplistically, it seems that only the NA of detection/emission  
> matters....
> since the fluorescence absorption/excitation and emission processes  
> are separated in time, and the excited fluorophore emits light in  
> all directions...?
>
> Is it something to do with the fact that there is anisotropy in the  
> emission process direction, and that the fluorophore can move  
> between excitation and emission?
>
> Is it explained in Pawley's confocal handbook? If so, what page?
>
> cheers
>
> Dan

[George McNamara]

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Hi Mark,

Since it is possible to illuminate the specimen from directions other
than the objective lens: resolution of a single fluorophore is only
dependent on the emission path numerical aperture. As for excitation
power and anisotropy (polarization states), more power from more
orientations, can get the same amount of excitation events from low NA
lenses. Or don't excite through the objective lens as in the references
below.

Too bad not many experiments involve looking at only one fluorophore.


Some (hopefully) useful literature:

Tran, P.T., Chang F. Transmitted Light Fluorescence Microscopy
Revisited. 2001 Biol. Bull.  201: 235.
http://www.biolbull.org/content/201/2/235.long

Brunstein M 2013 Combined evanescent-wave excitation and
supercritical-angle fluorescence detection improves optical sectioning.
arXiv.org
http://arxiv.org/ftp/arxiv/papers/1302/1302.1615.pdf
Addresses some of your original query.
Also, their table 2 shows that the effective NA (measured NA) of 5 high
NA objective lenses are not the values inscribed on the lens. the last
entry is inscribed 1.49, measured 1.474 +/- 0.007. So: don't believe
what you read on the web page, brochure, quote or side of the barrel.

http://www.tirf-labs.com/  and its founder, Alex Asanov's research
papers (Alex told me about the Brunstein work). Most useful may be
Alex's article on stray light causing errors in objective lens TIRF
http://www.tirf-labs.com/news.html
http://www.tirf-labs.com/Stray_light_cuases_errors_oTI...pdf
Not currently well explained on his web site it that his light guide
TIRF (lg-TIRF) can also illuminate a specimen with a thin sheet of
illumination. Contact Alex directly for details or check out
http://www.tirf-labs.com/_1st_pages_7reprints.pdf



Enjoy,

George


On 2/2/2014 10:22 AM, Mark Adelman (Work) wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Some follow-on questions.  Asked by a relative novice, so please be
> gentle.
>
> 1.  Assuming we are talking about epifluorescence, isn't there just
> one NA in play - i.e. that of the objective?  Or are we also
> considering the aperture of the illumination path by which the bfp of
> the objective is 'filled'?
>
> 2.  Why should high NA illumination give a higher S/N ratio?  It seems
> to me (recall - very naive) that a lower NA illumination will excite
> fluorophores adequately (so long as one is not using a REALLY small
> cone of illumination) and that anything beyond that would just give
> more 'stray' light (unless the dichroics are 'perfect') and hence a
> lower S/N ratio.
>
> 3.  More to the point that Dan originally raised.  How 'certain' is it
> that high NA illumination gives better resolution in fluorescence
> microscopy?  This is perhaps bridging Dan's question and Guy's reply,
> but are we talking about theoretical resolution or 'practical'
> resolution.  Can someone provide a reference to this?
>
> Mark Adelman
>
> On Feb 2, 2014, at 9:09 AM, Guy Cox wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Geometrically, obviously (as you say) it doesn't.  But practically
>> attainable resolution depend on the signal to nose ratio, and that is
>> typically a limiting factor in fluorescence.  High NA illumination
>> will improve the S/N ratio, and therefore the real world resolution.
>>
>>                                        Guy
>>
>> Guy Cox, Honorary Associate Professor
>> School of Medical Sciences
>>
>> Australian Centre for Microscopy and Microanalysis,
>> Madsen, F09, University of Sydney, NSW 2006
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Daniel White
>> Sent: Sunday, 2 February 2014 11:57 PM
>> To: [hidden email]
>> Subject: why does high NA excitation illumination give better
>> resolution in fluorescence microscopy?
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Can can anyone describe, as if to an idiot, in simple terms:
>>
>> Why does high NA excitation illumination give better resolution in
>> fluorescence microscopy?
>>
>> Simplistically, it seems that only the NA of detection/emission
>> matters....
>> since the fluorescence absorption/excitation and emission processes
>> are separated in time, and the excited fluorophore emits light in all
>> directions...?
>>
>> Is it something to do with the fact that there is anisotropy in the
>> emission process direction, and that the fluorophore can move between
>> excitation and emission?
>>
>> Is it explained in Pawley's confocal handbook? If so, what page?
>>
>> cheers
>>
>> Dan
>

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/26/

[Guy Cox-2]

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1.  The high illumination NA will only be realized if the bfp of the objective is filled by the epi-illuminator.  Most illuminators have the appropriate optics for this.  It's the achieved NA that is relevant, not the actual NA of the objective.  These should be the same unless you have seriously maladjusted the illuminator.  

2.  If you are saturating the excitation of the fluorophores then getting more light there won't help, as you say.  I was assuming that working with real biological samples at the limits of resolution you are not.  If inserting a neutral-density filter, or reducing the illumination NA with an iris, does not reduce the brightness of your fluorescence, leave the filter in!  It's not just the risk of light beating the dichroic and the barrier filter, it's also going to bleach your fluorochrome.  

3.  The signal to noise ratio required to practically achieve a given resolution is certainly documented but I'm not in my office so can't cite a reference.  Jim Pawley can, I'm sure.  Jim?   I'll just remind you that at the Rayeigh limit the contrast is very low - the intensity only drops to 3/4 of the peak between the two points.

Apologies for writing signal to nose ratio in my previous post, but I'm sure it gave many folks a laugh.

                                                     Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mark Adelman (Work)
Sent: Monday, 3 February 2014 3:23 AM
To: [hidden email]
Subject: Re: why does high NA excitation illumination give better resolution in fluorescence microscopy?

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

Some follow-on questions.  Asked by a relative novice, so please be gentle.

1.  Assuming we are talking about epifluorescence, isn't there just one NA in play - i.e. that of the objective?  Or are we also considering the aperture of the illumination path by which the bfp of the objective is 'filled'?

2.  Why should high NA illumination give a higher S/N ratio?  It seems to me (recall - very naive) that a lower NA illumination will excite fluorophores adequately (so long as one is not using a REALLY small cone of illumination) and that anything beyond that would just give more 'stray' light (unless the dichroics are 'perfect') and hence a lower S/N ratio.

3.  More to the point that Dan originally raised.  How 'certain' is it that high NA illumination gives better resolution in fluorescence microscopy?  This is perhaps bridging Dan's question and Guy's reply, but are we talking about theoretical resolution or 'practical'  
resolution.  Can someone provide a reference to this?

Mark Adelman

On Feb 2, 2014, at 9:09 AM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Geometrically, obviously (as you say) it doesn't.  But practically
> attainable resolution depend on the signal to nose ratio, and that is
> typically a limiting factor in fluorescence.  High NA illumination
> will improve the S/N ratio, and therefore the real world resolution.
>
>                                        Guy
>
> Guy Cox, Honorary Associate Professor
> School of Medical Sciences
>
> Australian Centre for Microscopy and Microanalysis, Madsen, F09,
> University of Sydney, NSW 2006
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]
> ] On Behalf Of Daniel White
> Sent: Sunday, 2 February 2014 11:57 PM
> To: [hidden email]
> Subject: why does high NA excitation illumination give better
> resolution in fluorescence microscopy?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Can can anyone describe, as if to an idiot, in simple terms:
>
> Why does high NA excitation illumination give better resolution in
> fluorescence microscopy?
>
> Simplistically, it seems that only the NA of detection/emission
> matters....
> since the fluorescence absorption/excitation and emission processes
> are separated in time, and the excited fluorophore emits light in all
> directions...?
>
> Is it something to do with the fact that there is anisotropy in the
> emission process direction, and that the fluorophore can move between
> excitation and emission?
>
> Is it explained in Pawley's confocal handbook? If so, what page?
>
> cheers
>
> Dan