View source for Total internal reflection microscopy

[[File:Total internal reflection scattering.svg|thumb|300px|Scattering of an evanescent field by a probe particle.]]
'''Total internal reflection microscopy''' is a specialized optical imaging technique for object tracking and detection utilizing the light scattered from an [[evanescent field]] in the vicinity of a [[dielectric]] interface.  Its advantages are a high [[signal-to-noise ratio]] and a high spatial resolution in the vertical dimension.

==Background==

[[Total internal reflection]] of light occurs at the interface between materials of differing indices of refraction at incident angles greater than the [[Total internal reflection#Critical angle|critical angle]], <math>\theta_c</math>, where
:<math>\theta_c = \sin^{-1}(n_2/n_1)</math>
and <math>n_1</math> is the index of the incident medium and <math>n_2</math> the index of the transmission medium and <math>\theta_c</math> is measured from the normal to the interface.

Under conditions of total internal reflection, the [[electromagnetic field]] in the transmission medium takes on the form of an [[evanescent wave]], whose intensity <math>I(z)</math> decays exponentially with distance from the interface such that,
:<math>I(z) = I_0 e^{-\beta z}</math>
with <math>\beta = \frac{4\pi}{\lambda}\sqrt{(n_1 \sin(\theta))^2 - n_2^2}</math>.   For practical purposes, the transmission medium is often chosen to be a fluid—usually water—in which a microscopic object can be immersed.  The object, when brought close to the interface, is expected to scatter light proportional to the intensity of the field at its height, <math>z</math>.<ref name="r1">Prieve, Dennis C., and Nasser A. Frej. "Total internal reflection microscopy: a quantitative tool for the measurement of colloidal forces." Langmuir 6.2 (1990): 396-403.</ref>  Since the penetration depth of the evanescent field is on the order of hundreds of nanometers, this technique is among the most sensitive for tracking displacements in the direction perpendicular to a surface.<ref>Prieve, Dennis C. "Measurement of colloidal forces with TIRM." Advances in Colloid and Interface Science 82.1 (1999): 93-125.</ref>

==Applications==

===Imaging===
The thin excitation region of an evanescent field allows for wide-field imaging of a select sample area with high [[signal-to-noise ratio]].  Rather than relying on optical scattering, however, often fluorophores are introduced into the sample for more selective visualization in biological applications.  This popular imaging technique is known as a [[Total internal reflection fluorescence microscopy]].

===Particle Tracking===
Using a calibrated evanescent wave,<ref name="r1" /> the position of a colloidal particle or microscopic probe may be tracked with nanometer precision by monitoring the intensity of light scattered via [[Total internal reflection#Frustrated total internal reflection|frustrated total internal reflection]]. Detailed dynamics of the probe or particle can then be obtained, either in thermal equilibrium or non-equilibrium conditions.

For instance, by collecting the time-independent position probability distribution of a probe particle in thermal equilibrium, and inverting the [[Maxwell–Boltzmann distribution]],
:<math>p(z)=\frac{1}{Z}e^{-\frac{V(z)}{kT}}</math>,
where <math>Z</math> is the [[Partition function (statistical mechanics)|partition function]], and <math>k</math> the [[Boltzmann constant]], one can obtain the potential energy profile of interactions between the particle and a surface.<ref>Walz, John Y. "Measuring particle interactions with total internal reflection microscopy." Current opinion in colloid & interface science 2.6 (1997): 600-606.</ref>  In this manner, sub-picoNewton forces may be detected.<ref>Flicker, Scott G., Jennifer L. Tipa, and Stacy G. Bike. "Quantifying double-layer repulsion between a colloidal sphere and a glass plate using total internal reflection microscopy." Journal of colloid and interface science 158.2 (1993): 317-325.</ref>

On the other hand, diffusive dynamics of a cell or a [[colloid]] can be deduced from its position time-series obtained via TIRM or another [[Single particle tracking|particle-tracking]] method.  Hydrodynamic coupling effects resulting in a particle's reduced [[diffusion]] in the vicinity of a solid interface have been studied in this way.<ref>Bevan, Michael A., and Dennis C. Prieve. "Hindered diffusion of colloidal particles very near to a wall: Revisited." The Journal of Chemical Physics 113.3 (2000): 1228-1236.</ref>

==See also==
* [[Dark-field microscopy]]
* [[Evanescent wave]]
* [[Total internal reflection]]
* [[Total internal reflection fluorescence microscopy]]

==References==
{{Reflist}}

[[Category:Microscopy]]