overfill or underfill the objective back aperture

[yuansheng sun]

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Dear Listers,

I have a question about filling the one-photon laser to the objective back
aperture in a confocal.  There is a paper (as shown below) which suggests
that underfill will yield a better Guassian excitation volume for FCS
measurements.  Underfill will also give more power.  However, would it be
tricky for the alignment when you multiple lasers and dicrhoics?  Thanks.

The paper - Focal Volume Optics and Experimental Artifacts in Confocal
Fluorescence Correlation Spectroscopy, Samuel T. Hess and Watt W. Webb,  BJ
83: 2300-2317, 2002.

Sheng

[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 Listers,
>
>I have a question about filling the one-photon laser to the objective back
>aperture in a confocal.  There is a paper (as shown below) which suggests
>that underfill will yield a better Guassian excitation volume for FCS
>measurements.  Underfill will also give more power.  However, would it be
>tricky for the alignment when you multiple lasers and dicrhoics?  Thanks.
>
>The paper - Focal Volume Optics and Experimental Artifacts in Confocal
>Fluorescence Correlation Spectroscopy, Samuel T. Hess and Watt W. Webb,  BJ
>83: 2300-2317, 2002.
>
>Sheng

Dear Sheng,

It is true that a Gaussian distribution of light
in the BFP will produce a Gaussian distribution
of light in the image plane.

Of course, as a Gaussian distrubution goes to
infinity, no matter how you choose the "size" of
the central spot to be in the BFP, you will
always cut off some of the Gaussian and so not
quite have a Gaussian in the image plane.

But the full-width-at-half-maximum of the
Gaussian in the image plane will be larger than
the FWHM of the almost-Airy spot that you would
get by overfilling the BFP (The BFP intensity
would have to be completely uniform to be a true
Airy. This is only approached by massive
overfilling.).

Of course, the AIry Disk has some light (about
20%) in the outer rings and these will reduce
contrast but as the intensity (photons/µm2) is
only about 1% of that in the central peak, they
don't excite many photons.

In terms of getting more light through the
objective, of course light that hits glass may be
transmitted while that which hits metal will not
be. However, we seldom have the problem of not
enough light. Indeed, even 1mw in a 0.5 µm FWHM
spot it close to singlet-state saturation with
most dies and you should try to stay at least 10
(preferably 100x) below that.

I would over fill about 2x.

Best,

JP
--
***************************************************************************
Prof. James B. Pawley,                
Ph.  608-238-3953              
21. N. Prospect Ave. Madison, WI 53726 USA
[hidden email]
3D Microscopy of Living Cells Course, June 9-21, 2012, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/ 
Application deadline 3/16/2012
               "If it ain't diffraction, it must be statistics." Anon. 11/16/12

[Sudipta Maiti]

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Also, for FCS, the easy way to get rid of the artifacts caused by overfilling  is to use a confical
pinhole that is small. The benefit is higher resolution, lower background and the ability to perform
FCS at higher concentrations.
Sudipta
Fri, 16 Dec 2011 09:51:52 -0600, James Pawley wrote

> *****
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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 Listers,
> >
> >I have a question about filling the one-photon laser to the objective back
> >aperture in a confocal.  There is a paper (as shown below) which suggests
> >that underfill will yield a better Guassian excitation volume for FCS
> >measurements.  Underfill will also give more power.  However, would it be
> >tricky for the alignment when you multiple lasers and dicrhoics?  Thanks.
> >
> >The paper - Focal Volume Optics and Experimental Artifacts in Confocal
> >Fluorescence Correlation Spectroscopy, Samuel T. Hess and Watt W. Webb,  BJ
> >83: 2300-2317, 2002.
> >
> >Sheng
>
> Dear Sheng,
>
> It is true that a Gaussian distribution of light
> in the BFP will produce a Gaussian distribution
> of light in the image plane.
>
> Of course, as a Gaussian distrubution goes to
> infinity, no matter how you choose the "size" of
> the central spot to be in the BFP, you will
> always cut off some of the Gaussian and so not
> quite have a Gaussian in the image plane.
>
> But the full-width-at-half-maximum of the
> Gaussian in the image plane will be larger than
> the FWHM of the almost-Airy spot that you would
> get by overfilling the BFP (The BFP intensity
> would have to be completely uniform to be a true
> Airy. This is only approached by massive
> overfilling.).
>
> Of course, the AIry Disk has some light (about
> 20%) in the outer rings and these will reduce
> contrast but as the intensity (photons/µm2) is
> only about 1% of that in the central peak, they
> don't excite many photons.
>
> In terms of getting more light through the
> objective, of course light that hits glass may be
> transmitted while that which hits metal will not
> be. However, we seldom have the problem of not
> enough light. Indeed, even 1mw in a 0.5 µm FWHM
> spot it close to singlet-state saturation with
> most dies and you should try to stay at least 10
> (preferably 100x) below that.
>
> I would over fill about 2x.
>
> Best,
>
> JP
> --
> ***************************************************************************
> Prof. James B. Pawley,                
> Ph.  608-238-3953
> 21. N. Prospect Ave. Madison, WI 53726 USA
> [hidden email]
> 3D Microscopy of Living Cells Course, June 9-21, 2012, UBC,
>  Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Application
> deadline 3/16/2012       "If it ain't diffraction, it must be
> statistics." Anon. 11/16/12

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

[yuansheng sun]

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Dear JP and Sudipta,

Thanks a lot for your suggestions.  Due to the cut-off at BFP, the
excitation volume is not quite Guassian in the one-photon case, as
JP pointed out.  Most of FCS data fittings assume a Gaussian excitation
volume characterized by w0 and z0 (e-2).  The paper I mentioned earlier
suggested that underfilling could improve on this.  But certainly there are
problems with underfilling, e.g. uneven illumination.  Thus, I posted the
question whether underfilling would be a practical way of getting a better
Gaussian excitation volume in a single-photon confocal.  I feel that
Sudipta's small pinhole with overfilling is a better practical solution.

Sheng

On Fri, Dec 16, 2011 at 1:03 PM, Sudipta Maiti
<[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Also, for FCS, the easy way to get rid of the artifacts caused by
> overfilling  is to use a confical
> pinhole that is small. The benefit is higher resolution, lower background
> and the ability to perform
> FCS at higher concentrations.
> Sudipta
> Fri, 16 Dec 2011 09:51:52 -0600, James Pawley wrote
>  > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > >*****
> > >To join, leave or search the confocal microscopy listserv, go to:
> > >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >*****
> > >
> > >Dear Listers,
> > >
> > >I have a question about filling the one-photon laser to the objective
> back
> > >aperture in a confocal.  There is a paper (as shown below) which
> suggests
> > >that underfill will yield a better Guassian excitation volume for FCS
> > >measurements.  Underfill will also give more power.  However, would it
> be
> > >tricky for the alignment when you multiple lasers and dicrhoics?
>  Thanks.
> > >
> > >The paper - Focal Volume Optics and Experimental Artifacts in Confocal
> > >Fluorescence Correlation Spectroscopy, Samuel T. Hess and Watt W. Webb,
>  BJ
> > >83: 2300-2317, 2002.
> > >
> > >Sheng
> >
> > Dear Sheng,
> >
> > It is true that a Gaussian distribution of light
> > in the BFP will produce a Gaussian distribution
> > of light in the image plane.
> >
> > Of course, as a Gaussian distrubution goes to
> > infinity, no matter how you choose the "size" of
> > the central spot to be in the BFP, you will
> > always cut off some of the Gaussian and so not
> > quite have a Gaussian in the image plane.
> >
> > But the full-width-at-half-maximum of the
> > Gaussian in the image plane will be larger than
> > the FWHM of the almost-Airy spot that you would
> > get by overfilling the BFP (The BFP intensity
> > would have to be completely uniform to be a true
> > Airy. This is only approached by massive
> > overfilling.).
> >
> > Of course, the AIry Disk has some light (about
> > 20%) in the outer rings and these will reduce
> > contrast but as the intensity (photons/痠2) is
> > only about 1% of that in the central peak, they
> > don't excite many photons.
> >
> > In terms of getting more light through the
> > objective, of course light that hits glass may be
> > transmitted while that which hits metal will not
> > be. However, we seldom have the problem of not
> > enough light. Indeed, even 1mw in a 0.5 痠 FWHM
> > spot it close to singlet-state saturation with
> > most dies and you should try to stay at least 10
> > (preferably 100x) below that.
> >
> > I would over fill about 2x.
> >
> > Best,
> >
> > JP
> > --
> >
> ***************************************************************************
> > Prof. James B. Pawley,
> > Ph.  608-238-3953
> > 21. N. Prospect Ave. Madison, WI 53726 USA
> > [hidden email]
> > 3D Microscopy of Living Cells Course, June 9-21, 2012, UBC,
> >  Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Application
> > deadline 3/16/2012           "If it ain't diffraction, it must be
> > statistics." Anon. 11/16/12
>
>
> 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
>