Weird transient in a 2P microscope (not sample bleaching)

[Peter Rupprecht-2]

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Dear list,

Today, I've seen an effect that is so strange that I can not even come up with a possible explanation. It is not really a huge problem, but I'd still be interested in understanding what is going on.

I'm working on a two-photon point scanning microscope. Each time I open the shutter, I see a higher fluorescence value in the beginning, which decreases to a steady state after few seconds. First, I thought this might simply have been photobleaching of the sample, but I could rule this out (see below).

To give you some context, the pulsed laser is first passed through a lambda/2 waveplate (Thorlabs), a polarizing beam splitter (PBS; Thorlabs), a shutter and a Pockels cell (Conoptics), before it enters the microscope - pretty much standard. See here for a schema: https://i.imgur.com/zPckeJN.png (shutter located at position C).

However, if I move the shutter to different positions (or use a manual blanker at the respective positions A, C or D), I do not see this transient any more, or in a strongly reduced version: https://imgur.com/gallery/aNgXhJE 

How is this even possible? This would suggest that Pockels cell and PBS are interacting in a strange way that only comes into play under certain conditions: 1) The laser hits the PBS both before and after the switch-on event 2) the laser does not hit the Pockels cell before the event 3) the laser hist the Pockels cell after the switch-on event.

Has anybody seen something like this before? Any ideas what this could be?

Best,
Peter

[Peter Rupprecht-2]

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Sorry, there was a typo in the second link. It should be https://imgur.com/gallery/aNgXhJE

Peter    Am Freitag, 22. Februar 2019, 14:55:58 MEZ hat Peter Rupprecht <[hidden email]> Folgendes geschrieben:  
 
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Dear list,

Today, I've seen an effect that is so strange that I can not even come up with a possible explanation. It is not really a huge problem, but I'd still be interested in understanding what is going on.

I'm working on a two-photon point scanning microscope. Each time I open the shutter, I see a higher fluorescence value in the beginning, which decreases to a steady state after few seconds. First, I thought this might simply have been photobleaching of the sample, but I could rule this out (see below).

To give you some context, the pulsed laser is first passed through a lambda/2 waveplate (Thorlabs), a polarizing beam splitter (PBS; Thorlabs), a shutter and a Pockels cell (Conoptics), before it enters the microscope - pretty much standard. See here for a schema: https://i.imgur.com/zPckeJN.png (shutter located at position C).

However, if I move the shutter to different positions (or use a manual blanker at the respective positions A, C or D), I do not see this transient any more, or in a strongly reduced version: https://imgur.com/gallery/aNgXhJE 

How is this even possible? This would suggest that Pockels cell and PBS are interacting in a strange way that only comes into play under certain conditions: 1) The laser hits the PBS both before and after the switch-on event 2) the laser does not hit the Pockels cell before the event 3) the laser hist the Pockels cell after the switch-on event.

Has anybody seen something like this before? Any ideas what this could be?

Best,
Peter  

[Bruno Pichler]

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Someone recently asked me about a fluorescence transient that occurs immediately at the start of a recording and decays over a second or so. Sounds very similar (possibly identical) to yours, but they hadn't yet tried to vary the shutter positions like you did. But they found one other piece of information that may help to solve the puzzle: their artefact disappears when the Pockels Cell is operated in Direct Mode in ScanImage (http://scanimage.vidriotechnologies.com/display/SI2018/Power+Controls#PowerControls-SelectedBeamPanel), ie. when the Pockels Cell command voltage is at a constant DC level for some time before the shutter opens.
Do you know whether this is true for your artefact as well?