Leica TCS SP2, LCS and Win7

[Schebique]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear all.

We are going to upgrade our acquisition computer serving our Leica TCS SP2
system with a new motherboard, Intel i5 core, two new SSD disks and Win 7
OS. My question is if there is anybody who has any experience with running
acquistion software LCS on either 32-bit or 64-bit Windows 7? We realized
that drivers for needed Adaptec SCSI boards should work and we do not see
any obvious problem to shift LCS onto supported OS Win7. However, nobody
knows if there could be any hidden problems, so any experience to share is
welcome.

Thank you very much.

Best Regards
Ondrej Sebesta

-- 
------------------------------------------
Mgr. Ondřej Šebesta
Laboratory of Confocal and Fluorescence Microscopy
Faculty of Science, Charles University in Prague
Vinicna 7
128 44 Prague
Czech Republic

Phone: [phone]
e-mail: [hidden email](mailto:[hidden email])

[Csúcs Gábor-3]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear Ondrej,

I would be extremely careful with this project. Although we never tried to
switch to Win 7 but a couple of years ago we tried to exchange a computer
under XP and failed (and if I remember correctly, other people on this
listed reported similar difficulties). Theoretically everything should
have worked, still we couldn't get the system running. So at the end we
ended up by paying Leica approx. 10000 EUR for a no-name computer the
value of which was less then 700 EUR. The same upgrade (if we would have
chosen a HP computer) would have costed 13000 EUR. I would estimate that a
switch to Windows 7 will be even more complicated.

Greetings     Gabor



On 11/7/14 3:33 PM, "Ondřej Šebesta" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>Dear all.
>
>We are going to upgrade our acquisition computer serving our Leica TCS
>SP2
>system with a new motherboard, Intel i5 core, two new SSD disks and Win 7
>OS. My question is if there is anybody who has any experience with
>running
>acquistion software LCS on either 32-bit or 64-bit Windows 7? We realized
>that drivers for needed Adaptec SCSI boards should work and we do not see
>any obvious problem to shift LCS onto supported OS Win7. However, nobody
>knows if there could be any hidden problems, so any experience to share
>is
>welcome.
>
>Thank you very much.
>
>Best Regards
>Ondrej Sebesta
>
>--
>------------------------------------------
>Mgr. Ondřej Šebesta
>Laboratory of Confocal and Fluorescence Microscopy
>Faculty of Science, Charles University in Prague
>Vinicna 7
>128 44 Prague
>Czech Republic
>
>Phone: [phone]
>e-mail: [hidden email](mailto:[hidden email])

Hi Ondrej,
In the past spring (April, 2014) we just upgraded SP5 II from win XP to win7 with new computer. The computer was tested by Leica before installation. But the engineer has to find right version software for our system. The whole process takes one day and so far everything works great.

Long

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ondrej Šebesta
Sent: Friday, November 07, 2014 9:33 AM
To: [hidden email]
Subject: Leica TCS SP2, LCS and Win7

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear all.

We are going to upgrade our acquisition computer serving our Leica TCS SP2 system with a new motherboard, Intel i5 core, two new SSD disks and Win 7 OS. My question is if there is anybody who has any experience with running acquistion software LCS on either 32-bit or 64-bit Windows 7? We realized that drivers for needed Adaptec SCSI boards should work and we do not see any obvious problem to shift LCS onto supported OS Win7. However, nobody knows if there could be any hidden problems, so any experience to share is welcome.

Thank you very much.

Best Regards
Ondrej Sebesta

--
------------------------------------------
Mgr. Ondřej Šebesta
Laboratory of Confocal and Fluorescence Microscopy Faculty of Science, Charles University in Prague Vinicna 7
128 44 Prague
Czech Republic

Phone: [phone]
e-mail: [hidden email](mailto:[hidden email])

[Thomas Trusk]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

When we upgraded our SP5 to a new computer with Windows7, I asked about
doing the same for our SP2 system.  Although we could (and did) upgrade to a
better computer, Leica informed us that we could not leave WindowsXP as there
were insurmountable hardware and driver issues that prevent it.  I would keep the
old computer system intact in case you find that this is true.  On the other hand, if
you DO find a way to upgrade to Win7 (32 or 64bit), we would be very interested
in specific details.

Thomas Trusk
Josh Spruill Image Facility
Medical University of South Carolina
Charleston, SC

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Thomas,

Before we upgraded to Win 7, we often had system crash with winXP system. However, when I asked for win7, I was informed that similar thing, the hardware/driver issues. So I called some cores and found that there are some SP5 II with win 7 system. Since winXP is no longer supported by Microsoft any more, Leica finally helped us to upgrade the system. Feel free to email me if you have any question, I will try my best to help you.

Best,
Long
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Thomas Trusk [[hidden email]]
Sent: Saturday, November 08, 2014 4:11 PM
To: [hidden email]
Subject: Re: Leica TCS SP2, LCS and Win7

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

When we upgraded our SP5 to a new computer with Windows7, I asked about
doing the same for our SP2 system.  Although we could (and did) upgrade to a
better computer, Leica informed us that we could not leave WindowsXP as there
were insurmountable hardware and driver issues that prevent it.  I would keep the
old computer system intact in case you find that this is true.  On the other hand, if
you DO find a way to upgrade to Win7 (32 or 64bit), we would be very interested
in specific details.

Thomas Trusk
Josh Spruill Image Facility
Medical University of South Carolina
Charleston, SC

[Torsten.Fregin]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi,

I had the same issue several years ago when upgrading an Leica TCS NT  
to XP. Leica Service said "not possible", but I think they just wanted  
to sell us a new system... So I upgraded it myself and it was working  
fine with XP (but not with W2k for some unknown reason).

So, just try it out! Make a backup of your hard disks, and from within  
your running XP start an upgrade to Win Vista, then to Win 7.

:-) Torsten



Zitat von Ond?ej ?ebesta <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear all.
>
> We are going to upgrade our acquisition computer serving our Leica TCS SP2
> system with a new motherboard, Intel i5 core, two new SSD disks and Win 7
> OS. My question is if there is anybody who has any experience with running
> acquistion software LCS on either 32-bit or 64-bit Windows 7? We realized
> that drivers for needed Adaptec SCSI boards should work and we do not see
> any obvious problem to shift LCS onto supported OS Win7. However, nobody
> knows if there could be any hidden problems, so any experience to share is
> welcome.
>
> Thank you very much.
>
> Best Regards
> Ondrej Sebesta
>
> -- 
> ------------------------------------------
> Mgr. Ond?ej ?ebesta
> Laboratory of Confocal and Fluorescence Microscopy
> Faculty of Science, Charles University in Prague
> Vinicna 7
> 128 44 Prague
> Czech Republic
>
> Phone: [phone]
> e-mail: [hidden email](mailto:[hidden email])

[Ondřej Šebesta]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear List

At first thank you a lot for all the advices. I found one eliminated older
computer (Pentium4, 1Gb RAM) with Win XP at our faculty, so I used it for a
test fresh installation of LCS under win XP. As was adviced I would like to
have a reserve computer in stock so it would be possible to turn back in
case of any troubles with win7 and LCS.
So far I was able to install it properly and I would like to share my
experience with you.


1) First of all it is really good idea to back up complete system including
your data, of course.
You will need a hardware configuration files stored in the LCS program
folder "Calibration Data". Back up these settings carefully somewhere
else(e.g. flash drive). You will find usefull to backup complete LCS program
folder (in Program Files) and LCS folder in "Documents and Settings/user,
where IPS profiles are stored. This is important.

2) You will need the installation file of your current version of LCS.
Alternatively you may use the LCS backuped files and copy them on the new
computer, however I did not test this.

3) It is very good idea carrefully examine your hardware and current
versions of your hardware - especially SCSI driver, serial bus (COM)
drivers, LPT drivers and multifuncton I/O adapters. In my case it was
Adaptec SCSI Card 29160 -ultra 160 SCSI (SCSI port where scanner is
connected); Decision PC COM PCI 4 port (it is multiport serial adapter where
on which laser table is connected); netMOS9835 PCI Multi-I/O controller
(driving my additional serial port where a motorized stage controller is
connected). Note down numbers of used COM ports and their setting (bitrate,
parity, stopbits etc.) and versions of used drivers.

4) In case you are spending money on a new computer take care about these
important things you will need:
a)at least 3 free PCI slots on your motherboard (or more - see into your
current setup) -first for SCSI PCI card, second for multiport card adapter
and third for additional serial and/or LPT ports.
b)matherboard should have a "native" LPT1 port, where licence dongle is
connected. This is neccessary to run LCS!
c)Two or three harddisks or logical disks. It is usefull to use one quick
harddisk as a swap disk/temporary disk and LCS program installation disk.
(disk where temporary data are stored between acquistion and saving-we have
SCSI disk, however modern SATAII and III disks has sufficient data bandwidth).

5) I had exchange all PCI cards, cables and LPT dongle onto new computer.
Some of them was automatically recognized and windows installed proper
drivers. I had download a driver for multiport (from here:
http://www.pccompci.com/driver/4p232-SMD.html). It is possible you will need
find your drivers yourself. SCSI drivers can be found here:
http://www.adaptec.com/en-us/support/scsi/u160/asc-29160/. I realized that
all needed hardware has support under win 7, so the swithc on new OS seems
possible to me.

6) Now it is time to try install LCS program. I get installation file of my
current version LCS 2.61.Build 1537 from my local Leica vendor. To avoid
troubles I recomend to use your current LCS version. There is a step during
installation when a new firmware may be upladed into the scanner (and the
version of firmware must fit with the version of LCS!). To ensure backstep
possibility I would avoid the scanner firmware overwriting!
I performed installation onto disk C:/Program Files (and it works), however
as I told before, you can choose the installation folder on the quick
swap/temporary disk.
During the process you will be asked on the name of "prefabrikated" IPS
profile folder. This looks something like:
SP2_4ChT_AOBS_MultiRGBB8_UV_405_AOTF. It is absolutely not important what
you choose, because you will overwrite default Leica IPS profiles with your
backuped IPS profiles. After the installation you are forced to restart
computer.

7) Before the first start of LCS you have to copy back your hardware
settings into the Program Files/LCS/Hardware Control folder and your
backuped personal settings files into Documents and Settings/LCS/ folder.

8)In the Control panel>system>hardware settings I had to actualize driver
for Leica TCS-x. I found it in the LCS program folder/tools. Now SP2 is
recognized as Imaging device. I also had to change naming of COM ports
according to where LCS seek serial devices like motorized stage etc.

9) At this moment it is possible to run LCS and start to work ;-)

I am not sure ofcourse, if this will works as nice on Win7, but I am
optimistic. There can be one issue with LPT port which is connected via USB
ports on modern motherboards and there could be a problem. I want to test
this later.

Best Regards
Ondrej.

------------------------------------------
Mgr. Ond&#345;ej Šebesta
Laboratory of Confocal and Fluorescence Microscopy
Faculty of Science, Charles University in Prague
Vinicna 7
128 44 Prague
Czech Republic

Phone: [phone]
e-mail: [hidden email]

[Zdenek Svindrych]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Good job, Ondrej!



However, don't be too optimistic about Win7.

I've tried LCS in simulator mode, and it was unable to open files. Maybe you
could be able to save them, I can't test it with the simulator, anyway I
doubt it.

I had to setup a virtual machine with XP to run the software (simulator),
but this is not going to work with all the hardware. Also note that only
VMware (no commercial interest) is able to virtualize parallel port, the
other option is to emulate the dongle (which is illegal, not necessarily
unethical).




Fortunately, XP is a good OS and there is still lot of supported hardware.
Only the PCI slots are becoming scarce...




Best,

zdenek svindrych

KCCI, University of Virginia







---------- Původní zpráva ----------
Od: Ondrej Sebesta <[hidden email]>
Komu: [hidden email]
Datum: 14. 11. 2014 11:27:52
Předmět: Re: Leica TCS SP2, LCS and Win7

"*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear List

At first thank you a lot for all the advices. I found one eliminated older
computer (Pentium4, 1Gb RAM) with Win XP at our faculty, so I used it for a
test fresh installation of LCS under win XP. As was adviced I would like to
have a reserve computer in stock so it would be possible to turn back in
case of any troubles with win7 and LCS.
So far I was able to install it properly and I would like to share my
experience with you.


1) First of all it is really good idea to back up complete system including
your data, of course.
You will need a hardware configuration files stored in the LCS program
folder "Calibration Data". Back up these settings carefully somewhere
else(e.g. flash drive). You will find usefull to backup complete LCS program
folder (in Program Files) and LCS folder in "Documents and Settings/user,
where IPS profiles are stored. This is important.

2) You will need the installation file of your current version of LCS.
Alternatively you may use the LCS backuped files and copy them on the new
computer, however I did not test this.

3) It is very good idea carrefully examine your hardware and current
versions of your hardware - especially SCSI driver, serial bus (COM)
drivers, LPT drivers and multifuncton I/O adapters. In my case it was
Adaptec SCSI Card 29160 -ultra 160 SCSI (SCSI port where scanner is
connected); Decision PC COM PCI 4 port (it is multiport serial adapter where
on which laser table is connected); netMOS9835 PCI Multi-I/O controller
(driving my additional serial port where a motorized stage controller is
connected). Note down numbers of used COM ports and their setting (bitrate,
parity, stopbits etc.) and versions of used drivers.

4) In case you are spending money on a new computer take care about these
important things you will need:
a)at least 3 free PCI slots on your motherboard (or more - see into your
current setup) -first for SCSI PCI card, second for multiport card adapter
and third for additional serial and/or LPT ports.
b)matherboard should have a "native" LPT1 port, where licence dongle is
connected. This is neccessary to run LCS!
c)Two or three harddisks or logical disks. It is usefull to use one quick
harddisk as a swap disk/temporary disk and LCS program installation disk.
(disk where temporary data are stored between acquistion and saving-we have
SCSI disk, however modern SATAII and III disks has sufficient data
bandwidth).

5) I had exchange all PCI cards, cables and LPT dongle onto new computer.
Some of them was automatically recognized and windows installed proper
drivers. I had download a driver for multiport (from here:
http://www.pccompci.com/driver/4p232-SMD.html). It is possible you will need
find your drivers yourself. SCSI drivers can be found here:
http://www.adaptec.com/en-us/support/scsi/u160/asc-29160/. I realized that
all needed hardware has support under win 7, so the swithc on new OS seems
possible to me.

6) Now it is time to try install LCS program. I get installation file of my
current version LCS 2.61.Build 1537 from my local Leica vendor. To avoid
troubles I recomend to use your current LCS version. There is a step during
installation when a new firmware may be upladed into the scanner (and the
version of firmware must fit with the version of LCS!). To ensure backstep
possibility I would avoid the scanner firmware overwriting!
I performed installation onto disk C:/Program Files (and it works), however
as I told before, you can choose the installation folder on the quick
swap/temporary disk.
During the process you will be asked on the name of "prefabrikated" IPS
profile folder. This looks something like:
SP2_4ChT_AOBS_MultiRGBB8_UV_405_AOTF. It is absolutely not important what
you choose, because you will overwrite default Leica IPS profiles with your
backuped IPS profiles. After the installation you are forced to restart
computer.

7) Before the first start of LCS you have to copy back your hardware
settings into the Program Files/LCS/Hardware Control folder and your
backuped personal settings files into Documents and Settings/LCS/ folder.

8)In the Control panel>system>hardware settings I had to actualize driver
for Leica TCS-x. I found it in the LCS program folder/tools. Now SP2 is
recognized as Imaging device. I also had to change naming of COM ports
according to where LCS seek serial devices like motorized stage etc.

9) At this moment it is possible to run LCS and start to work ;-)

I am not sure ofcourse, if this will works as nice on Win7, but I am
optimistic. There can be one issue with LPT port which is connected via USB
ports on modern motherboards and there could be a problem. I want to test
this later.

Best Regards
Ondrej.

------------------------------------------
Mgr. Ond&#345;ej Šebesta
Laboratory of Confocal and Fluorescence Microscopy
Faculty of Science, Charles University in Prague
Vinicna 7
128 44 Prague
Czech Republic

Phone: [phone]
e-mail: [hidden email]"

[Craig Brideau]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

When I have to remember port settings, I take several screen captures of
the Device Manager window with the various resources shown. The virtual
comm ports in particular can get excessive.

Craig
On Nov 14, 2014 9:00 AM, "Ondrej Sebesta" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear List
>
> At first thank you a lot for all the advices. I found one eliminated older
> computer (Pentium4, 1Gb RAM) with Win XP at our faculty, so I used it for a
> test fresh installation of LCS under win XP. As was adviced I would like to
> have a reserve computer in stock so it would be possible to turn back in
> case of any troubles with win7 and LCS.
> So far I was able to install it properly and I would like to share my
> experience with you.
>
>
> 1) First of all it is really good idea to back up complete system including
> your data, of course.
> You will need a hardware configuration files stored in the LCS program
> folder "Calibration Data". Back up these settings carefully somewhere
> else(e.g. flash drive). You will find usefull to backup complete LCS
> program
> folder (in Program Files) and LCS folder in "Documents and Settings/user,
> where IPS profiles are stored. This is important.
>
> 2) You will need the installation file of your current version of LCS.
> Alternatively you may use the LCS backuped files and copy them on the new
> computer, however I did not test this.
>
> 3) It is very good idea carrefully examine your hardware and current
> versions of your hardware - especially SCSI driver, serial bus (COM)
> drivers, LPT drivers and multifuncton I/O adapters. In my case it was
> Adaptec SCSI Card 29160 -ultra 160 SCSI (SCSI port where scanner is
> connected); Decision PC COM PCI 4 port (it is multiport serial adapter
> where
> on which laser table is connected); netMOS9835 PCI Multi-I/O controller
> (driving my additional serial port where a motorized stage controller is
> connected). Note down numbers of used COM ports and their setting (bitrate,
> parity, stopbits etc.) and versions of used drivers.
>
> 4) In case you are spending money on a new computer take care about these
> important things you will need:
> a)at least 3 free PCI slots on your motherboard (or more - see into your
> current setup) -first for SCSI PCI card, second for multiport card adapter
> and third for additional serial and/or LPT ports.
> b)matherboard should have a "native" LPT1 port, where licence dongle is
> connected. This is neccessary to run LCS!
> c)Two or three harddisks or logical disks. It is usefull to use one quick
> harddisk as a swap disk/temporary disk and LCS program installation disk.
> (disk where temporary data are stored between acquistion and saving-we have
> SCSI disk, however modern SATAII and III disks has sufficient data
> bandwidth).
>
> 5) I had exchange all PCI cards, cables and LPT dongle onto new computer.
> Some of them was automatically recognized and windows installed proper
> drivers. I had download a driver for multiport (from here:
> http://www.pccompci.com/driver/4p232-SMD.html). It is possible you will
> need
> find your drivers yourself. SCSI drivers can be found here:
> http://www.adaptec.com/en-us/support/scsi/u160/asc-29160/. I realized that
> all needed hardware has support under win 7, so the swithc on new OS seems
> possible to me.
>
> 6) Now it is time to try install LCS program. I get installation file of my
> current version LCS 2.61.Build 1537 from my local Leica vendor. To avoid
> troubles I recomend to use your current LCS version. There is a step during
> installation when a new firmware may be upladed into the scanner (and the
> version of firmware must fit with the version of LCS!). To ensure backstep
> possibility I would avoid the scanner firmware overwriting!
> I performed installation onto disk C:/Program Files (and it works), however
> as I told before, you can choose the installation folder on the quick
> swap/temporary disk.
> During the process you will be asked on the name of "prefabrikated" IPS
> profile folder. This looks something like:
> SP2_4ChT_AOBS_MultiRGBB8_UV_405_AOTF. It is absolutely not important what
> you choose, because you will overwrite default Leica IPS profiles with your
> backuped IPS profiles. After the installation you are forced to restart
> computer.
>
> 7) Before the first start of LCS you have to copy back your hardware
> settings into the Program Files/LCS/Hardware Control folder and your
> backuped personal settings files into Documents and Settings/LCS/ folder.
>
> 8)In the Control panel>system>hardware settings I had to actualize driver
> for Leica TCS-x. I found it in the LCS program folder/tools. Now SP2 is
> recognized as Imaging device. I also had to change naming of COM ports
> according to where LCS seek serial devices like motorized stage etc.
>
> 9) At this moment it is possible to run LCS and start to work ;-)
>
> I am not sure ofcourse, if this will works as nice on Win7, but I am
> optimistic. There can be one issue with LPT port which is connected via USB
> ports on modern motherboards and there could be a problem. I want to test
> this later.
>
> Best Regards
> Ondrej.
>
> ------------------------------------------
> Mgr. Ond&#345;ej Šebesta
> Laboratory of Confocal and Fluorescence Microscopy
> Faculty of Science, Charles University in Prague
> Vinicna 7
> 128 44 Prague
> Czech Republic
>
> Phone: [phone]
> e-mail: [hidden email]
>

[Steffen Dietzel]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear Confocalists,

for teaching purposes, I would like to have a graph with the following:

1) Intensity profile through the center of the Airy pattern (x,y) of a
fluorescent point for a wavelength of 500 nm (as, say, mean between 488
exc. and 512 em) in widefield, with the distance from the maximum on the
x-axis in nano- or micrometers and the normalized intensity on the
y-axis, say for a NA 1.4 Objective.

2) Same for confocal, closed pinhole.

Bonus:
3) Same for confocal/two photon with 900 nm excitation
4) All of the above along the optical axis instead of the x,y plane.


One purpose of this graph (more precisely: comparison of 1 and 2) would
be to visualize with typical parameters why in the confocal/two-photon
case with the steeper fall in intensity two Airy patterns can be closer
together and still have a 26% dip in intensity between them (called
extended Rayleigh-criterion, I believe?) although the ring with the
first minimum has the same diameter.

I am sure I could find that or a similar version in some book or
article, however I would like to include this in material published on
the web. And to avoid copyright issues the graphs have to be in the
public domain or with a Creative Commons Attribution License (CC-by).
Which probably means that they have to be made new. I would be perfectly
happy to make such graphs myself if I had the respective data (i.e.
columns with numbers for the x- and Y-values) to generate them in Excel,
Calc or Graphpad. However, as a mere biologist I don't know how to
generate such columns.

Therefore I would like to ask if someone would be willing to help out
and generate such columns with numbers. I assume that it is not that
much work for somebody with good insight in Airy pattern formulas and
the right program at hands - and hope I am not wrong with that
assumption. In particular data for graphs 1 and 2 above would be very
helpful.

I am thinking that covering the distance from -5 to +5 µm in 10 nm steps
would probably be more than sufficient.

Cheers

Steffen


--
------------------------------------------------------------
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

[Guy Cox-2]

Colin Sheppard did such plots for me and I'm sure he could do them for you.  Andreas Kriete is another one who has produced such plots.
 
I think they both read this list.

                                           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 Steffen Dietzel
Sent: Monday, 17 November 2014 9:40 PM
To: [hidden email]
Subject: Numbers for Airy pattern graphs?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear Confocalists,

for teaching purposes, I would like to have a graph with the following:

1) Intensity profile through the center of the Airy pattern (x,y) of a fluorescent point for a wavelength of 500 nm (as, say, mean between 488 exc. and 512 em) in widefield, with the distance from the maximum on the x-axis in nano- or micrometers and the normalized intensity on the y-axis, say for a NA 1.4 Objective.

2) Same for confocal, closed pinhole.

Bonus:
3) Same for confocal/two photon with 900 nm excitation
4) All of the above along the optical axis instead of the x,y plane.


One purpose of this graph (more precisely: comparison of 1 and 2) would be to visualize with typical parameters why in the confocal/two-photon case with the steeper fall in intensity two Airy patterns can be closer together and still have a 26% dip in intensity between them (called extended Rayleigh-criterion, I believe?) although the ring with the first minimum has the same diameter.

I am sure I could find that or a similar version in some book or article, however I would like to include this in material published on the web. And to avoid copyright issues the graphs have to be in the public domain or with a Creative Commons Attribution License (CC-by).
Which probably means that they have to be made new. I would be perfectly happy to make such graphs myself if I had the respective data (i.e.
columns with numbers for the x- and Y-values) to generate them in Excel, Calc or Graphpad. However, as a mere biologist I don't know how to generate such columns.

Therefore I would like to ask if someone would be willing to help out and generate such columns with numbers. I assume that it is not that much work for somebody with good insight in Airy pattern formulas and the right program at hands - and hope I am not wrong with that assumption. In particular data for graphs 1 and 2 above would be very helpful.

I am thinking that covering the distance from -5 to +5 µm in 10 nm steps would probably be more than sufficient.

Cheers

Steffen


--
------------------------------------------------------------
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

[jerie]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear Steffen,

the  online app http://www.svi.nl/NyquistCalculator would provide these
data so far as I can see.

Greetings,  Jens

http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/
Skype jens.Rietdorf
Am 17.11.2014 09:27 schrieb "Steffen Dietzel" <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Confocalists,
>
> for teaching purposes, I would like to have a graph with the following:
>
> 1) Intensity profile through the center of the Airy pattern (x,y) of a
> fluorescent point for a wavelength of 500 nm (as, say, mean between 488
> exc. and 512 em) in widefield, with the distance from the maximum on the
> x-axis in nano- or micrometers and the normalized intensity on the
> y-axis, say for a NA 1.4 Objective.
>
> 2) Same for confocal, closed pinhole.
>
> Bonus:
> 3) Same for confocal/two photon with 900 nm excitation
> 4) All of the above along the optical axis instead of the x,y plane.
>
>
> One purpose of this graph (more precisely: comparison of 1 and 2) would
> be to visualize with typical parameters why in the confocal/two-photon
> case with the steeper fall in intensity two Airy patterns can be closer
> together and still have a 26% dip in intensity between them (called
> extended Rayleigh-criterion, I believe?) although the ring with the
> first minimum has the same diameter.
>
> I am sure I could find that or a similar version in some book or
> article, however I would like to include this in material published on
> the web. And to avoid copyright issues the graphs have to be in the
> public domain or with a Creative Commons Attribution License (CC-by).
> Which probably means that they have to be made new. I would be perfectly
> happy to make such graphs myself if I had the respective data (i.e.
> columns with numbers for the x- and Y-values) to generate them in Excel,
> Calc or Graphpad. However, as a mere biologist I don't know how to
> generate such columns.
>
> Therefore I would like to ask if someone would be willing to help out
> and generate such columns with numbers. I assume that it is not that
> much work for somebody with good insight in Airy pattern formulas and
> the right program at hands - and hope I am not wrong with that
> assumption. In particular data for graphs 1 and 2 above would be very
> helpful.
>
> I am thinking that covering the distance from -5 to +5 µm in 10 nm steps
> would probably be more than sufficient.
>
> Cheers
>
> Steffen
>
>
> --
> ------------------------------------------------------------
> 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
>

[Arne Seitz]

Dear Steffen,

PSFLab is a freely available software with which you can create the excitation point spread function of a confocal microscope.
http://onemolecule.chem.uwm.edu/index.php/software

The PSF generator can be used in order to calculate a WF point spread function (which can be considered to be similar with the emission PSF of a confocal microscope).

http://bigwww.epfl.ch/algorithms/psfgenerator/

If you are interested in a "simple" airy pattern in the focal plane I can provide you with an ImageJ plugin.

Regards
Arne
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: lundi 17 novembre 2014 11:40
To: [hidden email]
Subject: Numbers for Airy pattern graphs?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear Confocalists,

for teaching purposes, I would like to have a graph with the following:

1) Intensity profile through the center of the Airy pattern (x,y) of a fluorescent point for a wavelength of 500 nm (as, say, mean between 488 exc. and 512 em) in widefield, with the distance from the maximum on the x-axis in nano- or micrometers and the normalized intensity on the y-axis, say for a NA 1.4 Objective.

2) Same for confocal, closed pinhole.

Bonus:
3) Same for confocal/two photon with 900 nm excitation
4) All of the above along the optical axis instead of the x,y plane.


One purpose of this graph (more precisely: comparison of 1 and 2) would be to visualize with typical parameters why in the confocal/two-photon case with the steeper fall in intensity two Airy patterns can be closer together and still have a 26% dip in intensity between them (called extended Rayleigh-criterion, I believe?) although the ring with the first minimum has the same diameter.

I am sure I could find that or a similar version in some book or article, however I would like to include this in material published on the web. And to avoid copyright issues the graphs have to be in the public domain or with a Creative Commons Attribution License (CC-by).
Which probably means that they have to be made new. I would be perfectly happy to make such graphs myself if I had the respective data (i.e.
columns with numbers for the x- and Y-values) to generate them in Excel, Calc or Graphpad. However, as a mere biologist I don't know how to generate such columns.

Therefore I would like to ask if someone would be willing to help out and generate such columns with numbers. I assume that it is not that much work for somebody with good insight in Airy pattern formulas and the right program at hands - and hope I am not wrong with that assumption. In particular data for graphs 1 and 2 above would be very helpful.

I am thinking that covering the distance from -5 to +5 µm in 10 nm steps would probably be more than sufficient.

Cheers

Steffen


--
------------------------------------------------------------
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

[mcammer]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

This sounds like a good exercise for a lab!  

_________________________________________
Michael Cammer, Optical Microscopy Specialist
http://ocs.med.nyu.edu/microscopy
Lab: [phone]  Cell: [phone]

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Steffen Dietzel [[hidden email]]
Sent: Monday, November 17, 2014 5:40 AM
To: [hidden email]
Subject: Numbers for Airy pattern graphs?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear Confocalists,

for teaching purposes, I would like to have a graph with the following:

1) Intensity profile through the center of the Airy pattern (x,y) of a
fluorescent point for a wavelength of 500 nm (as, say, mean between 488
exc. and 512 em) in widefield, with the distance from the maximum on the
x-axis in nano- or micrometers and the normalized intensity on the
y-axis, say for a NA 1.4 Objective.

2) Same for confocal, closed pinhole.

Bonus:
3) Same for confocal/two photon with 900 nm excitation
4) All of the above along the optical axis instead of the x,y plane.


One purpose of this graph (more precisely: comparison of 1 and 2) would
be to visualize with typical parameters why in the confocal/two-photon
case with the steeper fall in intensity two Airy patterns can be closer
together and still have a 26% dip in intensity between them (called
extended Rayleigh-criterion, I believe?) although the ring with the
first minimum has the same diameter.

I am sure I could find that or a similar version in some book or
article, however I would like to include this in material published on
the web. And to avoid copyright issues the graphs have to be in the
public domain or with a Creative Commons Attribution License (CC-by).
Which probably means that they have to be made new. I would be perfectly
happy to make such graphs myself if I had the respective data (i.e.
columns with numbers for the x- and Y-values) to generate them in Excel,
Calc or Graphpad. However, as a mere biologist I don't know how to
generate such columns.

Therefore I would like to ask if someone would be willing to help out
and generate such columns with numbers. I assume that it is not that
much work for somebody with good insight in Airy pattern formulas and
the right program at hands - and hope I am not wrong with that
assumption. In particular data for graphs 1 and 2 above would be very
helpful.

I am thinking that covering the distance from -5 to +5 µm in 10 nm steps
would probably be more than sufficient.

Cheers

Steffen


--
------------------------------------------------------------
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]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear Arne,

that does look indeed like an interesting piece of software, e.g. for
demonstrating the need to avoid refraction index mismatches. However,
although it says it is for confocal microscopy, I do not see an option
to play around with pinhole size, so I am afraid it will not work for my
specific purpose. The "getting started"-pdf does not contain the words
'pinhole' or 'Airy'. Did I overlook something?

Steffen

Am 17.11.2014 13:29, schrieb Seitz Arne:

> Dear Steffen,
>
> PSFLab is a freely available software with which you can create the excitation point spread function of a confocal microscope.
> http://onemolecule.chem.uwm.edu/index.php/software
>
> The PSF generator can be used in order to calculate a WF point spread function (which can be considered to be similar with the emission PSF of a confocal microscope).
>
> http://bigwww.epfl.ch/algorithms/psfgenerator/
>
> If you are interested in a "simple" airy pattern in the focal plane I can provide you with an ImageJ plugin.
>
> Regards
> Arne
>  
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
> Sent: lundi 17 novembre 2014 11:40
> To: [hidden email]
> Subject: Numbers for Airy pattern graphs?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Confocalists,
>
> for teaching purposes, I would like to have a graph with the following:
>
> 1) Intensity profile through the center of the Airy pattern (x,y) of a fluorescent point for a wavelength of 500 nm (as, say, mean between 488 exc. and 512 em) in widefield, with the distance from the maximum on the x-axis in nano- or micrometers and the normalized intensity on the y-axis, say for a NA 1.4 Objective.
>
> 2) Same for confocal, closed pinhole.
>
> Bonus:
> 3) Same for confocal/two photon with 900 nm excitation
> 4) All of the above along the optical axis instead of the x,y plane.
>
>
> One purpose of this graph (more precisely: comparison of 1 and 2) would be to visualize with typical parameters why in the confocal/two-photon case with the steeper fall in intensity two Airy patterns can be closer together and still have a 26% dip in intensity between them (called extended Rayleigh-criterion, I believe?) although the ring with the first minimum has the same diameter.
>
> I am sure I could find that or a similar version in some book or article, however I would like to include this in material published on the web. And to avoid copyright issues the graphs have to be in the public domain or with a Creative Commons Attribution License (CC-by).
> Which probably means that they have to be made new. I would be perfectly happy to make such graphs myself if I had the respective data (i.e.
> columns with numbers for the x- and Y-values) to generate them in Excel, Calc or Graphpad. However, as a mere biologist I don't know how to generate such columns.
>
> Therefore I would like to ask if someone would be willing to help out and generate such columns with numbers. I assume that it is not that much work for somebody with good insight in Airy pattern formulas and the right program at hands - and hope I am not wrong with that assumption. In particular data for graphs 1 and 2 above would be very helpful.
>
> I am thinking that covering the distance from -5 to +5 µm in 10 nm steps would probably be more than sufficient.
>
> Cheers
>
> Steffen
>
>
> --
> ------------------------------------------------------------
> 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

[Sergey Tauger]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear Steffen,

you can try http://cirl.memphis.edu/cosmos.php It supports WF and confocal
with all properties easy to specify, the only problem is PSF generation speed
(typically minutes). The output is .wu files that can be read with ImageJ using
Import -> Raw with properties:
Type 32-real
Offset is 1024 bytes
Gap 0 bytes
Little-endian byte order

You can easily extract a XZ cross-section both from COSMOS and ImageJ.

Best,
Sergey

[Arne Seitz]

Dear Steffen,

the PSF lab is "only" calculating the excitation point spread function.
To simulate the effect of the pinhole you needed to modify the emission PSF.

Best
Arne
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: lundi 17 novembre 2014 14:40
To: [hidden email]
Subject: Re: Numbers for Airy pattern graphs?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear Arne,

that does look indeed like an interesting piece of software, e.g. for demonstrating the need to avoid refraction index mismatches. However, although it says it is for confocal microscopy, I do not see an option to play around with pinhole size, so I am afraid it will not work for my specific purpose. The "getting started"-pdf does not contain the words 'pinhole' or 'Airy'. Did I overlook something?

Steffen

Am 17.11.2014 13:29, schrieb Seitz Arne:

> Dear Steffen,
>
> PSFLab is a freely available software with which you can create the excitation point spread function of a confocal microscope.
> http://onemolecule.chem.uwm.edu/index.php/software
>
> The PSF generator can be used in order to calculate a WF point spread function (which can be considered to be similar with the emission PSF of a confocal microscope).
>
> http://bigwww.epfl.ch/algorithms/psfgenerator/
>
> If you are interested in a "simple" airy pattern in the focal plane I can provide you with an ImageJ plugin.
>
> Regards
> Arne
>  
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Steffen Dietzel
> Sent: lundi 17 novembre 2014 11:40
> To: [hidden email]
> Subject: Numbers for Airy pattern graphs?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Confocalists,
>
> for teaching purposes, I would like to have a graph with the following:
>
> 1) Intensity profile through the center of the Airy pattern (x,y) of a fluorescent point for a wavelength of 500 nm (as, say, mean between 488 exc. and 512 em) in widefield, with the distance from the maximum on the x-axis in nano- or micrometers and the normalized intensity on the y-axis, say for a NA 1.4 Objective.
>
> 2) Same for confocal, closed pinhole.
>
> Bonus:
> 3) Same for confocal/two photon with 900 nm excitation
> 4) All of the above along the optical axis instead of the x,y plane.
>
>
> One purpose of this graph (more precisely: comparison of 1 and 2) would be to visualize with typical parameters why in the confocal/two-photon case with the steeper fall in intensity two Airy patterns can be closer together and still have a 26% dip in intensity between them (called extended Rayleigh-criterion, I believe?) although the ring with the first minimum has the same diameter.
>
> I am sure I could find that or a similar version in some book or article, however I would like to include this in material published on the web. And to avoid copyright issues the graphs have to be in the public domain or with a Creative Commons Attribution License (CC-by).
> Which probably means that they have to be made new. I would be perfectly happy to make such graphs myself if I had the respective data (i.e.
> columns with numbers for the x- and Y-values) to generate them in Excel, Calc or Graphpad. However, as a mere biologist I don't know how to generate such columns.
>
> Therefore I would like to ask if someone would be willing to help out and generate such columns with numbers. I assume that it is not that much work for somebody with good insight in Airy pattern formulas and the right program at hands - and hope I am not wrong with that assumption. In particular data for graphs 1 and 2 above would be very helpful.
>
> I am thinking that covering the distance from -5 to +5 µm in 10 nm steps would probably be more than sufficient.
>
> Cheers
>
> Steffen
>
>
> --
> ------------------------------------------------------------
> 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

[David Baddeley]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear Steffen,
just to add to the suggestions already made, confocal and two-photon PSFs are really easy to derive from an emission or excitation case, if you have a psf generation program for widefield.
To a 1st appoximation (i.e. infinitely small pinhole):
confocal PSF = widefield PSF at exctitation wavelength multiplied by widefield PSF at detection wavelength
Or for a large pinhole in 2-photon
2-photon PSF = widefield PSF at excitation wavelength squared.
These multiplications should be easy to do in ImageJ (convert the data type to floating point first)
If you want to model a finite pinhole size you can convolve the detection PSF with a flat disc with the pinhole diameter before doing the multiplication (doable in ImageJ, but somewhat harder, as the disc needs to be centred to avoid shifting the excitation and detection PSFS wrt each other).
cheers,David

[Steffen Dietzel]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Jens,

yes it does - but apparently only in 'normal pixel step size', i.e. in
the said case 89 nm. Not fine enough to extract a nice intensity plot

Steffen

Am 17.11.2014 13:03, schrieb jens rietdorf:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Steffen,
>
> the  online app http://www.svi.nl/NyquistCalculator would provide these
> data so far as I can see.
>
> Greetings,  Jens
>
> http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/
> Skype jens.Rietdorf
> Am 17.11.2014 09:27 schrieb "Steffen Dietzel" <[hidden email]>:
>

--
------------------------------------------------------------
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]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

David,

that is a good idea. So with the one-photon PSF data from PSFLab, image
multiplication in Fiji and normalization in Excel I should be getting
somewhere.

However, when I create a psf intensity distribution in the focal plane
with PSFlab for NA 1.4, lambda 0.5 µm (all Ris set to 1.518, calculation
in x from -1 µm to +1 µm in 10 nm steps, in y 30 nm wide), I get the
first minimum at 0.27 µm away from the maximum intensity. But according
to r=0.61*lambda/NA it should be at 0.22 µm.

Also, when I superimpose two intensity profiles with the max of the
second in the first min of the first (Rayleigh-criterion), the maximal
intensity drop between the two profiles is about 55% while it should be
only 26%. As Arne mentioned, PSFlab describes the excitation PSF only,
not the theoretical confocal 1/sqrt(2) case.

So apparently PSFlab does not calculate the simple theoretical Airy
pattern but some more complicated (and supposedly more realistic) case,
resulting in different results.

I am wondering, does that mean we should throw the Rayleigh formula out
the window, including the 26%-intensity dip if the max of one is on the
first min of the other pattern? Else, I am still looking for the right
numbers. I didn't yet get around to try Cosmos, as was suggested by Sergey.


Steffen



Am 17.11.2014 15:29, schrieb David Baddeley:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Steffen,
> just to add to the suggestions already made, confocal and two-photon PSFs are really easy to derive from an emission or excitation case, if you have a psf generation program for widefield.
> To a 1st appoximation (i.e. infinitely small pinhole):
> confocal PSF = widefield PSF at exctitation wavelength multiplied by widefield PSF at detection wavelength
> Or for a large pinhole in 2-photon
> 2-photon PSF = widefield PSF at excitation wavelength squared.
> These multiplications should be easy to do in ImageJ (convert the data type to floating point first)
> If you want to model a finite pinhole size you can convolve the detection PSF with a flat disc with the pinhole diameter before doing the multiplication (doable in ImageJ, but somewhat harder, as the disc needs to be centred to avoid shifting the excitation and detection PSFS wrt each other).
> cheers,David
>

--
------------------------------------------------------------
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