At 12.28 em 01-07-13 +0200, you wrote:
>Dear Alessandro,
>
>I have a simillar problem with our Leica SP2, but not always. I don't know 
>the reason for that.

Good afternoon,

the reflection pattern as described in an earlier mail can appear if you 
are focussing on the upper or lower side of the coverslip and if your 
detection wavelength band is to close to the laser lines in use. Dr. 
Schibler has
already outlined this. Also, if you are focussing onto the upper surface of the
microscope slide you can experience such an effect and if the distance 
between the lower surface of the cover slip and the upper surface of the 
slide is suitable, "interesting" interference patterns can be produced. 
Also laser light, reflected or scattered from the specimen can produce 
reflection effects, albeit generally much less pronounced since most 
biological preparations are barely reflecting and scattering.

Concerning the transmitted light detector: One has to arrange for a very 
proper Köhler illumination and set the filed diaphragm accordingly. 
Otherwise, stray light from outside can readily enter the light path and be 
detected as structureless background by the PMT. At high voltages, this is 
even valid in a dark room for the light emitted by the screen of your 
computer. I experienced that a quick-and-dirty ship-around is to produce a 
cylinder shaped "light tunnel" of dark cartboard - not too thick material, 
it takes by five to ten minutes to build one and you need nothing but 
cardboard, some good scissors and a stripe of tape - and carefully put it 
between the condensor and the microscope stand thus shielding stray light. 
You can easily verify how sensitive the setting of the field diaphragm is 
by first adjusting properly for Köhler and then scanning while modifying 
the opening of the field diaphragm. The problem is, of course, more 
pronounced if you are working on an inverted microscope with a long 
distance condensor.

Hope this helps.

Best regards,

Johannes Helm


>I have more often this problem with the transmitted light detector, even 
>with a continous scan, and in live cells and/or fixed samples.
>
>I have no idea for that. Any suggestion?
>
>
>Susanna Castel
>
>En/Na Alessandro Brero ha escrit:
>>Dear confocalists, since a few weeks we have a problem with our Leica TCS 
>>SP, which has become worse and worse. After a few hours of scanning in 
>>the sequential mode we get a totally overexposed picture. First it 
>>started to happen only in one photomultiplier, recently it happened also 
>>in the other and rarily on both. If one reduces the laser power and/or 
>>the photomultiplier drastically one can see an unregular still 
>>overexposed structure in the middle of the field of approximately 3-4µm 
>>with sometimes concentric rings around it or lines of brighter, but not 
>>overexposed pixels. The interesting part is that if we just restart our 
>>scanning software the structure is gone and the picture of our specimen 
>>apears normal, i.e. not overexposed. This phenomenon happened with our 
>>x63 and x100 objective and only in the sequential mode.Has anybody who 
>>works with the Leica ever had similar problems or ideas what the reason 
>>for this effect might be??? Thanks in advance for any suggestions or 
>>hints, Sandro. Alessandro Brero, Dipl. Biol.
>>AG Cremer
>>Institut fuer Anthropologie und Humangenetik
>>Richard-Wagnerstr.10/I
>>80333 Muenchen
>>Tel.:004989/ 2180-6713
>>Fax:004989/2180-6719 E-mail: 
>><mailto:[log in to unmask]>[log in to unmask]
>>Alessandro Brero, Dipl. Biol.
>>AG Cremer
>>Institut fuer Anthropologie und Humangenetik
>>Richard-Wagnerstr.10/I
>>80333 Muenchen
>>Tel.:004989/ 2180-6713
>>Fax:004989/2180-6719 E-mail: 
>><mailto:[log in to unmask]>[log in to unmask]

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Paul Johannes Helm, M.Sc., Ph.D.

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