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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
[log in to unmask]
www.tirf-labs.com www.TIRFmicroscopy.com
-----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] http://pubs.rsc.org/en/content/articlehtml/2011/sm/c1sm05232b
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Kai Schleicher, PhD | Research Associate in Advanced Light Microscopy | Biozentrum, University of Basel | Klingelbergstrasse 50/70 | CH-4056 Basel |
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