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If you take the critical angle at the water/SiOil interface as
sin(\theta_{H2O-SiOil}) = n_{H2O} / n_{SiOil}
and the refraction at the previous interface as
sin(\theta_{Glass}) / sin(\theta_{SiOil}) = n_{SiOil} / n_{Glass}
then you can substitute the first equation into the second (angle at
H2O-SiOil interface is equivalent to that on the SiOil side of the previous
interface). That yields
sin(\theta_{Glass}) / [ n_{H2O} / n_{SiOil} ] = n_{SiOil} / n_{Glass}
reducing to...
sin(\theta_{Glass}) = [ n_{SiOil} * n_{H2O}] / [ n_{Glass} * n_{SiOil} ]
...
sin(\theta_{Glass}) = [ n_{H2O}] / [ n_{Glass} ]
This is the usual expression for TIRF at a glass/water or immersion
oil/water interface. Angle is 61.63 degrees. Somewhat surprising, but the
fact that there is an intermediate layer of middle index drops out.
Changing the refractive index in steps does not lose anything. nor does it
gain anything in terms of increasing/decreasing angle at a further
interface. If that was the case I would imagine we wouldn't bother with
1.49 NA TIRF objectives and instead use coverslips with a very well-defined
layer of plastic or other middle-refractive-index material on top to get
the same effect.
Double-check the math, of course. And no idea what this will do to the
spherical aberrations on your system (sure not anything good, but likely
non-fatal).
Thanks,
Rusty
On Tue, Oct 17, 2017 at 8:56 AM, MODEL, MICHAEL <[log in to unmask]> wrote:
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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
>
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> --
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