Evanescent Wave Microscopy

Toomre, D. and Manstein, D. J. (2001). Lighting up the cell surface with evanescent wave microscopy. Trends Cell Biol. 11, 298-303. 

Khan, S., Pierce, D. and Vale, R.D. (2000). Interactions of the chemotaxis signal protein CheY with bacterial flagellar motors visualized by evanescent wave microscopy. Cum Biol. 10, 927-930. 

As fluid mechanics reaches down to the micro-and nanometer spatial scales, interfacial phenomena become increasingly important, and several diagnostic techniques have been adapted from other fields to quantify fluid phenomena at these scales. In addition to transmitted light and fluorescence microscopy, an important technique for fluid-surface studies is evanescent wave microscopy. 

Evanescent wave microscopy or total internal reflection microscopy (TIRM) has been employed in the fields of biology and chemistry since the 1970s. The TIRM technique has long been used in cell biology studies and more recently cell-substrate contacts, vesicle fusion, and single-molecule observation. Here, cells on a microscope cover glass are illuminated by an... 

Although all of the incident energy is reflected, the solution of Maxwell s equations predicts the existence of an electromagnetic field in the less dense medium with an intensity decaying exponentially away from the interface, termed evanescent waves. The full details to the solution of Maxwell s equations are outlined in several other resources [1]. Here, only the results relevant to evanescent wave microscopy are... 

Evanescent wave microscopy has already yielded a number of contributions to the fields of micro-and nanoscale fluid and mass transport, including investigation of the no-slip boundary condition, applications to electrokinetic flows, and verification of hindered Brownian motion. With more experimental data and improvements to TIRE techniques, the accuracy and resolution of these techniques are certain to improve. Areas of potential improvements include development of rmiform-sized and bright tracer particles, creation of high-NA imaging optics and high-sensitivity camera systems, and further development of variable index materials for better control of the penetration-depth characteristics. 

Evanescent-wave microscopy uses the evanescent wave produced by the total intemal reflection of light at a dielectric interface to illuminate a layer of material within the penetration depth of the evanescent field. The material of interest is imaged using a microscope objective typically within 100-200 nm of the interface. 

Total internal reflection fluorescence microscopy (TIRFM), or simply TIRF, is the application of fluorescence imaging to evanescent-wave microscopy. The material of interest is tagged with fluorescent material that emits light at wavelengths greater than the excitation photons from the evanescent field, which additionally improves the detectability of the evanescent-wave microscopy technique. 

Total internal reflection velocimetry combines evanescent-wave microscopy with established micro- and nanoscale velocimetry techniques. 

Evanescent Wave Microscopy, Figurel A comparison of (a) direct flood illumination versus (b) TIRF illumination of 200 nm diameter fluorescence polystyrene particles in water with a single 5 ns pulse from a Nd YA6 laser through a 10OX objective with a NA = 1.45... 

Evanescent Wave Microscopy, Rgure 2 Illustration of geometry and parameters for an evanescent wave produced by the total Internal reflection of plane waves Incident on a dielectric interface... 

Evanescent Wave Microscopy, Rgure 3 Behavior of the penetration depth d normalized by the incident wavelength as a tunction of the incident angle 0- for a typical glass-water interface with n = 0.878... 

Evanescent Wave Microscopy, Rgure 4 Amplitude of the evanescent... 

Due to the length scales involved, most interesting phenomena probed by the evanescent wave require microscope optics for imaging. In the past, several TIRM studies have been conducted using only scattering from objects in the evanescent field for imaging. More recently, fluorescence microscopy systems have become the standard imaging technique for evanescent wave microscopy... 

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