Optical Section Thickness-Leica SP5

[Linda Barthel]

Seems to be a simple enough question-but having trouble getting a simple
answer.  Operating a Leica SP5 Confocal microscope, software: LAS-AF ver.
2.0.0 software.  How do we determine the optical sections thickness of our
images?  This must be a simple oversight on our part.
Thanks,
Linda Barthel
Gregg Sobocinski
University of Michigan
Dept of MCDB
Ann Arbor

[James Pawley]

>Seems to be a simple enough question-but having trouble getting a simple
>answer.  Operating a Leica SP5 Confocal microscope, software: LAS-AF ver.
>2.0.0 software.  How do we determine the optical sections thickness of our
>images?  This must be a simple oversight on our part.
>Thanks,
>Linda Barthel
>Gregg Sobocinski
>University of Michigan
>Dept of MCDB
>Ann Arbor

Hi Linda,

Optical thickness is the same as z-resolution. It varies directly
with wavelength and inversely with (NA) squared. So the actual number
depends...

Besides this Abbe equation-like dependence just noted, z-resolution
can also be strongly degraded by the presence of spherical
aberration, the extent which the full numerical aperture is filled
with laser light, and how big your pinhole diameter is.

In simple terms, SA greatly extends the point-spread function in the
z-direction, even when it presence is hardly evident when looking at
an XY image.

Short version, unless you have taken precautions to detect and
correct SA, the actual z-resolution of any image of a living cell is
likely to be much worse (at least 2x) than that which you would
calculate from the equations.

Cheers,

Jim Pawley
--
*********************************************************************************
Prof. James B. Pawley,                          Ph.  608-263-3147
Room 223, Zoology Research Building,              
FAX  [phone]
1117 Johnson Ave., Madison, WI, 53706  
[hidden email]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/                 Applications still
being accepted
               "If it ain't diffraction, it must be statistics." Anon.

[Patrick Van Oostveldt]

If you really want to determine the thickness of a section you should  
take account of the fact that the refractive index of the medium can  
change the estimated thickness. Cfr. Paper of Visser, Oud and  
Brakenhoff in the early days of CSLM. Just google to "visser  
refractive index" and you will find the pdf print.

My experience is that fixation can also affect effective thickness of  
the section due to shrinking or swelling.

A test is possible by using a fluorescent dye mounted under a  
coverglass with specific spacers and measure the number of steps  
necessary to image the whole object.

A simple test is the use of some fluorescentive index and axial  
...Quoting "Linda Barthel" <[hidden email]>: textile fiber of PE  
which is round and where you can directly check if XY diameter  
corresponds to XZ distances.

Bye

Patrick Van Oostveldt

> Seems to be a simple enough question-but having trouble getting a simple
> answer.  Operating a Leica SP5 Confocal microscope, software: LAS-AF ver.
> 2.0.0 software.  How do we determine the optical sections thickness of our
> images?  This must be a simple oversight on our part.
> Thanks,
> Linda Barthel
> Gregg Sobocinski
> University of Michigan
> Dept of MCDB
> Ann Arbor
>

--
Dep. Moleculaire Biotechnologie
Coupure links 653
B 9000 GENT

tel [phone]
fax [phone]

[Ignatius, Mike-2]

For specific "spacers" our 15 micron fluorescent beads were designed for CLSM based Z (depth) calibration.  We also have 6 micron ones if you prefer.  

These also show the effect of color and RI mismatch on Z depth accuracy.  Simple protocol included.  

FocalCheck™ microspheres, 15 µm, fluorescent green ring stain⁄dark red throughout
Cat. No. F-7238
Or
FocalCheck™ microspheres, 15 µm, fluorescent dark-red ring stain⁄green throughout
Cat. No. F-7239

Kind Regards,

Mike Ignatius
Molecular Probes/Life Technologies

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Patrick Van Oostveldt
Sent: Tuesday, May 11, 2010 10:24 AM
To: [hidden email]
Subject: Re: Optical Section Thickness-Leica SP5

If you really want to determine the thickness of a section you should  
take account of the fact that the refractive index of the medium can  
change the estimated thickness. Cfr. Paper of Visser, Oud and  
Brakenhoff in the early days of CSLM. Just google to "visser  
refractive index" and you will find the pdf print.

My experience is that fixation can also affect effective thickness of  
the section due to shrinking or swelling.

A test is possible by using a fluorescent dye mounted under a  
coverglass with specific spacers and measure the number of steps  
necessary to image the whole object.

A simple test is the use of some fluorescentive index and axial  
...Quoting "Linda Barthel" <[hidden email]>: textile fiber of PE  
which is round and where you can directly check if XY diameter  
corresponds to XZ distances.

Bye

Patrick Van Oostveldt

> Seems to be a simple enough question-but having trouble getting a simple
> answer.  Operating a Leica SP5 Confocal microscope, software: LAS-AF ver.
> 2.0.0 software.  How do we determine the optical sections thickness of our
> images?  This must be a simple oversight on our part.
> Thanks,
> Linda Barthel
> Gregg Sobocinski
> University of Michigan
> Dept of MCDB
> Ann Arbor
>

--
Dep. Moleculaire Biotechnologie
Coupure links 653
B 9000 GENT

tel [phone]
fax [phone]

[Mario-2]

My inclination is to use subresolution size beads
(< 200 nm) of different colors. As Jim P. pointed
out the z-res. is easily twice as bad as the
theoretical values. Further, objectives that are
nominally of the same high NA (1.4 and above) can
yield quite different z-resolutions even factors
of 2. In addition, the z-axis PSF can be very
asymmetric, which becomes obvious when using
sub-res. beads and may be less obvious using very
large beads. Making sure you use the correct
coverslip thickness and be aware that the z-res.
is strongly dependent on the mediums RI.
Depending on the amount of SA of you particular
objective it will also get worse as you focus
deeper into the sample, particularly as you get
more than 10 um from the coverslip. Some
deconvolution software can at least partially
correct for the z-axis spread caused by SA if
good calibration data is available. It just
depends on how fussy you want to be.

Mario

>For specific "spacers" our 15 micron fluorescent
>beads were designed for CLSM based Z (depth)
>calibration.  We also have 6 micron ones if you
>prefer.
>
>These also show the effect of color and RI
>mismatch on Z depth accuracy.  Simple protocol
>included.
>
>FocalCheck microspheres, 15 µm, fluorescent
>green ring stainŽdark red throughout
>Cat. No. F-7238
>Or
>FocalCheck microspheres, 15 µm, fluorescent
>dark-red ring stainŽgreen throughout
>Cat. No. F-7239
>
>Kind Regards,
>
>Mike Ignatius
>Molecular Probes/Life Technologies
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On
>Behalf Of Patrick Van Oostveldt
>Sent: Tuesday, May 11, 2010 10:24 AM
>To: [hidden email]
>Subject: Re: Optical Section Thickness-Leica SP5
>
>If you really want to determine the thickness of a section you should
>take account of the fact that the refractive index of the medium can
>change the estimated thickness. Cfr. Paper of Visser, Oud and
>Brakenhoff in the early days of CSLM. Just google to "visser
>refractive index" and you will find the pdf print.
>
>My experience is that fixation can also affect effective thickness of
>the section due to shrinking or swelling.
>
>A test is possible by using a fluorescent dye mounted under a
>coverglass with specific spacers and measure the number of steps
>necessary to image the whole object.
>
>A simple test is the use of some fluorescentive index and axial
>...Quoting "Linda Barthel" <[hidden email]>: textile fiber of PE
>which is round and where you can directly check if XY diameter
>corresponds to XZ distances.
>
>Bye
>
>Patrick Van Oostveldt
>
>
>
>
>>  Seems to be a simple enough question-but having trouble getting a simple
>>  answer.  Operating a Leica SP5 Confocal microscope, software: LAS-AF ver.
>>  2.0.0 software.  How do we determine the optical sections thickness of our
>>  images?  This must be a simple oversight on our part.
>>  Thanks,
>>  Linda Barthel
>>  Gregg Sobocinski
>>  University of Michigan
>>  Dept of MCDB
>>  Ann Arbor
>>
>
>
>
>--
>Dep. Moleculaire Biotechnologie
>Coupure links 653
>B 9000 GENT
>
>tel [phone]
>fax [phone]

--
________________________________________________________________________________
Mario M. Moronne, Ph.D.
[hidden email]
[hidden email]

[Julio Vazquez]

Hi Linda, 

I'm guessing you want to know how to set up optimal z spacing based on your pinhole aperture and objective. I am not familiar with SP5, but I believe when you go to the z stack settings panel, there should be a button named "system optimized" that will match your pinhole aperture and section distance. This will be typically about half of the z resolution (or z focal depth). If you can't figure that out, you may search Google for leica las-af manual or leica sp5 manual, and you will get to some tutorials that may have the desired info. Also, a call or email to your Leica rep should give you the answer. Just ask them where in te hsoftware do you set up the optimal z section spacing or thickness. 

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

1100 Fairview Ave. N.,  mailstop DE-512

Seattle, WA 98109-1024

http://www.fhcrc.org/

On May 11, 2010, at 9:05 AM, Linda Barthel wrote:

Seems to be a simple enough question-but having trouble getting a simple

answer.  Operating a Leica SP5 Confocal microscope, software: LAS-AF ver.

2.0.0 software.  How do we determine the optical sections thickness of our

images?  This must be a simple oversight on our part.

Thanks, 

Linda Barthel 

Gregg Sobocinski

University of Michigan

Dept of MCDB

Ann Arbor

[Xuelin]

Hi james

You can ask leica for  software version2.2.1，

Goodluck

Xuelin



发自我的 iPod

在 2010-5-12，0:05，Linda Barthel <[hidden email]> 写到：

> Seems to be a simple enough question-but having trouble getting a  
> simple
> answer.  Operating a Leica SP5 Confocal microscope, software: LAS-AF  
> ver.
> 2.0.0 software.  How do we determine the optical sections thickness  
> of our
> images?  This must be a simple oversight on our part.
> Thanks,
> Linda Barthel
> Gregg Sobocinski
> University of Michigan
> Dept of MCDB
> Ann Arbor

[Adrian Smith-6]

The version of LAS AF that we have (2.2.x) has "optimised" z-slicing in two locations.

One is as Julio describes below, ie in the z-stack dialog. To get the z-step it takes the value reported in the objective information dialog and applies an 2-step Nyquist approximation (so the z-step size is half the z-resolution reported in the objective information pane). The objective dialog seem to use a very "optimistic" formula for z-resolution.

The other location is in the extra controls that come up when you click the pinhole radio box (ie disable automatic pinhole settings) - this reveals a new button which brings up a new window with a more conservsative formula with the option to enter the wavelength you are interested in. The calculated thickness changes with the pinhole setting which is really helpful when you have opened up the pinhole for some reason. There is another button to copy this to the z-stack dialog. z-steps set this way are about 2-fold larger than those using the first method.

I don't know which is the "correct" forumula but based on what I understand/have heard about z-resolution the older, smaller numbers in the objective dialog seem unrealistic.

I can't remember which version (it was sometime after 2.0) added the extra controls in the pinhole dialog.

Hope that helps,

Regards,

Adrian Smith

Centenary Institute, Sydney, Australia

On 12/05/2010, at 5:05 AM, Julio Vazquez wrote:

Hi Linda, 

I'm guessing you want to know how to set up optimal z spacing based on your pinhole aperture and objective. I am not familiar with SP5, but I believe when you go to the z stack settings panel, there should be a button named "system optimized" that will match your pinhole aperture and section distance. This will be typically about half of the z resolution (or z focal depth). If you can't figure that out, you may search Google for leica las-af manual or leica sp5 manual, and you will get to some tutorials that may have the desired info. Also, a call or email to your Leica rep should give you the answer. Just ask them where in te hsoftware do you set up the optimal z section spacing or thickness. 

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

1100 Fairview Ave. N.,  mailstop DE-512

Seattle, WA 98109-1024

http://www.fhcrc.org/

On May 11, 2010, at 9:05 AM, Linda Barthel wrote:

Seems to be a simple enough question-but having trouble getting a simple

answer.  Operating a Leica SP5 Confocal microscope, software: LAS-AF ver.

2.0.0 software.  How do we determine the optical sections thickness of our

images?  This must be a simple oversight on our part.

Thanks, 

Linda Barthel 

Gregg Sobocinski

University of Michigan

Dept of MCDB

Ann Arbor

[kirk-20]

Hi All,

Our University has not historically considered leasing as an option, but
we have a number of immediate major equipment needs that might be
well-served by this mechanism.

Does anyone have experience either 3rd party or directly with the
manufacturer in a University environment? Any special issues? Were
leasing conditions favorable? Any 3rd party companies that were good to
deal with?

Any and all comments and suggestions appreciated. I will compile results
and share with the list for others out there wondering how to keep-up
with the pace of technology and instrumentation.

Best Regards, Kirk

Kirk J. Czymmek, Ph.D.
Associate Professor
Department of Biological Sciences
Delaware Biotechnology Institute Bio-Imaging Center, Director
University of Delaware
Newark, DE 19716

[David Panzarella]

Hi Kirk,

We have had our clients use Heartland Business Credit in the past and
they seem to be a pretty good company to deal with.  As a general rule
of thumb you would get a better interest rate from a third party leasing
company vs. a vendor as in general a vendor's cost of capital is higher
and therefore there interest rate charge to you would be higher.

Hope this helps.

Dave

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of kirk
Sent: Wednesday, May 12, 2010 3:33 AM
To: [hidden email]
Subject: Major Equipment Leasing

Hi All,

Our University has not historically considered leasing as an option, but

we have a number of immediate major equipment needs that might be
well-served by this mechanism.

Does anyone have experience either 3rd party or directly with the
manufacturer in a University environment? Any special issues? Were
leasing conditions favorable? Any 3rd party companies that were good to
deal with?

Any and all comments and suggestions appreciated. I will compile results

and share with the list for others out there wondering how to keep-up
with the pace of technology and instrumentation.

Best Regards, Kirk

Kirk J. Czymmek, Ph.D.
Associate Professor
Department of Biological Sciences
Delaware Biotechnology Institute Bio-Imaging Center, Director
University of Delaware
Newark, DE 19716


====================================================================================

The information contained in this e-mail message is intended only
for the personal and confidential use of the recipient(s) named above. If the reader of this message is not the intended recipient
or an agent responsible for delivering it to the intended recipient,
you are hereby notified that you have received this document in
error and that any review, dissemination, distribution, or copying
of this message is strictly prohibited. If you have received this
communication in error, please notify us immediately by e-mail, and
delete the original message.

====================================================================================

[Gregg Sobocinski]

Thanks to those who responded to our inquiry about determining optical thickness of images captured on the Leica SP5. We’re happy with the responses from both this Listserver community and the Leica support specialists.

 

Between the Listserver responses, and the helpful response of Leica, we now have the latest software (which calculates optical thickness for us) as well as a complete understanding of the variables involved in reaching that number.

 

With gratitude,

~Gregg

 

Gregg Sobocinski

Microscope Imaging Specialist

University of Michigan, MCDB Dept.

Ann Arbor, Michigan

USA

 

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Adrian Smith
Sent: Wednesday, May 12, 2010 2:54 AM
To: [hidden email]
Subject: Re: Optical Section Thickness-Leica SP5

 

The version of LAS AF that we have (2.2.x) has "optimised" z-slicing in two locations.

 

One is as Julio describes below, ie in the z-stack dialog. To get the z-step it takes the value reported in the objective information dialog and applies an 2-step Nyquist approximation (so the z-step size is half the z-resolution reported in the objective information pane). The objective dialog seem to use a very "optimistic" formula for z-resolution.

 

The other location is in the extra controls that come up when you click the pinhole radio box (ie disable automatic pinhole settings) - this reveals a new button which brings up a new window with a more conservsative formula with the option to enter the wavelength you are interested in. The calculated thickness changes with the pinhole setting which is really helpful when you have opened up the pinhole for some reason. There is another button to copy this to the z-stack dialog. z-steps set this way are about 2-fold larger than those using the first method.

 

I don't know which is the "correct" forumula but based on what I understand/have heard about z-resolution the older, smaller numbers in the objective dialog seem unrealistic.

 

I can't remember which version (it was sometime after 2.0) added the extra controls in the pinhole dialog.

 

Hope that helps,

 

Regards,

 

Adrian Smith

Centenary Institute, Sydney, Australia

 

 

 

 

 

On 12/05/2010, at 5:05 AM, Julio Vazquez wrote:

Hi Linda, 

 

I'm guessing you want to know how to set up optimal z spacing based on your pinhole aperture and objective. I am not familiar with SP5, but I believe when you go to the z stack settings panel, there should be a button named "system optimized" that will match your pinhole aperture and section distance. This will be typically about half of the z resolution (or z focal depth). If you can't figure that out, you may search Google for leica las-af manual or leica sp5 manual, and you will get to some tutorials that may have the desired info. Also, a call or email to your Leica rep should give you the answer. Just ask them where in te hsoftware do you set up the optimal z section spacing or thickness. 

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

1100 Fairview Ave. N.,  mailstop DE-512

Seattle, WA 98109-1024

 

 

http://www.fhcrc.org/

 

 

On May 11, 2010, at 9:05 AM, Linda Barthel wrote:

Seems to be a simple enough question-but having trouble getting a simple

answer.  Operating a Leica SP5 Confocal microscope, software: LAS-AF ver.

2.0.0 software.  How do we determine the optical sections thickness of our

images?  This must be a simple oversight on our part.

Thanks, 

Linda Barthel 

Gregg Sobocinski

University of Michigan

Dept of MCDB

Ann Arbor