Evanescent field excitation

Hayazawa, N Inouye, Y. and Kawata, S. (1999) Evanescent field excitation and measurement of dye fluorescence using a high N.A. objective lens in a metallic probe near-field scanning optical microscopy J. Microsc., 194, 472-476. 

Figure 2. Regular reflectance Replication of Snellius law for reflected and refracted radiation at interface in dependence on the refractive indices of the media adjacent to this interface, demonstrating total internal reflectance and evanescent field, exciting fluorophores close to the waveguide or even surface plasmon resonance.

A. L. Stout and D. Axelrod, Evanescent field excitation of fluorescence by epi-illumination microscopy, Appl. Opt 28, 5237-5242 (1989). 

For FFS the confocal illumination scheme led to a highly confined excitation and detection volume. Alternative optical schemes for confining the excitation volume, for instance evanescent field excitation, were demonstrated already in the 80 s (13, 14), However, they did not show a performance comparable to confocal FFS for single molecule detection. 

In the following sub-section, we describe, compare and analyze in detail the sampling volume, in particular for an evanescent field excitation. The dipole response and its interaction with a dielectric substrate within the framework of the molecule detection efficiency will be considered in detail as a relevant model for the overall FFS process. Finally, fluorescence correlation spectroscopy (FCS) as a prominent member of FFS techniques will be described in the context of SMD. 

Marcel Leutenegger, Kai Hassler, Rudolf Rigler, Alberto Bilenca, and Theo Lasser describe dual-color fluorescence fluctuation spectroscopy based on an evanescent field excitation scheme as an alternative concept for single molecule detection at surfaces. 

The tuneable nature of the evanescent field penetration depth is critical to the effective operation of this sensor as it facilitates surface-specific excitation of fluorescence. This means that only those fluorophores attached to the surface via the antibody-antigen-labelled antibody recognition event... 

While planar optical sensors exist in various forms, the focus of this chapter has been on planar waveguide-based platforms that employ evanescent wave effects as the basis for sensing. The advantages of evanescent wave interrogation of thin film optical sensors have been discussed for both optical absorption and fluorescence-based sensors. These include the ability to increase device sensitivity without adversely affecting response time in the case of absorption-based platforms and the surface-specific excitation of fluorescence for optical biosensors, the latter being made possible by the tuneable nature of the evanescent field penetration depth. 

The background problem can be further overcome when using a surface-confined fluorescence excitation and detection scheme at a certain angle of incident light, total internal reflection (TIR) occurs at the interface of a dense (e.g. quartz) and less dense (e.g. water) medium. An evanescent wave is generated which penetrates into the less dense medium and decays exponentially. Optical detection of the binding event is restricted to the penetration depth of the evanescent field and thus to the surface-bound molecules. Fluorescence from unbound molecules in the bulk solution is not detected. In contrast to standard fluorescence scanners, which detect the fluorescence after hybridization, evanescent wave technology allows the measurement of real-time kinetics (www.zeptosens.com, www.affinity-sensors.com). 

To use the OFRR as a biosensing device, the optical resonant mode is excited and the resonant frequency is measured continuously in real time. The conceptual measurement setup is illustrated in Fig. 14.3. Laser light from a distributed feedback (DFB) laser is delivered to the OFRR using fiber optic cable. One method that has been used to excite the resonant modes is to place a tapered fiber optic cable with a diameter less than 4 pm in contact with the OFRR. The evanescent field of the tapered fiber overlaps with the evanescent field outside of the capillary wall, which enables mode coupling between the two media24. 

A disposable, patterned, planar waveguide with a number of individual wells has been reported for a one-step homogeneous immunoassay of IgG.<133) The device is fabricated by an ion-exchange process, etching, and covalent reagent immobilization. The sample fills the waveguide by capillary action. The sample well, as well as fluorescent and nonfluorescent control wells are excited by an evanescent field, and individually scanned. The IgG detection limit is in the 10range. 

If the excitation electric field is an s-polanzed evanescent field instead of the above p-polarized example, then wH 11 [ = wHJI(z)] does not depend upon p. Therefore, an approximate C(z) can be calculated from the observed fluorescence (P) (obtained experimentally by varying 0) by ignoring the z dependence in the bracketed term in Eq. (7.45) and by inverse Laplace transforming Eq. (7.44) after the ,(0, /J) 2 term has been factored out.,37 39)... 

Excitation of a fluorophore that is close to the surface of the waveguide can be achieved via the evanescent field. The resulting fluorescence emission is isotropically distributed, however, some small component of the emitted light... 

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