Re: TIR critical angle re-adjustent
Posted by
John Oreopoulos on
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Hi Vitaly,
I'm not sure if you have a commercial instrument or a home-built TIRF microscope on an optical bench, but generally this is an issue that always comes up anytime you have multi-color TIRF. The problem stems from the fact that the critical angle for each laser line is different because of the characteristics and wavelength dependence of the substrate-sample refractive interface (ie: the critical angle is slightly different for each laser line) and also the wavelength dependence of the glass inside the objective. Note also that the equation for the evanescent penetration depth is wavelength dependent as well such that even if you had all laser lines impinging on the interface at the same angle, each penetration depth would be different. (There's an article on this topic here:
http://www.photonics.com/content/bio/2008/May/features/91651.aspx)
I know that this effect was nearly impossible to deal with in the early commercial TIRF systems, but I think now that all or some of the major microscope vendors have developed a system where each laser line is directed towards the microscope down a different path and then each line merges after passing through or reflecting off a dichroic mirror before entering the microscope. Each laser sits on an adjustable platform so that you can line them all up differently and independently. For an example of this, see page 15 of this .pdf document:
I've seen home-built systems at other labs that are essentially the same thing. If you're in the situation where all laser lines are funneled down the same optical path (ie: through a single optical fiber) and there is only one way to adjust the angle of incidence for all three laser lines at the same time, then the best you can do is replace the manual micrometer screw with some kind of fast motorized screw and synchronize this to your image acquisition software somehow (maybe using something like Micro-Manager -
http://www.micro-manager.org/ ?). Thorlabs sells motorized actuators that could do the job:
http://www.thorlabs.com/Navigation.cfm?Guide_ID=83I tried this once on our system but the actuator I was using was too slow.
I think it's also important to emphasize that even after setting up all laser lines the way you want them to go into the scope or after you've found a way to adjust the beam incident angle rapidly, you should measure the actual penetration depth somehow. In objective-based TIRF systems, the penetration depth can sometimes deviate significantly far away from the predicted depth given by the equation. I think about a year or two ago there was a listing on the server about this topic which I posted a reply to. Here it is:
Confocal listserver item 027568 posted on 2007-02-22
John Oreopoulos, BSc,
PhD Candidate
University of Toronto
Institute For Biomaterials and Biomedical Engineering
Centre For Studies in Molecular Imaging
Tel: W:416-946-5022
On 24-Jul-08, at 10:48 AM, Vitaly Boyko wrote:
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Dear All,
Yesterday I have tested our new 585 nm laser in conjuction with 514 nm argon and 437 nm diode under TIR conditions.
Even though I have not noticed the need for the critical angle re-adjustment between 437 nm and 514 nm lines (77 nm difference), however, when the critical angle was set for the yellow laser (585 nm), switching from 585 nm to 514 nm line (71 nm difference) required the (manual) resetting of the critical angle.
I used the 89006 triple beamsplitter (A/R coated), which seemed to reflect the 514 argon line (up to 70%).
I am looking for help with the device that would allow quick, low msec range, critical angle re-adjustment or any other suggestions will be appreciated.
Thanks a lot in advance,
Vitaly
Vitaly P. Boyko,
NCI-Frederick,
301-846-6575