Question for TIRF specialists

[Kai Schleicher]

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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] http://pubs.rsc.org/en/content/articlehtml/2011/sm/c1sm05232b

--
>>Please note my NEW PHONE NUMBERS: [phone] (direct) [phone] (central)<<
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[mmodel]

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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:[hidden email]] On Behalf Of Kai Schleicher
Sent: Tuesday, October 17, 2017 11:07 AM
To: [hidden email]
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

--
>>Please note my NEW PHONE NUMBERS: [phone] (direct) +41 61 207
>>22 50 (central)<<
Kai Schleicher, PhD | Research Associate in Advanced Light Microscopy | Biozentrum, University of Basel | Klingelbergstrasse 50/70 | CH-4056 Basel |
Phone: [phone] (direct) [phone] (central) | [hidden email] | https://na01.safelinks.protection.outlook.com/?url=www.biozentrum.unibas.ch&data=01%7C01%7Cmmodel%40KENT.EDU%7C43e53a73782c49c1d03108d515722a98%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=z%2FhwxOuGkqswrHj49FSg6rLk88AthLyRzoXGs8C4RVg%3D&reserved=0 | https://na01.safelinks.protection.outlook.com/?url=www.microscopynetwork.unibas.ch&data=01%7C01%7Cmmodel%40KENT.EDU%7C43e53a73782c49c1d03108d515722a98%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=e1SELTEmL88Zqv1J82igjdLpBnv7htKqy5MqKawpjrw%3D&reserved=0

[mmodel]

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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


________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of MODEL, MICHAEL <[hidden email]>
Sent: Tuesday, October 17, 2017 11:56 AM
To: [hidden email]
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:[hidden email]] On Behalf Of Kai Schleicher
Sent: Tuesday, October 17, 2017 11:07 AM
To: [hidden email]
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

--
>>Please note my NEW PHONE NUMBERS: [phone] (direct) +41 61 207
>>22 50 (central)<<
Kai Schleicher, PhD | Research Associate in Advanced Light Microscopy | Biozentrum, University of Basel | Klingelbergstrasse 50/70 | CH-4056 Basel |
Phone: [phone] (direct) [phone] (central) | [hidden email] | https://na01.safelinks.protection.outlook.com/?url=www.biozentrum.unibas.ch&data=01%7C01%7Cmmodel%40KENT.EDU%7C43e53a73782c49c1d03108d515722a98%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=z%2FhwxOuGkqswrHj49FSg6rLk88AthLyRzoXGs8C4RVg%3D&reserved=0 | https://na01.safelinks.protection.outlook.com/?url=www.microscopynetwork.unibas.ch&data=01%7C01%7Cmmodel%40KENT.EDU%7C43e53a73782c49c1d03108d515722a98%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=e1SELTEmL88Zqv1J82igjdLpBnv7htKqy5MqKawpjrw%3D&reserved=0

[James D. Manton]

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Dear Kai,

That should work. The critical angle for TIR between the silicon oil and
the glass is 67.3 degrees, while the critical angle for TIR between the
oil and the PBS is 71.8 degrees and so it may initially seem like it is
not possible. However, the light will bend further away from the normal
when travelling from the glass to the oil and so the 'effective'
critical angle for TIR at the PBS boundary is actually 61.2 degrees
(i.e. the light needs to be travelling through the glass at this angle
or greater). Hence, illumination with an incident angle between 62 and
67 degrees should cause TIR only at the PBS boundary, as shown in a
quick plot at [1]. This can be confirmed with a quick MEEP [2]
simulation, which solves Maxwell's equations directly for the dielectric
materials you specified [3] (light enters from the left and travels to
the right — the first set of vertical bands correspond to the glass-oil
interface and the second to the oil-PBS interface, from which there is
no travelling wave).

If you're interested in playing around with the code yourself, the plot
code is available from [4], with the MEEP code at [5].

Good luck aligning,
James


[1] https://i.imgur.com/ujBZzGQ.png
[2] https://meep.readthedocs.io/en/latest/
[3] https://i.imgur.com/uNvzhzj.png
[4] http://rpubs.com/jdmanton/multilayertirf
[5] https://pastebin.com/m88ns6d7

[John Oreopoulos]

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Kai,

The following is clipped from Dan Axelrod's 2008 review on TIRF microscopy:

F. Intermediate Dielectric Layers

In actual experiments in biophysics, chemistry, or cell biology, the  
interface may not be a simple interface between two media, but rather  
a stratified multilayersystem. One example is the case of a biological  
membrane or lipid bilayer interposed between glass and an aqueous  
medium. Another example is a thin metal film coating, which can be  
used to quench fluorescence within the first 10 nm of the surface. We  
discuss here the TIR evanescent wave in a three-layer system in which  
incident light travels from medium 3 (refractive index n3) through the  
intermediate layer (n2) toward medium 1 (n1). Qualitatively, several  
features can be noted:

Insertion of an intermediate layer never thwarts TIR, regardless of  
the inter-mediate layer’s refractive index n2 The only question is  
whether TIR takes place at the n3:n2 interface or the n2:n1 interface.  
Since the intermediate layer is likely to be very thin (no deeper than  
several tens of nanometers) in many applications, precisely which  
interface supports TIR is not important for qualitative studies.

Regardless of n2 and the thickness of the intermediate layer, the  
evanescent wave’s profile in medium 1 will be exponentially decaying  
with a characteristic decay distance given by Eq. (3). However, the  
intermediate layer affects intensity at the interface with medium 1  
and the overall distance of penetration of the field as measured from  
the surface of medium 3.


I think you should also take a look at the following papers:

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0023807

http://jcs.biologists.org/content/87/5/677.long

The second paper is a very theoretical approach to the treatment of  
multi-layer TIRF situations.

My only concern is trying to image through 18 um of the silicone oil  
layer. The refractive index mismatch might highly distort the images.  
You might be better off using the older prism-based TIRF approach  
instead of the objective based TIRF. Prism-TIRF rigs are fairly easy  
to build and there are lots of instructional papers out there on how  
to do that safely and properly.

Sincerely,

John Oreopoulos


Quoting "James D. Manton" <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Kai,
>
> That should work. The critical angle for TIR between the silicon oil
> and the glass is 67.3 degrees, while the critical angle for TIR between
> the oil and the PBS is 71.8 degrees and so it may initially seem like
> it is not possible. However, the light will bend further away from the
> normal when travelling from the glass to the oil and so the 'effective'
> critical angle for TIR at the PBS boundary is actually 61.2 degrees
> (i.e. the light needs to be travelling through the glass at this angle
> or greater). Hence, illumination with an incident angle between 62 and
> 67 degrees should cause TIR only at the PBS boundary, as shown in a
> quick plot at [1]. This can be confirmed with a quick MEEP [2]
> simulation, which solves Maxwell's equations directly for the
> dielectric materials you specified [3] (light enters from the left and
> travels to the right ? the first set of vertical bands correspond to
> the glass-oil interface and the second to the oil-PBS interface, from
> which there is no travelling wave).
>
> If you're interested in playing around with the code yourself, the plot
> code is available from [4], with the MEEP code at [5].
>
> Good luck aligning,
> James
>
>
> [1] https://i.imgur.com/ujBZzGQ.png
> [2] https://meep.readthedocs.io/en/latest/
> [3] https://i.imgur.com/uNvzhzj.png
> [4] http://rpubs.com/jdmanton/multilayertirf
> [5] https://pastebin.com/m88ns6d7

[Rusty Nicovich]

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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 <[hidden email]> wrote:

> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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:[hidden email]]
> On Behalf Of Kai Schleicher
> Sent: Tuesday, October 17, 2017 11:07 AM
> To: [hidden email]
> Subject: Question for TIRF specialists
>
> *****
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> and include the link in your posting.
> *****
>
> 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%7Ce5a06f4a1ec44d018f73e7dd15f2
> 6134%7C1&sdata=YlEJa8%2F7%2BwRwGMXuJN3BHaRQiy6JStOtYi4hA
> VTGL00%3D&reserved=0
>
> --
> >>Please note my NEW PHONE NUMBERS: [phone] (direct) +41 61 207
> >>22 50 (central)<<
> Kai Schleicher, PhD | Research Associate in Advanced Light Microscopy |
> Biozentrum, University of Basel | Klingelbergstrasse 50/70 | CH-4056 Basel |
> Phone: [phone] (direct) [phone] (central) |
> [hidden email] | https://na01.safelinks.
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[Alex Asanov]

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Hi Kai,

Yes, in theory, you will have total internal reflection at the oil/water interface (1.4/1.33) at angles of incidence >72 degrees. I assume that you meant a 170-micron glass coverslip and a 18-micron oil layer. I am guessing that you have a hydrophobic sublayer to keep a layer of oil onto hydrophilic glass. Is it correct? Perhaps, you are using Sigmacote or something similar. How thick and how optically perfect are these layers? How much scatter and auto-fluorescence they produce?   These are the issues that will affect your TIRF experiments.

In practice, the excitation light behaves more complex than in TIRF theory. In TIRF objective, deeper inside your microscope, and at the glass/sublayer/oil layer/water interfaces there are inevitable scatter, reflections, refractions, and auto-fluorescence that produce undesirable stray light. In the case of objective-type TIRF geometry, significant deviations from exponential decay have been reported [1-5]. Typically, objective-TIRF is contaminated with 10-15% of stray light or more. 10-15% is the intensity at TIRF interface; relative contamination increases exponentially as the evanescent wave decays with the distance.

In prism- and lightguide-TIRF geometries the amount of stray light is much smaller, because the excitation lightpath is independent from the emission channel. See White Paper for details: http://tirf-labs.com/Select_TIRF_geometry_WP.pdf.


1. Ambrose W, Goodwin P, Nolan J. Single-molecule detection with total internal reflection excitation: comparing
signal-to-background and total signals in different geometries. Cytometry 1999, 36(3), 224.

2. Brunstein M, Teremetz M, Hérault K, Tourain C, Oheim M. Eliminating unwanted far-field excitation in objective-type
TIRF. Part I. Biophys J. 2014; 106(5): 1020.

3. Brunstein M, Hérault K, Oheim M. Eliminating unwanted far-field excitation in objective-type TIRF. Part II.
Biophys J. 2014; 106(5): 1044.

4. Mattheyses A, Axelrod D. Direct measurement of the evanescent field profile produced by objective-based
TIRF. J Biomed Opt, 2006, 11: 014006A.

5. Conibear P, Bagshaw C. A comparison of optical geometries for combined flash photolysis and TIRF
microscopy. J Microsc, 2000, 200(3): 218-29.



Best regards,
Alexander N. Asanov, Ph.D.
President,  TIRF Labs
[hidden email]
www.tirf-labs.com   www.TIRFmicroscopy.com



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kai Schleicher
Sent: Tuesday, October 17, 2017 11:07 AM
To: [hidden email]
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] http://pubs.rsc.org/en/content/articlehtml/2011/sm/c1sm05232b

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Dear all,

thank you very much for you replys, these are incredibly helpful!

I wont have the option to try a prism based TIRF at the moment, but thanks to your input I feel confident to try it our with the ring-based one that we have.

Again, thanks for your highly valuable input!
Cheers,
Kai