Light Lab by Carl Zeiss Microscopy for iOS devices

[Cameron Nowell]

One other thing, the calculator asks for the back projected pinhole size. You can calculate that here http://www.svi.nl/BackprojectedPinholeCalculator


Cheers

Cam



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: Tuesday, 17 January 2012 12:06 AM
To: [hidden email]
Subject: Re: Light Lab by Carl Zeiss Microscopy for iOS devices

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

Well, for my part I wouldn't mind an (Android) app for

- z-resolution, prefereably one that includes 2- and 3-photon excitation and

- resolution and brightness at various depths under refraction index mismatch conditions (e.g. sample embedded in glycerol instead of immersion oil or underneath a coverslip with a dipping objective).

Life isn't always as simple as 0.61 lambda/NA.


Even better though than a bunch of apps for various systems would a calculator running in a web browser based on common standards so that it would be accessible on every system.


Steffen


On 16.01.2012 02:08, Guy Cox wrote:

> There isn't any difference between widefield and confocal resolution in any normal fluorescence mode.   Do you really need an app to work out 0.61 lambda/NA?  And then to divide by 2.3 to get Nyquist?  If you are in multiphoton it's a little more complicated since root 2 (1.414) comes in, but you can just use the formula 0.43 lambda / NA.
>
> Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor&  Francis
>       http://www.guycox.com/optical.htm 
> ______________________________________________
> Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for
> Microscopy&  Microanalysis, Madsen Building F09, University of Sydney,
> NSW 2006
>
> Phone [phone]     Fax [phone]
>               Mobile [phone]
> ______________________________________________
>        http://www.guycox.net
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

[Cameron Nowell]

Ok for some reason it didn’t let me post my first response so here it is again

Hi Steffen,

The SVI website has a Nyquist calculator that does what you want

http://www.svi.nl/NyquistCalculator


It calculates optimal Nyquist for various settings. It can also generate a PSF of a refractive mismatch to show you what goes wrong if you want. It seems to be limited to <10um into your sample though for some reason.

The calculations for resolution at different depths is easy enough to code up for a webpage, but the brightness shift is rather tricky. There are so many parameters that will effect the brightness of your fluorophore as you go deeper into your sample (even in a perfectly matched situation) that I don't think it would be possible to calculate it. You could get a theoretical value but I doubt it would reflect reality in any way in an actual tissue or clump of cells.

If anyone knows of some formulas to do these sort of calculations send em through and I can put a calculator up on my facilities webpage.


Cheers

Cam


Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research Melbourne - Parkville Branch PO Box 2008 Royal Melbourne Hospital Victoria, 3050 AUSTRALIA
Office: [phone]
Mobile: [phone]
Fax: [phone]
Facility Website
Linked In Profile



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cameron Nowell
Sent: Tuesday, 17 January 2012 8:26 AM
To: [hidden email]
Subject: Re: Light Lab by Carl Zeiss Microscopy for iOS devices

One other thing, the calculator asks for the back projected pinhole size. You can calculate that here http://www.svi.nl/BackprojectedPinholeCalculator


Cheers

Cam



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: Tuesday, 17 January 2012 12:06 AM
To: [hidden email]
Subject: Re: Light Lab by Carl Zeiss Microscopy for iOS devices

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

Well, for my part I wouldn't mind an (Android) app for

- z-resolution, prefereably one that includes 2- and 3-photon excitation and

- resolution and brightness at various depths under refraction index mismatch conditions (e.g. sample embedded in glycerol instead of immersion oil or underneath a coverslip with a dipping objective).

Life isn't always as simple as 0.61 lambda/NA.


Even better though than a bunch of apps for various systems would a calculator running in a web browser based on common standards so that it would be accessible on every system.


Steffen


On 16.01.2012 02:08, Guy Cox wrote:

> There isn't any difference between widefield and confocal resolution in any normal fluorescence mode.   Do you really need an app to work out 0.61 lambda/NA?  And then to divide by 2.3 to get Nyquist?  If you are in multiphoton it's a little more complicated since root 2 (1.414) comes in, but you can just use the formula 0.43 lambda / NA.
>
> Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor&  Francis
>       http://www.guycox.com/optical.htm 
> ______________________________________________
> Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for
> Microscopy&  Microanalysis, Madsen Building F09, University of Sydney,
> NSW 2006
>
> Phone [phone]     Fax [phone]
>               Mobile [phone]
> ______________________________________________
>        http://www.guycox.net
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

[Adrian Smith-6]

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


On 16/01/2012, at 12:08 PM, Guy Cox wrote:

> There isn't any difference between widefield and confocal resolution in any normal fluorescence mode.   Do you really need an app to work out 0.61 lambda/NA?  

On 17/01/2012, at 12:05 AM, Steffen Dietzel wrote:

> - z-resolution, prefereably one that includes 2- and 3-photon excitation and



z-resolution was more what I had in mind - I should have been more specific :)

Regards,

Adrian

[Steffen Dietzel]

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

Cameron,

thank you for sharing this link, a nice tool indeed.

Maybe I didn't have enough coffee yet, but it appears that although the
PSF is visualized and Nyquist is calculatet, the FWHM (or 'resolution')
is not given.

A pitty, in particular since their Nyquist values multiplied by 2.3 and
what I have calculated don't really match (in multi-photon examples).

But I don't have the time right now to check which formulas they have
applied.

Steffen


On 16.01.2012 22:57, Cameron Nowell wrote:

> Ok for some reason it didn’t let me post my first response so here it is again
>
> Hi Steffen,
>
> The SVI website has a Nyquist calculator that does what you want
>
> http://www.svi.nl/NyquistCalculator
>
>
> It calculates optimal Nyquist for various settings. It can also generate a PSF of a refractive mismatch to show you what goes wrong if you want. It seems to be limited to<10um into your sample though for some reason.
>
> The calculations for resolution at different depths is easy enough to code up for a webpage, but the brightness shift is rather tricky. There are so many parameters that will effect the brightness of your fluorophore as you go deeper into your sample (even in a perfectly matched situation) that I don't think it would be possible to calculate it. You could get a theoretical value but I doubt it would reflect reality in any way in an actual tissue or clump of cells.
>
> If anyone knows of some formulas to do these sort of calculations send em through and I can put a calculator up on my facilities webpage.
>
>
> Cheers
>
> Cam
>
>
> Cameron J. Nowell
> Microscopy Manager
> Centre for Advanced Microscopy
> Ludwig Institute for Cancer Research Melbourne - Parkville Branch PO Box 2008 Royal Melbourne Hospital Victoria, 3050 AUSTRALIA
> Office: [phone]
> Mobile: [phone]
> Fax: [phone]
> Facility Website
> Linked In Profile
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cameron Nowell
> Sent: Tuesday, 17 January 2012 8:26 AM
> To: [hidden email]
> Subject: Re: Light Lab by Carl Zeiss Microscopy for iOS devices
>
> One other thing, the calculator asks for the back projected pinhole size. You can calculate that here http://www.svi.nl/BackprojectedPinholeCalculator
>
>
> Cheers
>
> Cam
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
> Sent: Tuesday, 17 January 2012 12:06 AM
> To: [hidden email]
> Subject: Re: Light Lab by Carl Zeiss Microscopy for iOS devices
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Well, for my part I wouldn't mind an (Android) app for
>
> - z-resolution, prefereably one that includes 2- and 3-photon excitation and
>
> - resolution and brightness at various depths under refraction index mismatch conditions (e.g. sample embedded in glycerol instead of immersion oil or underneath a coverslip with a dipping objective).
>
> Life isn't always as simple as 0.61 lambda/NA.
>
>
> Even better though than a bunch of apps for various systems would a calculator running in a web browser based on common standards so that it would be accessible on every system.
>
>
> Steffen
>
>
> On 16.01.2012 02:08, Guy Cox wrote:
>> There isn't any difference between widefield and confocal resolution in any normal fluorescence mode.   Do you really need an app to work out 0.61 lambda/NA?  And then to divide by 2.3 to get Nyquist?  If you are in multiphoton it's a little more complicated since root 2 (1.414) comes in, but you can just use the formula 0.43 lambda / NA.
>>
>> Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox    CRC Press / Taylor&   Francis
>>        http://www.guycox.com/optical.htm
>> ______________________________________________
>> Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for
>> Microscopy&   Microanalysis, Madsen Building F09, University of Sydney,
>> NSW 2006
>>
>> Phone [phone]     Fax [phone]
>>                Mobile [phone]
>> ______________________________________________
>>         http://www.guycox.net
>>
>
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy
>
> Mail room:
> Marchioninistr. 15, D-81377 München
>
> Building location:
> Marchioninistr. 27,  München-Großhadern

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

[Gitta Hamel]

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

/Commercial response/

Hello Steffen and Cameron,

For calculating the ideal sampling
<http://www.svi.nl/NyquistCalculator>for multi-photon data you must
indeed select  the Confocal mode and then set the number of excitation
photons on '2'.

You can calculate the Full Width Half Maximum by downloading the Huygens
software <http://www.svi.nl/Download>, then load your image in either
the Huygens Essential or Professional and select the Twin-slicer either
by right-clicking on your image or selecting it via Visualization in the
taskbar.

If you want to get the FWHM of the actual PSF image you can use the
Huygens Professional with a (test)license. You also load your image and
select Tools and in the next window you choose Theoretical PSF and press
the green button. Your PSF will be visualized in the main window and you
can open it as well in the twin-slicer.

By moving your mouse over your image in the twin-slicer you'll see a
graph appear in the other window with the intensities. You can enlarge
the graph and you'll get the FWHM if you move your mouse halfway over
the various parts of the graph.

Indeed the PSF will change deeper in your sample if there is a
refractive mismatch. Huygens takes that fully into account by measuring
the different PSF's also much deeper in the sample. For multi-photon
data that depth is up to hundreds of microns. So the 10 micron limit in
this calculator has only a practical reason for not overloading our
servers. Feel free to try.


Kind wishes,

Gitta Hamel

****************************************
Gitta Hamel
Managing Director Scientific Volume Imaging bv
Developers of the *HUYGENS* software
Laapersveld 63
1213 VB Hilversum
The Netherlands
phone: ++ 31 35 6 42 16 26
Skype: gittahamel
Fax: ++ 31 35 6 83 79 71
*****************************************


^SVI Customer support: mail us your questions [hidden email]
<mailto:[hidden email]>or find answers online in our Huygens WIKI  
www.svi.nl/FrontPage <http://www.svi.nl/FrontPage>




On 01/17/2012 09:17 AM, Steffen Dietzel wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Cameron,
>
> thank you for sharing this link, a nice tool indeed.
>
> Maybe I didn't have enough coffee yet, but it appears that although
> the PSF is visualized and Nyquist is calculatet, the FWHM (or
> 'resolution') is not given.
>
> A pitty, in particular since their Nyquist values multiplied by 2.3
> and what I have calculated don't really match (in multi-photon examples).
>
> But I don't have the time right now to check which formulas they have
> applied.
>
> Steffen
>
>
> On 16.01.2012 22:57, Cameron Nowell wrote:
>> Ok for some reason it didn’t let me post my first response so here it
>> is again
>>
>> Hi Steffen,
>>
>> The SVI website has a Nyquist calculator that does what you want
>>
>> http://www.svi.nl/NyquistCalculator
>>
>>
>> It calculates optimal Nyquist for various settings. It can also
>> generate a PSF of a refractive mismatch to show you what goes wrong
>> if you want. It seems to be limited to<10um into your sample though
>> for some reason.
>>
>> The calculations for resolution at different depths is easy enough to
>> code up for a webpage, but the brightness shift is rather tricky.
>> There are so many parameters that will effect the brightness of your
>> fluorophore as you go deeper into your sample (even in a perfectly
>> matched situation) that I don't think it would be possible to
>> calculate it. You could get a theoretical value but I doubt it would
>> reflect reality in any way in an actual tissue or clump of cells.
>>
>> If anyone knows of some formulas to do these sort of calculations
>> send em through and I can put a calculator up on my facilities webpage.
>>
>>
>> Cheers
>>
>> Cam
>>
>>
>> Cameron J. Nowell
>> Microscopy Manager
>> Centre for Advanced Microscopy
>> Ludwig Institute for Cancer Research Melbourne - Parkville Branch PO
>> Box 2008 Royal Melbourne Hospital Victoria, 3050 AUSTRALIA
>> Office: [phone]
>> Mobile: [phone]
>> Fax: [phone]
>> Facility Website
>> Linked In Profile
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Cameron Nowell
>> Sent: Tuesday, 17 January 2012 8:26 AM
>> To: [hidden email]
>> Subject: Re: Light Lab by Carl Zeiss Microscopy for iOS devices
>>
>> One other thing, the calculator asks for the back projected pinhole
>> size. You can calculate that here
>> http://www.svi.nl/BackprojectedPinholeCalculator
>>
>>
>> Cheers
>>
>> Cam
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Steffen Dietzel
>> Sent: Tuesday, 17 January 2012 12:06 AM
>> To: [hidden email]
>> Subject: Re: Light Lab by Carl Zeiss Microscopy for iOS devices
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Well, for my part I wouldn't mind an (Android) app for
>>
>> - z-resolution, prefereably one that includes 2- and 3-photon
>> excitation and
>>
>> - resolution and brightness at various depths under refraction index
>> mismatch conditions (e.g. sample embedded in glycerol instead of
>> immersion oil or underneath a coverslip with a dipping objective).
>>
>> Life isn't always as simple as 0.61 lambda/NA.
>>
>>
>> Even better though than a bunch of apps for various systems would a
>> calculator running in a web browser based on common standards so that
>> it would be accessible on every system.
>>
>>
>> Steffen
>>
>>
>> On 16.01.2012 02:08, Guy Cox wrote:
>>> There isn't any difference between widefield and confocal resolution
>>> in any normal fluorescence mode.   Do you really need an app to work
>>> out 0.61 lambda/NA?  And then to divide by 2.3 to get Nyquist?  If
>>> you are in multiphoton it's a little more complicated since root 2
>>> (1.414) comes in, but you can just use the formula 0.43 lambda / NA.
>>>
>>>                     Guy
>>>
>>> Optical Imaging Techniques in Cell Biology
>>> by Guy Cox    CRC Press / Taylor&   Francis
>>> http://www.guycox.com/optical.htm
>>> ______________________________________________
>>> Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for
>>> Microscopy&   Microanalysis, Madsen Building F09, University of Sydney,
>>> NSW 2006
>>>
>>> Phone [phone]     Fax [phone]
>>>                Mobile [phone]
>>> ______________________________________________
>>> http://www.guycox.net
>>>
>>
>>
>> --
>> ------------------------------------------------------------
>> Steffen Dietzel, PD Dr. rer. nat
>> Ludwig-Maximilians-Universität München
>> Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of
>> light microscopy
>>
>> Mail room:
>> Marchioninistr. 15, D-81377 München
>>
>> Building location:
>> Marchioninistr. 27,  München-Großhadern
>
>

[Cameron Nowell]

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

Hi List,

To get FWHM you can also use the MetroloJ plugin for ImageJ/Fiji (http://imagejdocu.tudor.lu/doku.php?id=plugin:analysis:metroloj:start ) it is really simple to use and generates very nice PDF reports. It's also totally free:)


Cheers

Cam

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gitta Hamel
Sent: Tuesday, 17 January 2012 9:08 PM
To: [hidden email]
Subject: Re: Light Lab by Carl Zeiss Microscopy for iOS devices - SVI Nyquist Calculator

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

/Commercial response/

Hello Steffen and Cameron,

For calculating the ideal sampling
<http://www.svi.nl/NyquistCalculator>for multi-photon data you must indeed select  the Confocal mode and then set the number of excitation photons on '2'.

You can calculate the Full Width Half Maximum by downloading the Huygens software <http://www.svi.nl/Download>, then load your image in either the Huygens Essential or Professional and select the Twin-slicer either by right-clicking on your image or selecting it via Visualization in the taskbar.

If you want to get the FWHM of the actual PSF image you can use the Huygens Professional with a (test)license. You also load your image and select Tools and in the next window you choose Theoretical PSF and press the green button. Your PSF will be visualized in the main window and you can open it as well in the twin-slicer.

By moving your mouse over your image in the twin-slicer you'll see a graph appear in the other window with the intensities. You can enlarge the graph and you'll get the FWHM if you move your mouse halfway over the various parts of the graph.

Indeed the PSF will change deeper in your sample if there is a refractive mismatch. Huygens takes that fully into account by measuring the different PSF's also much deeper in the sample. For multi-photon data that depth is up to hundreds of microns. So the 10 micron limit in this calculator has only a practical reason for not overloading our servers. Feel free to try.


Kind wishes,

Gitta Hamel

****************************************
Gitta Hamel
Managing Director Scientific Volume Imaging bv Developers of the *HUYGENS* software Laapersveld 63
1213 VB Hilversum
The Netherlands
phone: ++ 31 35 6 42 16 26
Skype: gittahamel
Fax: ++ 31 35 6 83 79 71
*****************************************


^SVI Customer support: mail us your questions [hidden email] <mailto:[hidden email]>or find answers online in our Huygens WIKI www.svi.nl/FrontPage <http://www.svi.nl/FrontPage>




On 01/17/2012 09:17 AM, Steffen Dietzel wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Cameron,
>
> thank you for sharing this link, a nice tool indeed.
>
> Maybe I didn't have enough coffee yet, but it appears that although
> the PSF is visualized and Nyquist is calculatet, the FWHM (or
> 'resolution') is not given.
>
> A pitty, in particular since their Nyquist values multiplied by 2.3
> and what I have calculated don't really match (in multi-photon examples).
>
> But I don't have the time right now to check which formulas they have
> applied.
>
> Steffen
>
>
> On 16.01.2012 22:57, Cameron Nowell wrote:
>> Ok for some reason it didn’t let me post my first response so here it
>> is again
>>
>> Hi Steffen,
>>
>> The SVI website has a Nyquist calculator that does what you want
>>
>> http://www.svi.nl/NyquistCalculator
>>
>>
>> It calculates optimal Nyquist for various settings. It can also
>> generate a PSF of a refractive mismatch to show you what goes wrong
>> if you want. It seems to be limited to<10um into your sample though
>> for some reason.
>>
>> The calculations for resolution at different depths is easy enough to
>> code up for a webpage, but the brightness shift is rather tricky.
>> There are so many parameters that will effect the brightness of your
>> fluorophore as you go deeper into your sample (even in a perfectly
>> matched situation) that I don't think it would be possible to
>> calculate it. You could get a theoretical value but I doubt it would
>> reflect reality in any way in an actual tissue or clump of cells.
>>
>> If anyone knows of some formulas to do these sort of calculations
>> send em through and I can put a calculator up on my facilities webpage.
>>
>>
>> Cheers
>>
>> Cam
>>
>>
>> Cameron J. Nowell
>> Microscopy Manager
>> Centre for Advanced Microscopy
>> Ludwig Institute for Cancer Research Melbourne - Parkville Branch PO
>> Box 2008 Royal Melbourne Hospital Victoria, 3050 AUSTRALIA
>> Office: [phone]
>> Mobile: [phone]
>> Fax: [phone]
>> Facility Website
>> Linked In Profile
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Cameron Nowell
>> Sent: Tuesday, 17 January 2012 8:26 AM
>> To: [hidden email]
>> Subject: Re: Light Lab by Carl Zeiss Microscopy for iOS devices
>>
>> One other thing, the calculator asks for the back projected pinhole
>> size. You can calculate that here
>> http://www.svi.nl/BackprojectedPinholeCalculator
>>
>>
>> Cheers
>>
>> Cam
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Steffen
>> Dietzel
>> Sent: Tuesday, 17 January 2012 12:06 AM
>> To: [hidden email]
>> Subject: Re: Light Lab by Carl Zeiss Microscopy for iOS devices
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Well, for my part I wouldn't mind an (Android) app for
>>
>> - z-resolution, prefereably one that includes 2- and 3-photon
>> excitation and
>>
>> - resolution and brightness at various depths under refraction index
>> mismatch conditions (e.g. sample embedded in glycerol instead of
>> immersion oil or underneath a coverslip with a dipping objective).
>>
>> Life isn't always as simple as 0.61 lambda/NA.
>>
>>
>> Even better though than a bunch of apps for various systems would a
>> calculator running in a web browser based on common standards so that
>> it would be accessible on every system.
>>
>>
>> Steffen
>>
>>
>> On 16.01.2012 02:08, Guy Cox wrote:
>>> There isn't any difference between widefield and confocal resolution
>>> in any normal fluorescence mode.   Do you really need an app to work
>>> out 0.61 lambda/NA?  And then to divide by 2.3 to get Nyquist?  If
>>> you are in multiphoton it's a little more complicated since root 2
>>> (1.414) comes in, but you can just use the formula 0.43 lambda / NA.
>>>
>>>                     Guy
>>>
>>> Optical Imaging Techniques in Cell Biology
>>> by Guy Cox    CRC Press / Taylor&   Francis
>>> http://www.guycox.com/optical.htm
>>> ______________________________________________
>>> Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for
>>> Microscopy&   Microanalysis, Madsen Building F09, University of Sydney,
>>> NSW 2006
>>>
>>> Phone [phone]     Fax [phone]
>>>                Mobile [phone]
>>> ______________________________________________
>>> http://www.guycox.net
>>>
>>
>>
>> --
>> ------------------------------------------------------------
>> Steffen Dietzel, PD Dr. rer. nat
>> Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für
>> experimentelle Medizin (WBex) Head of light microscopy
>>
>> Mail room:
>> Marchioninistr. 15, D-81377 München
>>
>> Building location:
>> Marchioninistr. 27,  München-Großhadern
>
>

This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.

[lgelman]

Hi everybody,

Just for your info and along the same lines...
We have also developed a free ImageJ macro which generates nice PSF reports and automatically calculates FWHM. You can find more info at
http://www.imaging-git.com/science/light-microscopy/routine-assessment-fluorescence-microscope-performance
The macro is available upon request.

We have been routinely monitoring the performance of our microscopes by taking 3 PSFs per objective and per scope every week for the last 4 years. We have collected over 4000 PSFs, all displayed in a comparable manner with the FWHM in xy and z thanks to this macro, and have thereby detected and solved several problems, not always noticed/reported by our users.

Very best regards,

Laurent.

___________________________
Laurent Gelman, PhD
Friedrich Miescher Institut
Facility for Advanced Imaging and Microscopy
Head of Light Microscopy
WRO 1066.2.16
Maulbeerstrasse 66
CH-4058 Basel
[phone]



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cameron Nowell
Sent: mercredi 18 janvier 2012 04:42
To: [hidden email]
Subject: Re: Light Lab by Carl Zeiss Microscopy for iOS devices - SVI Nyquist Calculator

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

Hi List,

To get FWHM you can also use the MetroloJ plugin for ImageJ/Fiji (http://imagejdocu.tudor.lu/doku.php?id=plugin:analysis:metroloj:start ) it is really simple to use and generates very nice PDF reports. It's also totally free:)


Cheers

Cam

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gitta Hamel
Sent: Tuesday, 17 January 2012 9:08 PM
To: [hidden email]
Subject: Re: Light Lab by Carl Zeiss Microscopy for iOS devices - SVI Nyquist Calculator

*****
To join, leave or search the confocal microscopy listserv, go to:
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/Commercial response/

Hello Steffen and Cameron,

For calculating the ideal sampling
<http://www.svi.nl/NyquistCalculator>for multi-photon data you must indeed select  the Confocal mode and then set the number of excitation photons on '2'.

You can calculate the Full Width Half Maximum by downloading the Huygens software <http://www.svi.nl/Download>, then load your image in either the Huygens Essential or Professional and select the Twin-slicer either by right-clicking on your image or selecting it via Visualization in the taskbar.

If you want to get the FWHM of the actual PSF image you can use the Huygens Professional with a (test)license. You also load your image and select Tools and in the next window you choose Theoretical PSF and press the green button. Your PSF will be visualized in the main window and you can open it as well in the twin-slicer.

By moving your mouse over your image in the twin-slicer you'll see a graph appear in the other window with the intensities. You can enlarge the graph and you'll get the FWHM if you move your mouse halfway over the various parts of the graph.

Indeed the PSF will change deeper in your sample if there is a refractive mismatch. Huygens takes that fully into account by measuring the different PSF's also much deeper in the sample. For multi-photon data that depth is up to hundreds of microns. So the 10 micron limit in this calculator has only a practical reason for not overloading our servers. Feel free to try.


Kind wishes,

Gitta Hamel

****************************************
Gitta Hamel
Managing Director Scientific Volume Imaging bv Developers of the *HUYGENS* software Laapersveld 63
1213 VB Hilversum
The Netherlands
phone: ++ 31 35 6 42 16 26
Skype: gittahamel
Fax: ++ 31 35 6 83 79 71
*****************************************


^SVI Customer support: mail us your questions [hidden email] <mailto:[hidden email]>or find answers online in our Huygens WIKI www.svi.nl/FrontPage <http://www.svi.nl/FrontPage>




On 01/17/2012 09:17 AM, Steffen Dietzel wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Cameron,
>
> thank you for sharing this link, a nice tool indeed.
>
> Maybe I didn't have enough coffee yet, but it appears that although
> the PSF is visualized and Nyquist is calculatet, the FWHM (or
> 'resolution') is not given.
>
> A pitty, in particular since their Nyquist values multiplied by 2.3
> and what I have calculated don't really match (in multi-photon examples).
>
> But I don't have the time right now to check which formulas they have
> applied.
>
> Steffen
>
>
> On 16.01.2012 22:57, Cameron Nowell wrote:
>> Ok for some reason it didn’t let me post my first response so here it
>> is again
>>
>> Hi Steffen,
>>
>> The SVI website has a Nyquist calculator that does what you want
>>
>> http://www.svi.nl/NyquistCalculator
>>
>>
>> It calculates optimal Nyquist for various settings. It can also
>> generate a PSF of a refractive mismatch to show you what goes wrong
>> if you want. It seems to be limited to<10um into your sample though
>> for some reason.
>>
>> The calculations for resolution at different depths is easy enough to
>> code up for a webpage, but the brightness shift is rather tricky.
>> There are so many parameters that will effect the brightness of your
>> fluorophore as you go deeper into your sample (even in a perfectly
>> matched situation) that I don't think it would be possible to
>> calculate it. You could get a theoretical value but I doubt it would
>> reflect reality in any way in an actual tissue or clump of cells.
>>
>> If anyone knows of some formulas to do these sort of calculations
>> send em through and I can put a calculator up on my facilities webpage.
>>
>>
>> Cheers
>>
>> Cam
>>
>>
>> Cameron J. Nowell
>> Microscopy Manager
>> Centre for Advanced Microscopy
>> Ludwig Institute for Cancer Research Melbourne - Parkville Branch PO
>> Box 2008 Royal Melbourne Hospital Victoria, 3050 AUSTRALIA
>> Office: [phone]
>> Mobile: [phone]
>> Fax: [phone]
>> Facility Website
>> Linked In Profile
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Cameron Nowell
>> Sent: Tuesday, 17 January 2012 8:26 AM
>> To: [hidden email]
>> Subject: Re: Light Lab by Carl Zeiss Microscopy for iOS devices
>>
>> One other thing, the calculator asks for the back projected pinhole
>> size. You can calculate that here
>> http://www.svi.nl/BackprojectedPinholeCalculator
>>
>>
>> Cheers
>>
>> Cam
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Steffen
>> Dietzel
>> Sent: Tuesday, 17 January 2012 12:06 AM
>> To: [hidden email]
>> Subject: Re: Light Lab by Carl Zeiss Microscopy for iOS devices
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Well, for my part I wouldn't mind an (Android) app for
>>
>> - z-resolution, prefereably one that includes 2- and 3-photon
>> excitation and
>>
>> - resolution and brightness at various depths under refraction index
>> mismatch conditions (e.g. sample embedded in glycerol instead of
>> immersion oil or underneath a coverslip with a dipping objective).
>>
>> Life isn't always as simple as 0.61 lambda/NA.
>>
>>
>> Even better though than a bunch of apps for various systems would a
>> calculator running in a web browser based on common standards so that
>> it would be accessible on every system.
>>
>>
>> Steffen
>>
>>
>> On 16.01.2012 02:08, Guy Cox wrote:
>>> There isn't any difference between widefield and confocal resolution
>>> in any normal fluorescence mode.   Do you really need an app to work
>>> out 0.61 lambda/NA?  And then to divide by 2.3 to get Nyquist?  If
>>> you are in multiphoton it's a little more complicated since root 2
>>> (1.414) comes in, but you can just use the formula 0.43 lambda / NA.
>>>
>>>                     Guy
>>>
>>> Optical Imaging Techniques in Cell Biology
>>> by Guy Cox    CRC Press / Taylor&   Francis
>>> http://www.guycox.com/optical.htm
>>> ______________________________________________
>>> Guy Cox, MA, DPhil(Oxon), Honorary Associate, Australian Centre for
>>> Microscopy&   Microanalysis, Madsen Building F09, University of Sydney,
>>> NSW 2006
>>>
>>> Phone [phone]     Fax [phone]
>>>                Mobile [phone]
>>> ______________________________________________
>>> http://www.guycox.net
>>>
>>
>>
>> --
>> ------------------------------------------------------------
>> Steffen Dietzel, PD Dr. rer. nat
>> Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für
>> experimentelle Medizin (WBex) Head of light microscopy
>>
>> Mail room:
>> Marchioninistr. 15, D-81377 München
>>
>> Building location:
>> Marchioninistr. 27,  München-Großhadern
>
>

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