PSF bead shot but X Y and Z dont match

[Matiar Jafari]

Hey Fellow ListServers,

I purchased a PSF kit from Invitrogen and imaged the beads and when i
loaded the beads to correct the Z blur i measured the XY of the bead
to make sure the diameter matched but it turns out it doesnt.  The
beads are supposedly .17um with +-.005 but whatever angle i measure
the bead at in xy i keep getting .4 um.  Any ideas as to why this is
happening?

Thank You
-------
Matiar Jafari

[Fred Mast]

Is this the diametre of your deconvolved bead? If it is not than you have diffraction to thank for the increased width. The .17 micron beads should be sub-resolution on your microscope and are representative of a point source of light. The resulting "blurred" image that you have captured is the result of the physical properties of light and the optical constraints of your microscope. A good source for further information can be found at the following links:

http://support.svi.nl/wiki/SubResolution

http://en.wikipedia.org/wiki/Diffraction

But also try the Handbook for Biological Confocal Microscopy, James Pawley is the editor, for further information.

Best,

Fred

On 17-Feb-10, at 8:14 PM, Matiar Jafari wrote:

Hey Fellow ListServers,

I purchased a PSF kit from Invitrogen and imaged the beads and when i
loaded the beads to correct the Z blur i measured the XY of the bead
to make sure the diameter matched but it turns out it doesnt.  The
beads are supposedly .17um with +-.005 but whatever angle i measure
the bead at in xy i keep getting .4 um.  Any ideas as to why this is
happening?

Thank You
-------
Matiar Jafari

Fred D. Mast

Department of Cell Biology

Medical Sciences Building Room 5-14

University of Alberta

Edmonton, Alberta, T6G 2H7

Canada

Tel: [phone]

[hidden email]

[Matiar Jafari]

how would i then go about correcting the Z blur for all my other
images. if the XY resolution is just distorted because of the
limitations of optics? sorry im really new to this kind of stuff.
Thank You
-------
Matiar Jafari



On Wed, Feb 17, 2010 at 7:29 PM, Fred Mast <[hidden email]> wrote:

> Is this the diametre of your deconvolved bead? If it is not than you have
> diffraction to thank for the increased width. The .17 micron beads should be
> sub-resolution on your microscope and are representative of a point source
> of light. The resulting "blurred" image that you have captured is the result
> of the physical properties of light and the optical constraints of your
> microscope. A good source for further information can be found at the
> following links:
> http://support.svi.nl/wiki/SubResolution
> http://en.wikipedia.org/wiki/Diffraction
> But also try the Handbook for Biological Confocal Microscopy, James Pawley
> is the editor, for further information.
> Best,
> Fred
> On 17-Feb-10, at 8:14 PM, Matiar Jafari wrote:
>
> Hey Fellow ListServers,
>
> I purchased a PSF kit from Invitrogen and imaged the beads and when i
> loaded the beads to correct the Z blur i measured the XY of the bead
> to make sure the diameter matched but it turns out it doesnt.  The
> beads are supposedly .17um with +-.005 but whatever angle i measure
> the bead at in xy i keep getting .4 um.  Any ideas as to why this is
> happening?
>
> Thank You
> -------
> Matiar Jafari
>
>
> Fred D. Mast
> Department of Cell Biology
> Medical Sciences Building Room 5-14
> University of Alberta
> Edmonton, Alberta, T6G 2H7
> Canada
> Tel: [phone]
> [hidden email]
>
>
>
>

[Fred Mast]

Email me off list with the specifics of your setup and I can assist you in getting everything up and running.

On 17-Feb-10, at 8:32 PM, Matiar Jafari wrote:

how would i then go about correcting the Z blur for all my other
images. if the XY resolution is just distorted because of the
limitations of optics? sorry im really new to this kind of stuff.
Thank You
-------
Matiar Jafari



On Wed, Feb 17, 2010 at 7:29 PM, Fred Mast <[hidden email]> wrote:

Is this the diametre of your deconvolved bead? If it is not than you have

diffraction to thank for the increased width. The .17 micron beads should be

sub-resolution on your microscope and are representative of a point source

of light. The resulting "blurred" image that you have captured is the result

of the physical properties of light and the optical constraints of your

microscope. A good source for further information can be found at the

following links:

http://support.svi.nl/wiki/SubResolution

http://en.wikipedia.org/wiki/Diffraction

But also try the Handbook for Biological Confocal Microscopy, James Pawley

is the editor, for further information.

Best,

Fred

On 17-Feb-10, at 8:14 PM, Matiar Jafari wrote:

Hey Fellow ListServers,

I purchased a PSF kit from Invitrogen and imaged the beads and when i

loaded the beads to correct the Z blur i measured the XY of the bead

to make sure the diameter matched but it turns out it doesnt.  The

beads are supposedly .17um with +-.005 but whatever angle i measure

the bead at in xy i keep getting .4 um.  Any ideas as to why this is

happening?

Thank You

-------

Matiar Jafari

Fred D. Mast

Department of Cell Biology

Medical Sciences Building Room 5-14

University of Alberta

Edmonton, Alberta, T6G 2H7

Canada

Tel: [phone]

[hidden email]

Fred D. Mast

Department of Cell Biology

Medical Sciences Building Room 5-14

University of Alberta

Edmonton, Alberta, T6G 2H7

Canada

Tel: [phone]

[hidden email]

[Chris Baumann]

Matiar,

What is happening is that you are up against the limit of resolution of the optics in your system.  To correct for this, you would use a technique called deconvolution.  There are a number of resources available on deconvolution but a good place to start will be http://micro.magnet.fsu.edu/primer/digitalimaging/deconvolution/deconvolutionhome.html. 

Good luck,

Chris

On 2/17/2010 10:32 PM, Matiar Jafari wrote:

how would i then go about correcting the Z blur for all my other
images. if the XY resolution is just distorted because of the
limitations of optics? sorry im really new to this kind of stuff.
Thank You
-------
Matiar Jafari



On Wed, Feb 17, 2010 at 7:29 PM, Fred Mast [hidden email] wrote:
  

Is this the diametre of your deconvolved bead? If it is not than you have
diffraction to thank for the increased width. The .17 micron beads should be
sub-resolution on your microscope and are representative of a point source
of light. The resulting "blurred" image that you have captured is the result
of the physical properties of light and the optical constraints of your
microscope. A good source for further information can be found at the
following links:
http://support.svi.nl/wiki/SubResolution
http://en.wikipedia.org/wiki/Diffraction
But also try the Handbook for Biological Confocal Microscopy, James Pawley
is the editor, for further information.
Best,
Fred
On 17-Feb-10, at 8:14 PM, Matiar Jafari wrote:

Hey Fellow ListServers,

I purchased a PSF kit from Invitrogen and imaged the beads and when i
loaded the beads to correct the Z blur i measured the XY of the bead
to make sure the diameter matched but it turns out it doesnt.  The
beads are supposedly .17um with +-.005 but whatever angle i measure
the bead at in xy i keep getting .4 um.  Any ideas as to why this is
happening?

Thank You
-------
Matiar Jafari


Fred D. Mast
Department of Cell Biology
Medical Sciences Building Room 5-14
University of Alberta
Edmonton, Alberta, T6G 2H7
Canada
Tel: [phone]
[hidden email]




    

[Guy Cox-2]

What NA objective are you using?  With an NA 1.4 oil immersion objective you should get a FWHM below 250nm.  If you are getting 400nm then something is optically wrong - probably spherical aberration caused by a refractive index mismatch - things get pretty sensitive at this sort of NA.  With a dry NA 0.75 NA objective you should do better than 450nm but only if the preparation is perfect - my advice is always to use an objective with a correction collar if you want to do serious quality imaging with a dry lens.  (Yes, I know this is not a cheap option.)

 

The other thing to bear in mind is that 170nm isn't really small enough to count as a point object for an NA 1.4 objective - so there will be a convolution of the bead with the psf probably taking the image to larger than the true psf.  I use 100nm or even 60nm beads to check with an oil lens - that way you can in practice ignore the contribution of the object.

 

I can't see much point in getting into deconvolution until your system is performing at its optical limit.  (And even then I wouldn't expect too much).

 

                                                                    Guy

 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
http://www.guycox.net

---

From: Confocal Microscopy List on behalf of Matiar Jafari
Sent: Thu 10/02/18 2:14 PM
To: [hidden email]
Subject: PSF bead shot but X Y and Z dont match

Hey Fellow ListServers,

I purchased a PSF kit from Invitrogen and imaged the beads and when i
loaded the beads to correct the Z blur i measured the XY of the bead
to make sure the diameter matched but it turns out it doesnt.  The
beads are supposedly .17um with +-.005 but whatever angle i measure
the bead at in xy i keep getting .4 um.  Any ideas as to why this is
happening?

Thank You
-------
Matiar Jafari