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I don't know why I said, impossible: in theory, it is very possible to have (n3/n1) < sin(a) < (n2/n1). But you would have to aim very accurately
Mike
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From: Confocal Microscopy List <[log in to unmask]> on behalf of MODEL, MICHAEL <[log in to unmask]>
Sent: Tuesday, October 17, 2017 11:56 AM
To: [log in to unmask]
Subject: Re: Question for TIRF specialists
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If you designate the angles as: a (incidence), b (refraction in oil), and c (refraction in PBS), the condition of total reflection from the second boundary would be sin(b) > n3/n2. To achieve that, you need to have sin(a) = sin(b)*n2/n1 = n3/n1. On the other hand, you want to avoid TIRF at the first boundary, so sin(a) should be smaller than n2/n1. But since n2 > n3, the condition (n3/n1) < sin(a) < (n2/n1) is impossible. (Please check, I may have easily messed up something)
Mike
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Kai Schleicher
Sent: Tuesday, October 17, 2017 11:07 AM
To: [log in to unmask]
Subject: Question for TIRF specialists
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Dear List,
I'd like to do a TIRF experiment and am thinking about how to best set it up. I have an Objective-based TIRF system (ring or multi-point).
Similarly to what I have seen in publications [1], the aim is to observe protein adsorption at a SiliconOil - aqu. buffer interface.
From the viewpoint of the light path, the experiment can be described as follows: Light exits the Objective and then enters
1. Immersion Oil (n=1.518)
2. coverslip (n=1.518, thickness 0.17 um) 3. SiliconOil film (n=1.4, film thickness 18 um) 4. PBS buffer (n=1.33)
My question from a theoretical point of view is, would you expect TIRF to work even when presenting this "gradient" of refractive indices and if yes, how can the TIRF effect be focused on the further away SiliconOil-PBS buffer interface rather then the immediate Glass-SiliconOil interface?
Thanks for your ideas and cheers,
Kai
[1] https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fpubs.rsc.org%2Fen%2Fcontent%2Farticlehtml%2F2011%2Fsm%2Fc1sm05232b&data=01%7C01%7Cmmodel%40KENT.EDU%7C43e53a73782c49c1d03108d515722a98%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=YlEJa8%2F7%2BwRwGMXuJN3BHaRQiy6JStOtYi4hAVTGL00%3D&reserved=0
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