Leica confocal- between lines/frames

[Matthew Pearson-3]

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Hi there,

I am currently using a Leica TCS-SP2 AOBS microscope running leica
confocal software.  When performing a sequential scan, the sequential
scan window opens where you drag and drop you're parameter settings and
you have to select between lines or between frames.

Basically i'm unsure as to the differences between the two (between
lines/frames) and also when i select between frames when i go to the
acquire window i can only click the series button to begin scanning,
single scan and continuous are not highlighted and unselectable, can
anyone shed any light on why this might be?


Many thanks,

Matt Pearson.

Institute of Ophthalmology
University College London

[Nowell, Cameron]

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Hi Matt,
           Between line sequential scanning means that alternating lines are scanned of each channel to build up the image. For example if you had a two colour scan (say 488 and 568) with 512x512 resolution and you selected between lines scan the microscope would scan one line with just 488 and then one line with just 568 and then repeat the whole thing as many times as it needed to to build up the image. In the example given that would be 512 times. It then continually repeats this until stopped. The effect is that you get a sequential image that you can see all the channels at the same time, it will just be refreshing slower depending on your scan speed, the resolution and the number of channels.
 
Between frame scanning means that the whole frame of one channel will be scanned before it goes onto the next one.
 
As to why you have buttons greyed out i am not sure as we do not have an SP2 in our facility (we have an SP5 though). I would guess that they are not selectable because you have between frame scanning set. There will be other people here that have more experience with the SP2 than me.
 
 
Hope that makes sense
 
 
Cheers
 
Cam
 
 
 
Cameron Nowell
Microscopy Research and Imaging Facility
Cell Cycle and Development
Peter MacCallum Cancer Centre
7 St Andrews Place
East Melbourne, 3002
Victoria AUSTRALIA

Phone: [phone]
Fax: [phone]
Mobile: [phone]

 

________________________________

From: Confocal Microscopy List on behalf of Matthew Pearson
Sent: Sat 5/01/2008 3:39 AM
To: [hidden email]
Subject: Leica confocal- between lines/frames



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

Hi there,

I am currently using a Leica TCS-SP2 AOBS microscope running leica
confocal software.  When performing a sequential scan, the sequential
scan window opens where you drag and drop you're parameter settings and
you have to select between lines or between frames.

Basically i'm unsure as to the differences between the two (between
lines/frames) and also when i select between frames when i go to the
acquire window i can only click the series button to begin scanning,
single scan and continuous are not highlighted and unselectable, can
anyone shed any light on why this might be?


Many thanks,

Matt Pearson.

Institute of Ophthalmology
University College London




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[Michael Weber-4]

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Dear Matt,

Cam already pointed out some differences between line and frame sequence.

Main advantage of line-by-line is the multi-color speed per line - you get
a better overlay (seen on pixel level) between multiple channels if
something is going on in the sample (movement, intensity changes etc.) or
in the system (stage instability etc.).

Main advantage of frame-by-frame is that you can change settings between
channels - i.e. position of sp-sliders or pinhole diameter. You can also
acquire more colors than the number of available detectors in this mode.
These kind of mechanical changes are not possible line-by-line.

The fact that LCS software doesn't allow continuous or single scan if
frame-by-frame is activated is just a technical limitation of the setup.
In principle it is possible, at least for one channel which is currently
selected. It's solved in a kind of useful way in LAS-AF for the SP5.

cheers,
Michael

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi Matt,
>            Between line sequential scanning means that alternating lines
> are scanned of each channel to build up the image. For example
> if you had a two colour scan (say 488 and 568) with 512x512
> resolution and you selected between lines scan the microscope
> would scan one line with just 488 and then one line with just
> 568 and then repeat the whole thing as many times as it needed
> to to build up the image. In the example given that would be
> 512 times. It then continually repeats this until stopped. The
> effect is that you get a sequential image that you can see all
> the channels at the same time, it will just be refreshing
> slower depending on your scan speed, the resolution and the
> number of channels.
>
> Between frame scanning means that the whole frame of one channel will be
> scanned before it goes onto the next one.
>
> As to why you have buttons greyed out i am not sure as we do not have an
> SP2 in our facility (we have an SP5 though). I would guess that they are
> not selectable because you have between frame scanning set. There will be
> other people here that have more experience with the SP2 than me.
>
>
> Hope that makes sense
>
>
> Cheers
>
> Cam
>
>
>
> Cameron Nowell
> Microscopy Research and Imaging Facility
> Cell Cycle and Development
> Peter MacCallum Cancer Centre
> 7 St Andrews Place
> East Melbourne, 3002
> Victoria AUSTRALIA
>
> Phone: [phone]
> Fax: [phone]
> Mobile: [phone]
>
>
>
> ________________________________
>
> From: Confocal Microscopy List on behalf of Matthew Pearson
> Sent: Sat 5/01/2008 3:39 AM
> To: [hidden email]
> Subject: Leica confocal- between lines/frames
>
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi there,
>
> I am currently using a Leica TCS-SP2 AOBS microscope running leica
> confocal software.  When performing a sequential scan, the sequential
> scan window opens where you drag and drop you're parameter settings and
> you have to select between lines or between frames.
>
> Basically i'm unsure as to the differences between the two (between
> lines/frames) and also when i select between frames when i go to the
> acquire window i can only click the series button to begin scanning,
> single scan and continuous are not highlighted and unselectable, can
> anyone shed any light on why this might be?
>
>
> Many thanks,
>
> Matt Pearson.
>
> Institute of Ophthalmology
> University College London
>
>
>
>
> This email (including any attachments) may contain
> confidential and/or legally privileged information and is
> intended only to be read or used by the addressee.  If you
> are not the intended addressee, any use, distribution,
> disclosure or copying of this email is strictly
> prohibited.
> Confidentiality and legal privilege attached to this email
> (including any attachments) are not waived or lost by
> reason of its mistaken delivery to you.
> If you have received this email in error, please delete it
> and notify us immediately by telephone or email.  Peter
> MacCallum Cancer Centre provides no guarantee that this
> transmission is free of virus or that it has not been
> intercepted or altered and will not be liable for any delay
> in its receipt.