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The Leica SP-2 does do ROI scanning both in the context of visible lines
using the AOTF and for IR using the EOM.  We do it all the time.  Try
changing the power level of the laser while the ROI scan is
rastering--you should expect to see the intensity outside of the ROI go
down when laser power is reduced (for the laser checkmarked in the
config dialog) and the reverse should happen as the laser power is
increased.  The ROI "remembers" the laser power settings when it was
drawn, so if you haven't changed the power settings (usually down)
before beginning the scan, you won't see a difference between intensity
in the ROI and the surrounding area. This is a little counter intuitive,
but once you know the trick it should work.
    With MP excitation, the area surrounding the ROI will be set to the
EOM "offset" avg. power, and the area within the ROI will be controlled
with the EOM gain setting.  With the offset adjusted to the maximum
extinction ratio, only the area inside the ROI will be irradiated once
the scan is started.
    When ROI's are defined, you can use the ROI "select" tool to select
an ROI; upon right clicking you have the option to save the ROI
definition or load a previously saved ROI definition.  This can be useful.
-Karl

John Runions wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi Russell, I have tried to do an ROI scan this way and the system
> still scans the entire field, i.e. I have defined the ROI and
> specified a laser that is not different from the one I was using when
> scanning the whole field.  Can you tell me exactly how you use the
> Config box?  I am trying to advise a collegue without actually looking
> at the software..  Is there perhaps a hidden button that is the Leica
> equivalent of Zeiss' 'use ROI' button?
>
> Thanks for your help.  John.
>
> Russell McConnell wrote:
>
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>Hi All,
>>The Leica software does have an ROI scanning functionality, the only
>>problem is that the button(s) that control it are usually not present on
>>the default GUI (graphic user interface).  Go to Tools-->Customize.
>>This opens a window with a list of different commands.  Select 'Basic
>>Scan' from the list and a number of buttons should appear in the right
>>part of the window.  The two that you want are the 'ROI' and 'Config'
>>buttons, just drag them onto the main GUI where you want them to be.
>>The 'ROI' button lets you define the areas you want to scan, while the
>>'Config' button lets you specify which laser lines you want to use in
>>each area (i.e. in ROI-1 you want to use 488 and 568, while in ROI-2 you
>>want only 488 etc.).
>>This feature is very useful for not only FRAP and FRET analyses, but
>>also for checking to see if using a particular laser power is bleaching
>>a sample; the AOTF blanks the laser light outside of the ROI so those
>>parts of the sample are not exposed to the excitation light.  Any
>>confocal with an AOTF should be able to do this type of scanning.
>>Cheers,
>>Russell
>>
>>Jill Pflugheber wrote:
>>
>>
>>>Search the CONFOCAL archive at
>>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>>Hi John,
>>>
>>>Having used both systems, I know EXACTLY what you mean.  I have not
>>>figured a way to use ROI scanning on the Leica as it is on the Zeiss.
>>>For those of you not understanding the Zeiss system, it allows the user
>>>to define a ROI to scan using a rectangle, oval, or draw-your-own.  I
>>>used that function, for instance, to collect a stack of images of one
>>>cell in a field.  I have not found any way to do the same with the
>>>Leica--only ROI for analysis.
>>>
>>>J
>>>
>>>andrea manazza wrote:
>>>
>>>
>>>
>>>>Search the CONFOCAL archive at
>>>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>
>>>>Hi John. I'm not quite sure I understood your question; anyhow, if I get
>>>>the point, the ROI on the SP2 might be used both for analysis and for
>>>>scanning (selective bleaching for FRAP, eg).
>>>>
>>>>I hope this is what you were looking for.
>>>>
>>>>Best wishes.
>>>>
>>>>Andrew
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>With the Zeiss confocal systems, it is posible to scan only within a
>>>>Region Of Interest.  Is this posible with the Leica SP2 software or is
>>>>the Leica ROI only intended for analysis?
>>>>
>>>>Thank you.  John.
>>>>
>>>>
>>>>
>>>>dott. Andrea Manazza
>>>>Dipartimento di Oncologia, Università di Torino
>>>>CeRMS, ospedale Molinette
>>>>Via Santena, 7 - 10126 Torino, Italia
>>>>lab: +39-11-6336859
>>>>fax: +39-11-6336887
>>>>
>>>>
>>>>
>>
>>--
>>Russell McConnell
>>Manager, Tufts-NEMC Imaging Facility
>>Department of Neuroscience
>>http://www.neurosci.tufts.edu/Imaging
>>
>>
>>
>>
>
> --
>
> *********************************
> C. John Runions, Ph.D.
> School of Biological and Molecular Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email: [log in to unmask] <mailto:[log in to unmask]>
> phone: +44 (0) 1865 483 964
>

--
Karl Garsha
Light Microscopy Specialist
Imaging Technology Group
Beckman Institute for Advanced Science and Technology
University of Illinois at Urbana-Champaign
405 North Mathews Avenue
Urbana, IL 61801
Office: B650J
Phone: 217.244.6292
Fax: 217.244.6219
Mobile: 217.390.1874
www.itg.uiuc.edu