Evanescent field fluorescence

Fig. 15 Detection principle of evanescent-field fluorescence on planar waveguides excitation light is coupled into a thin-fifin waveguide surface confined fluorescence of bound labeled molecules is detected by a CCD camera. The fluorescence could be also separated via the outcoupling grating and detected by a photodiode. S substrate, W waveguide, L lens, F filter.

Kuno A, Uchiyama N, Koseki-Kuno S, Ebe Y, Takashima S, Yamada M, Hirabayashi J (2005) Evanescent-field fluorescence-assisted lectin microarray a new strategy for glycan profiling. Nat Methods 2 851-856... 

Uchiyama N, Kuno A, Tateno H, Kubo Y, Mizuno M, Noguchi M, Hirabayashi J (2008) Optimization of evanescent-field fluorescence-assisted lectin microarray for high-sensitivity detection of monovalent oligosaccharides and glycoproteins. Proteomics 8 3042-3050... 

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. 

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. 

In addition, typical methods of sensing are total internal reflection fluorescence or monitoring of fluorescence resonance energy transfer6,7. The second class is a direct optical detection principle which relies either on reflectometry or refractometry. The latter is connected to evanescent field... 

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). 

Fig. 1 Real-time tracking of cell adhesion [42]. (a) Components of a total internal reflection fluorescent microscope (TIRFM). (b) The cell adhesion process (7) a cell approaches the surface, (2) the cell lands, (3) the cell attaches, and (4) the cell spreads out on the surface. The evanescent field was generated by total internal reflection of a laser beam at the glass-water interface. Cells with fluorescently labeled membranes (dashed lines) were plated on SAMs. Cell membranes within the evanescent field (solid line) were observed by TIRFM. Corresponding TIRFM images are shown below...

The second possibility is to make use of the continuous evanescent field that exists at the surface of an optical fiber. The cladding layer is replaced by a layer containing the fluorescent molecular sensor in a short portion of the optical fiber (Figure 10.45). At each reflection at the surface of the fiber core, the eva-... 

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)... 

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

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... 

A four-layer microchip has been constructed to generate total internal reflection (TIR) and an evanescent field (see Figure 7.8). Surface-adhered Nile red-labeled fluorescent microspheres (1 pm) are excited by the evanescent field for fluorescent measurement. An essential feature on the chip was the micromirror that was constructed by depositing Au/Cr on the slanted wall (54.7° due to anisotropic etch of Si). Operation near the critical angle 0C assures strong evanescent intensity [695]. 

After that the behaviour of the evanescent field rather than possible applications was of primary interest. In 1902 Hall (5) succeeded in photographing the evanescent wave and soon afterwards fluorescence (6) and scattering (7) excited by such waves were observed. In 1910 Schaefer and Gross (8) measured quantitatively the exponential decay of the amplitude of the field with microwaves. 

Fig. 15.1. Schematic representation of amyloid fibrils revealed by total internal reflection fluorescence microscopy, (a) The penetration depth of the evanescent field formed by the total internal reflection of laser light is 150nm for a laser light at 455 nm, so only amyloid fibrils lying parallel to the slide glass surface were observed. (b) Schematic diagram of a prism-type TIRFM system on an inverted microscope. ISIT image-intensifier-coupled silicone intensified target camera, CCD charge-coupled device camera...

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