Evanescent Wave Fluorescence Spectroscopy

However, if one investigates not only peak line intensities in EW spectroscopy but also the lineshapes, much more detailed physical information can 

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In these sensors, the intrinsic absorption of the analyte is measured directly. No indicator chemistry is involved. Thus, it is more a kind of remote spectroscopy, except that the instrument comes to the sample (rather than the sample to the instrument or cuvette). Numerous geometries have been designed for plain fiber chemical sensors, all kinds of spectroscopies (from IR to mid-IR and visible to the UV from Raman to light scatter, and from fluorescence and phosphorescence intensity to the respective decay times) have been exploited, and more sophisticated methods including evanescent wave spectroscopy and surface plasmon resonance have been applied. 

One can distinguish between methods in which absorption of the evanescent surface wave in different wavelength regions is measured (these are often called attenuated total reflection methods), and methods which use the evanescent wave to excite other, spectroscopic phenomena, like fluorescence and Raman scattering or light scattering. As the methods of conventional fluorescence spectroscopy have been shown to be exceptionally successful in studies of proteins and other biopolymers, their evanescent surface-sensitive counterparts will be reviewed first. 

This article shows how the evanescent wave can be used with advantage for spectroscopic purposes in the field of biomedical engineering. Three types of spectroscopy can be done with the evanescent wave in the UV-VIS range of the spectrum (a) attenuated total reflection (ATR) spectroscopy, which is well known in the infrared (b) the excitation of Raman scattering with the evanescent wave and (c) the excitation of fluorescence with the evanescent wave. The first two types will be discussed in this article the third is discussed for example by Hirschfeld U) and more recently by Watkins and Robertson (2). But before going into details a historical review may be of some interest. 

For TIR fluorescence spectroscopy on water/oil interfaces, the choice of a probe molecule is of primary importance. For example, the penetration depth (dp) of an incident evanescent wave at a 1,2-dichloroethane (DCE, refractive index (n) n = 1.44)/water (m2 = 1.33)interface is calculated to be 94nm on the basisofEquation(13),whereX = 580 nm and 0 = 80°. It has been reported that the thickness of a sharp water/oil interface represented by water/DCE is 1 nm [9], so that dp of the incident evanescent wave is thicker than the thickness of the interfacial layer, and the fluorescence characteristics of a probe molecule in the bulk phase are superimposed, more or less, on those at the interface [2]. Therefore, a probe molecule should be highly surface-active and adsorb on the interface, so as to exclude fluorescence of the probe molecule from the bulk phase. In the present experiments, we employed xanthene dyes as fluorescence probes throughout... 

Time resolved evanescent wave induced fluorescence spectroscopy is a powerful method for the investigation of dye molecules at interfaces. This technique has been used on studies on the popular photosensidzer aluminium phthalocyanine tetra/sulphonate absorbed at fused silica/methanol interfaces . 2nd harmonic detection of sinusoidally modulated two photon excited fluorescence can also be used to obtain luminescence spectra ". ... 

When light traversing an optically dense medium approaches an interface with a more optically rare medium at an angle exceeding a critical value, Bent = sin (rerare/ dens), total internal reflection occurs and an evanescent wave of exponentially deca5ung intensity penetrates the rarer medium. This phenomenon is at the heart of certain spectroscopic methods used to probe biomolecules at interfaces (199). In total internal reflection fluorescence (TIRF) spectroscopy (200-202), the evanescent wave excites fluorescent probes attached to the biomolecules, and detection of the emission associated with their decay provides information on the density, composition, and conformation of adsorbed molecules. In fourier transform infrared attenuated total reflection (FTIR-ATIR) spectroscopy (203,204), the evanescent wave excites certain molecular vibrational degrees of freedom, and the detected loss in intensity due to these absorbances can provide quantitative data on density, composition, and conformation. 

A large number of different biochemical systems have been studied by optical measurement on continuous surfaces, using ellipsometry, attenuated total refiection, interference techniques and total internal reflection fluorescence (reference 6 lists 45 different experiments). It is therefore likely that evanescent wave spectroscopy will become a widely applied technique in the future. 

While the SFA provided direct evidence as to the thickness of a polymer layer adhering to a surface, TIRF provides a measure of the surface area concentration, r. TIRF spectroscopy makes use of the total internal reflection of light at the interface between a solid adsorption substrate of relatively high refractive index and a polymer solution of lower refractive index. Ihe total internal reflection, however, generates a standing evanescent wave, a nonclassical penetration of the light into the lower refractive index phase. The evanescent wave may penetrate some 60 to 65 nm into the lower refractive index phase, be absorbed by it, fluoresce, and so on (72,73). 

We should point out that we will not be concerned with many other issues related to fluorescence spectroscopy that are also relevant in the case of evanescent wave excitation in general and SPFS, in particular. E.g., we will not deal with the details cf the distance dependence of fluorescence emission in a quantitative way, and are not concerned with orientational effects taking into account that surface modes have a particular... 

Two-photon fluorescence spectroscopy provides a much better signal-to-noise ratio compared with the one-photon excitation scheme. We have already seen its advantages in the course of measuring the fluorescence of atoms adsorbed near to a metal surface (Section 6.5). In this chapter we shall consider a similar technique based on two-photon evanescent wave excitation of a gas near a surface. Due to the reliability of the signal, this kind of spectroscopy allows one to study the fluorescence lineshapes in detail. 

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