Fluorescence internal reflectance

Fluorescence internal reflectance Many practical disadvantages (42) 

Fluorescence polarization as a method of FI A is quick and precise however, it does have disadvantages. The equipment for this type of assay is specialized and the polarization change tends to be small, resulting in assays with sensitivity limited to the umolL to the upper jttmolL range. 

This technique uses a surface to which antibodies are bound (an immuno- 

---

Protein adsorption has been studied with a variety of techniques such as ellipsome-try [107,108], ESCA [109], surface forces measurements [102], total internal reflection fluorescence (TIRE) [103,110], electron microscopy [111], and electrokinetic measurement of latex particles [112,113] and capillaries [114], The TIRE technique has recently been adapted to observe surface diffusion [106] and orientation [IIS] in adsorbed layers. These experiments point toward the significant influence of the protein-surface interaction on the adsorption characteristics [105,108,110]. A very important interaction is due to the hydrophobic interaction between parts of the protein and polymeric surfaces [18], although often electrostatic interactions are also influential [ 116]. Protein desorption can be affected by altering the pH [117] or by the introduction of a complexing agent [118]. 

Tokunaga M, Kitamura K, Saito K, Iwane A H and Yanagida T 1997 Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy Biochem. Biophys. Res. Commun. 235 47-53... 

If the surface of an acrylic sheet, rod or tube is roughened or carved, less light is internally reflected and the material is often rather brighter at these non-polished surfaces. The use of this effect enables highly attractive carvings to be produced. Similarly, lettering cut into sheet, particularly fluorescent sheet, becomes Tit-up and this effect is useful in display signs. 

Rotational dynamics of a fluorescent dye adsorbed at the interface provides useful information concerning the rigidity of the microenvironment of liquid-liquid interfaee in terms of the interfacial viscosity. The rotational relaxation time of the rhodamine B dye was studied by time-resolved total internal reflection fluorescent anisotropy. In-plane... 

Jones MA, Bohn PW (2001) Total internal reflection fluorescence and electrocapillary investigations of adsorption at the water-dichloroethane electrochemical interface. 2. Fluorescence-detected linear dichroism investigation of adsorption-driven reorientation of di-N-butylaminonaphthylethenylpyridiniumpropylsulfonate. J Phys Chem B 105(11) 2197-2204... 

Recently, a formalism has been developed to determine the second and the fourth order parameters of films using polarized total internal reflection fluorescence (TIRF) [71]. Similarly to IR-ATR spectroscopy (Section 4), the experiment makes use of p- and s-polarized excitation, but the fluorescence emission (analyzed either in p- or s-direction) is detected normal to the substrate. Two approaches are developed based on the measurements of two intensity ratios. In the first one, the S angle has to be known experimentally or theoretically, and the order parameters (P2) and (P4) can be determined. In the second one, the order parameter (R ) is obtained by another technique, for instance IR-ATR spectroscopy, which allows deducing the order parameter (P4) and (cos2<5). 

Total internal reflection fluorescence lifetime imaging microscopy... 

The upgrade of a frequency-domain fluorescence lifetime imaging microscope (FLIM) to a prismless objective-based total internal reflection-FLIM (TIR-FLIM) system is described. By off-axis coupling of the intensity-modulated laser from a fiber and using a high numerical aperture oil objective, TIR-FLIM can be readily achieved. The usefulness of the technique is demonstrated by a fluorescence resonance energy transfer study of Annexin A4 relocation and two-dimensional crystal formation near the plasma membrane of cultured mammalian cells. Possible future applications and comparison to other techniques are discussed. 

Axelrod, D. (1981). Cell-substrate contacts illuminated by total internal reflection fluorescence. J. Cell Biol. 89, 141-5. 

Axelrod, D. (2001). Total internal reflection fluorescence microscopy in cell biology. Traffic 2, 764-74. 

Beaumont, V. (2003). Visualizing membrane trafficking using total internal reflection fluorescence microscopy. Biochem. Soc. Trans. 31, 819-23. 

Khakh, B. S., Fisher, J. A., Nashmi, R., Bowser, D. N. and Lester, H. A. (2005). An angstrom scale interaction between plasma membrane ATP-gated P2X2 and alpha4beta2 nicotinic channels measured with fluorescence resonance energy transfer and total internal reflection fluorescence microscopy. J. Neurosci. 25, 6911-20. 

Figure 14 illustrates an example of a structure that can provide enhanced fluorescence capture. It consists of a truncated cone, on top of which the fluorescent species is deposited. The cone angle, a, is chosen in order to cause total internal reflection of the emitted fluorescence (the angular distribution of which is calculated from the model) and is therefore dependent on the refractive indices of the cone material and the environment. The emitted fluorescence is reflected onto a detector positioned directly beneath the cone. 

A sensor configuration employing these cones is shown in Figure 15 Fluorescence from the luminescent spots is excited from behind the platform using an appropriate source (LED s in this case), is subsequently emitted via total internal reflection through the sensor chip and is detected by a CMOS camera, which is positioned behind the chip. For the purposes of intensity comparisons, luminescent spots are also deposited directly onto the planar surface of the chip and excited along with those deposited on the cones. 

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... 

Figure 1. Schematic of the optical fiber system. Excitation light is launched into the fiber. Due to the refractive index differences between the fiber core and cladding materials, the light is internally reflected and travels through the fiber with minimal loss (see inset). The emitted light is carried back from the fluorescent sensor located on the tip of the fiber to a CCD camera detector. Reprinted with permission from Science, 2000, 287, 451-452. Copyright 2000 AAAS.

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...

Axelrod D, Hellen EH, Fulbright RM (1992) Total internal reflection fluorescence. In Lakowicz JR (ed) Topics in fluorescence microscopy. Plenum, New York... 

Burmeister JS, Olivier LA, Reichert WM, Truskey GA (1998) Application of total internal reflection fluorescence microscopy to study cell adhesion to biomaterials. Biomaterials 19 307-325... 

TIRF Total internal reflection fluorescence immunoassay WWTPs Wastewater treatment plants... 

Total internal reflection fluorescence (TIRF) microscopy, fluorescence in situ hybridization (FISH), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM). 

---