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Ekaterina,

I'm having trouble accessing the images you posted in your link. My browser says the following:

The requested URL /~papusheva/olympus_TIRF_30102013 was not found on this server.

Can you try reposting another way? Try sending them to me directly offline from the confocal listserver. 
Can you also list here exactly what dichroic mirror, emission filters, and excitation filters (if any) that are present in your system? This sounds like it could be a case of a bad filter with poor out-of-band rejection of your laser line. Without seeing the images and knowing what filters you're dealing with, it's hard to say.

John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca



On 2013-10-31, at 5:10 AM, Ekaterina PAPUSHEVA wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
> 
> Dear all,
> 
> We got a new Olympus TIRF delivered three weeks ago, and could not bring it to running.
> Problem: we observe strong background, seemingly coming from laser, in a TIRF mode, when we use 405 or 488 nm laser for excitation. Increasing laser power does not improve signal-to-noise ratio 561 nm is seemingly ok.
> Also, when we set 100 nm penetration depths through the software, and focus through the volume, 488 (and 405) nm background seems to shift from one side to the other very strongly. I know this is to be expected to some extent in objective-based TIRF, but  we do not have it so bad on our other TIRF system with 100x. My feeling is, software calibration of the beam angle is wrong, also because when I set it to 'critical angle' or '100 nm penetration depth' beam still exits at about 40 degree angle. But I could not get good TIRF also by manually adjusting the slider undtil it looks like what is critical angle for me! The background stays just the same.
> TIRF dischroics, emission filters and laser cleanup filters are correct and positioned correctly. Laser spectra are clean. PSF of the objective seems to be ok.  It also cannot be a wrong cover glass- ours are highly corrected, cleaned and sonicated coverglasses, and I see the background even when I focus up from the glass
> What else could I check? Is there anyone out who has Olympus TIRF and knows these problems?
> 
> I put few images here: widefield and TIRF images of Tetraspeck beads in water and a Zstack in TIRF
> (to show the background shift) obtained with 488 nm excitation, just to illustrate what I am talking about.
> 
> http://pub.ist.ac.at/~papusheva/olympus_TIRF_30102013
> 
> Would be grateful for any tips!
> Best regards,
> Ekaterina