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A couple of thoughts:

Firstly, I have seen this sort of problem due to back-reflection of the TIRF beam from an excitation filter in the cube.  Make sure the TIRF cube you are using doesn't have an excitation filter and that the laser is filtered independently prior to entering the TIRF launch (and more efficiently than simply an aotf alone).  

Secondly, the first thing I do on every TIRF scope is to move the beam straight up out of the objective and tilt back the condenser (you will need to defeat the interlock and take appropriate laser safety precautions).  That way you will see the beam on the ceiling.  It should be as small as possible (probably less than 1 inch in diameter depending on the height of your ceiling).  There should be some sort of divergence alignment within the system that you can use to make the beam smaller.

Once these two things are done, there are very few other reasons why TIRF wouldn't work (other than the catastrophic like a bad objective or a bad emission filter).  Your system should allow for alignment of the laser to full blocking position.  Then simply back off from that position until you see signal and it should be in TIRF.  Of course, getting the angle calibrated for depth is a whole other issue which has been discussed on this list before.

Jay

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of John Oreopoulos
Sent: Thursday, October 31, 2013 8:13 AM
To: [log in to unmask]
Subject: Re: Olympus TIRF

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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:

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> To join, leave or search the confocal microscopy listserv, go to:
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> 
> 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