LSM510 multiple locations macro

[yee z]

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

Dear all,

I am a newbie to Zeiss LSM510.

Recently I tried to run a multiple locations time series macro on LSM510.
The protocol I followed is the "Zeiss LSM 510 META NLO User's Manual" from
Fred Hutchinson Cancer Research Center which I got online.

After mark 4 different location and load my configuration, I did these step as
follows:

6. Start Multi-Time Macro (Main Menu>MACROS>MultiTime"R")
7. Click "Refresh" button.
8. Load Multi-Track configuration from pull-down menu in Multi-Time
window (Config, Time-Interval and Bleach), (e.g. aa1)
9. Make sure "number of scans" is set to "0", and z-stack is checked in the
"Config, Time-Interval and Bleach" panel.(I did not check the z-stack because
I only have one z-stack)
10. Under "List of Blocks", set number of repetitions to "0"(I changed this
number to 3 later in step 15.1)
11. Make sure autofocus is NOT checked, and bleach is NOT checked.
12. Click "Multiple Location" button on top of Multi-Time window.
13. Verify that the list of locations in the Multi-Time series window menu
matches the list in the stage control panel. If not, click Refresh.
14. Click "Options" (in Multi_Time window); Check "Keep Final Image
Open" and "Save Final Image"
15.1. Under List of Blocks, enter the number of Block repetitions (e.g. 3, for
three time-points)
15.2. Under Block 1 parameters, enter the wait interval.
16. Click "Start Time".

But after all these, no images were collected except the black one and
warning message as "Pinhole PH2 is not adjusted".  I also notice that in
configuration control panel, under list of tracks, the channels info changed to
another laser momentarily (it switched from this to my previous setting
continuously). In the end, I had to stop the recording manualy and no image
was saved.

I have attached three pics of my setting screen, and would you guys please
check my protocol and let me know which step I made a mistake?

Here is the link
[IMG]http://i143.photobucket.com/albums/r138/qqbmwcichlid/macro-1.jpg
[/IMG]

[IMG]http://i143.photobucket.com/albums/r138/qqbmwcichlid/macro-3.jpg
[/IMG]

[IMG]http://i143.photobucket.com/albums/r138/qqbmwcichlid/macro-2.jpg
[/IMG]

Thanks in advance for any suggestions.


Yi

[Pawel,Pasierbek]

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

Dear Yi,

We do quite some imaging using the Zeiss MTM. You can contact me off the
list so we could try to solve your problem together.

Best regards,
Pawel


-----------------------------------------------------------------
Pawel Pasierbek, PhD
Biooptics
IMP - Institute of Molecular Pathology
IMBA - Institute of Molecular Biotechnology
Dr. Bohr Gasse 7
A-1030 Vienna
Austria
 
Tel. [phone] - 3702 or 3866
Fax [phone]

e-mail: [hidden email]

www.imp.ac.at
www.imba.oeaw.ac.at

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Yi Zheng
Sent: Dienstag, 26. August 2008 05:32
To: [hidden email]
Subject: LSM510 multiple locations macro

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

Dear all,

I am a newbie to Zeiss LSM510.

Recently I tried to run a multiple locations time series macro on
LSM510.
The protocol I followed is the "Zeiss LSM 510 META NLO User's Manual"
from
Fred Hutchinson Cancer Research Center which I got online.

After mark 4 different location and load my configuration, I did these
step as
follows:

6. Start Multi-Time Macro (Main Menu>MACROS>MultiTime"R")
7. Click "Refresh" button.
8. Load Multi-Track configuration from pull-down menu in Multi-Time
window (Config, Time-Interval and Bleach), (e.g. aa1)
9. Make sure "number of scans" is set to "0", and z-stack is checked in
the
"Config, Time-Interval and Bleach" panel.(I did not check the z-stack
because
I only have one z-stack)
10. Under "List of Blocks", set number of repetitions to "0"(I changed
this
number to 3 later in step 15.1)
11. Make sure autofocus is NOT checked, and bleach is NOT checked.
12. Click "Multiple Location" button on top of Multi-Time window.
13. Verify that the list of locations in the Multi-Time series window
menu
matches the list in the stage control panel. If not, click Refresh.
14. Click "Options" (in Multi_Time window); Check "Keep Final Image
Open" and "Save Final Image"
15.1. Under List of Blocks, enter the number of Block repetitions (e.g.
3, for
three time-points)
15.2. Under Block 1 parameters, enter the wait interval.
16. Click "Start Time".

But after all these, no images were collected except the black one and
warning message as "Pinhole PH2 is not adjusted".  I also notice that in

configuration control panel, under list of tracks, the channels info
changed to
another laser momentarily (it switched from this to my previous setting
continuously). In the end, I had to stop the recording manualy and no
image
was saved.

I have attached three pics of my setting screen, and would you guys
please
check my protocol and let me know which step I made a mistake?

Here is the link
[IMG]http://i143.photobucket.com/albums/r138/qqbmwcichlid/macro-1.jpg
[/IMG]

[IMG]http://i143.photobucket.com/albums/r138/qqbmwcichlid/macro-3.jpg
[/IMG]

[IMG]http://i143.photobucket.com/albums/r138/qqbmwcichlid/macro-2.jpg
[/IMG]

Thanks in advance for any suggestions.


Yi

[Robert Peterson-3-3]

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

Make sure that you are saving and updating the config after every change
and before you open the MULTITIME macro. Also, on our system you have to
make sure that the stage is at the "correct" focal plane when starting a
z-stack. the system doesn't seem to know where to start, so it just
starts wherever you happen to be focused. I know you aren't doing a
z-stack, but if you are moving the focus plane after saving the config
it could cause problems.

I dread people asking me to use Multitime because things like this seem
to happen all the time.

Good luck,
Robert


Yi Zheng wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear all,
>
> I am a newbie to Zeiss LSM510.
>
> Recently I tried to run a multiple locations time series macro on LSM510.
> The protocol I followed is the "Zeiss LSM 510 META NLO User's Manual" from
> Fred Hutchinson Cancer Research Center which I got online.
>
> After mark 4 different location and load my configuration, I did these step as
> follows:
>
> 6. Start Multi-Time Macro (Main Menu>MACROS>MultiTime"R")
> 7. Click "Refresh" button.
> 8. Load Multi-Track configuration from pull-down menu in Multi-Time
> window (Config, Time-Interval and Bleach), (e.g. aa1)
> 9. Make sure "number of scans" is set to "0", and z-stack is checked in the
> "Config, Time-Interval and Bleach" panel.(I did not check the z-stack because
> I only have one z-stack)
> 10. Under "List of Blocks", set number of repetitions to "0"(I changed this
> number to 3 later in step 15.1)
> 11. Make sure autofocus is NOT checked, and bleach is NOT checked.
> 12. Click "Multiple Location" button on top of Multi-Time window.
> 13. Verify that the list of locations in the Multi-Time series window menu
> matches the list in the stage control panel. If not, click Refresh.
> 14. Click "Options" (in Multi_Time window); Check "Keep Final Image
> Open" and "Save Final Image"
> 15.1. Under List of Blocks, enter the number of Block repetitions (e.g. 3, for
> three time-points)
> 15.2. Under Block 1 parameters, enter the wait interval.
> 16. Click "Start Time".
>
> But after all these, no images were collected except the black one and
> warning message as "Pinhole PH2 is not adjusted".  I also notice that in
> configuration control panel, under list of tracks, the channels info changed to
> another laser momentarily (it switched from this to my previous setting
> continuously). In the end, I had to stop the recording manualy and no image
> was saved.
>
> I have attached three pics of my setting screen, and would you guys please
> check my protocol and let me know which step I made a mistake?
>
> Here is the link
> [IMG]http://i143.photobucket.com/albums/r138/qqbmwcichlid/macro-1.jpg
> [/IMG]
>
> [IMG]http://i143.photobucket.com/albums/r138/qqbmwcichlid/macro-3.jpg
> [/IMG]
>
> [IMG]http://i143.photobucket.com/albums/r138/qqbmwcichlid/macro-2.jpg
> [/IMG]
>
> Thanks in advance for any suggestions.
>
>
> Yi
>