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My guess is that your 50/50 beamsplitter is not splitting
at the IR wavelength - as Sophie said, you should be able
to check it with your IR viewer.
But I must add that what you're trying to do sounds like
an awfully bad idea unless you first un-modelock the beam
so that you have CW illumination. Otherwise you risk
doing very strange things to the specimens (put some
carbon black on a slide by holding it above a match and
then see what happens if you look at it in 2p mode).
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
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Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
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http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Jerry Sedgewick
Sent: Thursday, 14 February 2008 3:28 AM
To: [log in to unmask]
Subject: Re: LSM multiphoton confocal not picking up reflection
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I know this had been discussed in the past, but I didn't follow the thread. I seem to be getting more engineer types wanting to use our confocal. Because they often have ancillary equipment they need to set up to replace the stage, I am cornered into providing a spot for them on a custom-built multiphoton. They are forever wanting to see surfaces or reflection from particles: if my memory serves me well, reflection cannot be had unless the illumination wavelength matches the reflected wavelength. But I'm starting to wonder whether another phenomenon I am not aware of creeps in, for, on this custom multiphoton, I have replaced a cube with a 50/50 beamsplitter so that the reflected wavelength is the same as the illumination wavelength, and removed filters from in front of the PMT detector (sensitive out to 800nm or so), set the pulsed laser at 750nm, and even placed a 1/2 waveplate and glan polarizer in the laser path with another polarizer in front of the PMT to play with these (I believe the polarizer in front of the PMT, however, is a circular polarizer as the reflected light never quite extinguished with cross-polarization).
Inexplicably, I got no reflection off surfaces.
The custom multiphoton has two paths, one for the external detectors (where the 50/50 beamsplitter sent the reflected light) and internal PMTs in a Fluoview 300 confocal unit. Strangely, if I sent the reflected light to the internal PMTs, I was able to get a reflection, if I was willing to live with a bright spot in the middle of the field. This wasn't supposed to happen because the reflected light was (supposedly) blocked by bandpass filters, but, after placing another 650sp filter in the reflected light path, the reflection went away. (Clearly, a portion of the laser light bled through).
In any event, it is confounding to get the reflection in one instance, and to not get it in the other even when optimized. Any ideas?
Jerry Sedgewick
University of Minnesota
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