Leica between lines scan mode!

[Matthew Pearson-3]

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Hi there,

I am using a leica tcs_sp2 confocal with leica confocal software.  
Basically, when you do a three colour sequential scan for instance, you
drag your individual parameter settings into the sequential window. I
have one parameter setting per fluorophore, hence i have dragged three
settings into the sequential window:
                                                        -DAPI
                                                       -FITC
                                                       -TRITC
As I understand it I now save this in the sequential box as e.g RGB.
then press load to load this sequential configuration.

But this is where i'm stuck, as when you select between the lines you
can scan continuously unlike with between frames.  But when i do press
continuous i only get a one channel image in the viewing window e.g
tritc, the screen is divided into 4 as if all 3 channels should be
visible but only the TRITC channel is visible. And its not my individual
DAPI, FITC settings as when these are loaded and scanned individually I
get good signal.

It would seem to make sense that when you can use the continuous button
you should be able to click one of the display windows e.g FITC, change
the gain settings then perhaps click the DAPI window, chnage the gain
settings all as the system is scanning.  Maybe this cannot be done? But
this still doesnt explain why i cant see all three channels in the
viewing window.

One final thing, I dont know how i do it, but sometimes when i load a
sequential setting, the only options are between frames/between stacks,
there is no between lines, does anyone know why this option would have
disappeared and if it does how to  undo it. I have to close the software
and re-open to get between lines back!

Sorry for the long email guys, hope this makes sense.

I'd appreciate any advice on this as this software drives me crazy :s

Many thanks,

Matt Pearson
University College London

[Csucs Gabor]

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Hallo Matt,

Although I don't completely understand your problem, but one detail
which may help. If you work in the sequential mode, you need to check
whether you do line by line or frame by frame scanning. In the
continuous scanning mode you'll see the different colors together (same
time) only if you do line by line sequential scanning. Now, for this
line by line scanning it is essential that you set up the different
individual channels (DAPI FITC etc.) that between the different images
you are not moving any of the sliders. So you should just switch on and
off the corresponding laser lines and detectors. A small, but important
detail: switching off the laser line should be by setting the laser
intensity to zero and NOT by checking/unchecking the little box (this
controls a mechanical - rather slow shutter). If you try to use the line
by line mode without controlling the above mentioned features - you'll
get strange results. With the frame by frame scans this is not an issue.
Hopefully this helps.

Cheers    Gabor

--
Gabor Csucs
Light Microscopy Centre, ETH Zurich
Schafmattstrasse 18, HPM F16
CH-8093, Zurich, Switzerland

Web: www.lmc.ethz.ch
Phone: [phone]
Fax: [phone]
e-mail: [hidden email]

[Zoltan]

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Dear Matt,

 

Basically you have to make sure that the system can handle the hardware changes fast enough between lines, if you want to do this type of sequential scanning.  Otherwise you'll have to use frame by frame scanning, which is much less demanding in terms of fast switching your hardware.   E.g., you can rely on the AOTF / AOBS to switch very quickly. The software seems to be set up such that it disables options that the hardware couldn't handle.

 

Zoltan

On Jan 17, 2008 11:20 AM, Matthew Pearson <[hidden email]> wrote:

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi there,

I am using a leica tcs_sp2 confocal with leica confocal software.
Basically, when you do a three colour sequential scan for instance, you
drag your individual parameter settings into the sequential window. I
have one parameter setting per fluorophore, hence i have dragged three
settings into the sequential window:
                                                       -DAPI
                                                      -FITC
                                                      -TRITC
As I understand it I now save this in the sequential box as e.g RGB.
then press load to load this sequential configuration.

But this is where i'm stuck, as when you select between the lines you
can scan continuously unlike with between frames.  But when i do press
continuous i only get a one channel image in the viewing window e.g
tritc, the screen is divided into 4 as if all 3 channels should be
visible but only the TRITC channel is visible. And its not my individual
DAPI, FITC settings as when these are loaded and scanned individually I
get good signal.

It would seem to make sense that when you can use the continuous button
you should be able to click one of the display windows e.g FITC, change
the gain settings then perhaps click the DAPI window, chnage the gain
settings all as the system is scanning.  Maybe this cannot be done? But
this still doesnt explain why i cant see all three channels in the
viewing window.

One final thing, I dont know how i do it, but sometimes when i load a
sequential setting, the only options are between frames/between stacks,
there is no between lines, does anyone know why this option would have
disappeared and if it does how to  undo it. I have to close the software
and re-open to get between lines back!

Sorry for the long email guys, hope this makes sense.

I'd appreciate any advice on this as this software drives me crazy :s

Many thanks,

Matt Pearson
University College London

--
--
Zoltan Cseresnyes
Facility manager, Imaging Suite
Dept. of Zoology University of Cambridge
Downing Street, Cambridge
CB2 3EJ    UK

Tel.: (++44) (0)1223 769282
Fax.: (++44) (0)1223 336676

[Jon Ekman]

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Set up your DAPI/FITC/TRITC first with all lasers active at once and adjust
all other beam path settings according and save as dapi-fitc-tritc. Next,
turn off the individual lasers and PMTs not being used for DAPI acquisition
but do not change any sliders or dichroics save that setting as dapi. Repeat
this for FITC and TRITC. Load those saved settings into the Sequential scan
setup.

The goal for sequential line scanning is just switch on and off the lasers
and the PMTs, if you move a slider or any hardware you will have to use
"between the frames" mode.

You can't see the all three channels because you are moving a piece of
hardware between settings.

Does this help?

-Jon


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Matthew Pearson
Sent: Thursday, January 17, 2008 5:20 AM
To: [hidden email]
Subject: Leica between lines scan mode!

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi there,

I am using a leica tcs_sp2 confocal with leica confocal software.  
Basically, when you do a three colour sequential scan for instance, you
drag your individual parameter settings into the sequential window. I
have one parameter setting per fluorophore, hence i have dragged three
settings into the sequential window:
                                                        -DAPI
                                                       -FITC
                                                       -TRITC
As I understand it I now save this in the sequential box as e.g RGB.
then press load to load this sequential configuration.

But this is where i'm stuck, as when you select between the lines you
can scan continuously unlike with between frames.  But when i do press
continuous i only get a one channel image in the viewing window e.g
tritc, the screen is divided into 4 as if all 3 channels should be
visible but only the TRITC channel is visible. And its not my individual
DAPI, FITC settings as when these are loaded and scanned individually I
get good signal.

It would seem to make sense that when you can use the continuous button
you should be able to click one of the display windows e.g FITC, change
the gain settings then perhaps click the DAPI window, chnage the gain
settings all as the system is scanning.  Maybe this cannot be done? But
this still doesnt explain why i cant see all three channels in the
viewing window.

One final thing, I dont know how i do it, but sometimes when i load a
sequential setting, the only options are between frames/between stacks,
there is no between lines, does anyone know why this option would have
disappeared and if it does how to  undo it. I have to close the software
and re-open to get between lines back!

Sorry for the long email guys, hope this makes sense.

I'd appreciate any advice on this as this software drives me crazy :s

Many thanks,

Matt Pearson
University College London

[Christoph BAUER-5]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Three remarks that might be important for your problem.
1.If you want to combine parameter settings to do between-line scans you should make sure that for each of the channels you would like to combine you have identical detection windows. Although for each channel normally only one of the detection windows is checked, the position of the non-checked windows is important and problems might occur if they are not identical.
2. Any changes of for example gain, offset etc. need to be saved in the settings window. Only then they will be applied in the sequential scan.
3. When you open a saved sequential scan setting like RGB in you example, it helps to just once double click on the individual parameter settings in your list.This helps to position the mirrors of the spectral detector correctly I guess; but probably you did already not follow rule one if results occur like those you describe.
Cheers,
Christoph

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi there,

I am using a leica tcs_sp2 confocal with leica confocal software. 
Basically, when you do a three colour sequential scan for instance, you drag your individual parameter settings into the sequential window. I have one parameter setting per fluorophore, hence i have dragged three settings into the sequential window:
                                                       -DAPI
                                                      -FITC
                                                      -TRITC
As I understand it I now save this in the sequential box as e.g RGB. then press load to load this sequential configuration.

But this is where i'm stuck, as when you select between the lines you can scan continuously unlike with between frames.  But when i do press continuous i only get a one channel image in the viewing window e.g tritc, the screen is divided into 4 as if all 3 channels should be visible but only the TRITC channel is visible. And its not my individual DAPI, FITC settings as when these are loaded and scanned individually I get good signal.

It would seem to make sense that when you can use the continuous button you should be able to click one of the display windows e.g FITC, change the gain settings then perhaps click the DAPI window, chnage the gain settings all as the system is scanning.  Maybe this cannot be done? But this still doesnt explain why i cant see all three channels in the viewing window.

One final thing, I dont know how i do it, but sometimes when i load a sequential setting, the only options are between frames/between stacks, there is no between lines, does anyone know why this option would have disappeared and if it does how to  undo it. I have to close the software and re-open to get between lines back!

Sorry for the long email guys, hope this makes sense.

I'd appreciate any advice on this as this software drives me crazy :s

Many thanks,

Matt Pearson
University College London

NATIONAL CENTER OF COMPETENCE IN RESEARCH
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