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

When a Zeiss confocal (LSM510) is setup the system is calibrated in such 
a way that it gives the same intensity for all scan speeds. In your case 
you could ask Zeiss to do this calibration again. I once asked Zeiss if 
it was possible to take this out and this was not recommended (done). I 
did some comparisons by making 2 images of the same sample either with 
lower scan speed or with a average and if you make a 2D plot of the 
pixel intensities for both situations you will see that the average 
image gives less noise.

We also make sometimes saturated pictures but never observed the 
stripes, so this may be a machine dependent phenomenon.
I always thought that the detector protection that was mentioned in an 
earlier mail was only active when you activate a bleaching protocol.

Best regards,
Gert van Cappellen

on 11-3-2008 20:33 Teng-Leong Chew said the following:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi Maria,
>
> The increase in fluorescence intensity with slower scan speed
> is to be expected. The slower the scan speed, the higher the
> pixel dwell time of the laser, you will always get higher
> intensity with that no matter what confocal you use.
>
> I am not sure about the stripes. Are you doing serpentine scan
> by any chance, meaning scanning the sample back and forth and
> not always from left to right? Serpentine scan tends to create
> scan lines. I am not sure if that's what you are describing.
>
> Leong
>
>
> Teng-Leong Chew, Ph.D.
> Director,
> Cell Imaging Facility
> Nikon Imaging Center, Chicago
> Northwestern University Feinberg School of Medicine
> 303 E. Chicago Avenue
> Chicago, IL 60611
>
>
>
> Maria Jimena Ortega wrote:
>>  ...
>> First thing I notice is that the intensity level varies a lot with 
>> scan speed (from 3.2 usec/pixel to .64 usec/pixel).  So, parameters 
>> choosed at a speed doesn`t work in the other…  it really  annoying!
>>
>> The other situation is that when I am working at high speeds (0.8 – 
>> 1.64 usec/pix), average 4 line, if my image has saturating conditions 
>> ( I =255, 8 bit) I see some regular “stripes”. 
>> Zeiss tells there is a security system (to protect the PMT), but I do 
>> not understand why I see it in some speeds and not others, or why I 
>> don`t get them in my Pascal…  any clue?
>>
>> The situation is that I need to saturate some parts of my sample in 
>> order to see the parts I am interested in!!  What should I do?
>>
>> The also tell me not to make average and slow down speed… but I don`t 
>> get the same results… Can someone help me in this scan speed vs 
>> average discussion?

-- 
wigGert van Cappellen, [log in to unmask]
!!! NEW TEL +31-10-70 43578; FAX +31-10-7044736 !!!
Assistant professor
Dept. of Reproduction and Development; http://www.erasmusmc.nl/rede
Optical Imaging Centre; http://www.erasmusmc.nl/oic/
Erasmus MC, room Ee914, Dr. Molenwaterplein 50, 3015 GE ROTTERDAM, The Netherlands
Delivery adres:
Erasmus MC, Westzeedijk 353, 3015 AA Rotterdam, The Netherlands