Fluorescence Excitation and Detection

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). 

Table I. LASER FLUORESCENCE EXCITING AND DETECTING WAVELENGTHS...

Dichroic beam splitters (or filters) use the same principle as interference filters. They are placed in a collimated beam, typically at a 45-degree angle. There are long-pass, short-pass and bandpass versions. They are extremely suitable for building high-efficiency optical systems. Figure 7.17, left, shows how a laser is coupled into a high-NA fluorescence excitation and detection system. 

FCS is based on the universal formalism of correlation analysis, comparing time signals for series of lag times r. In the common case, the signal results from fluorescence excitation and detection within a confocal volume [22]. The nor-mahzed fluorescence autocorrelation function is generally defined by... 

Figure 3 Two fluorescence schemes. (A) Resonance fluorescence excitation and detection at the same wavelength. (B) Nonresonance fluorescence excitation and detection at different wavelengths, Adet > Aexc-...

AA fluorescence was measured with a Perkin-Elmer model MPF-3L spectro-fluoremeter, with 400 nm and 520 nm fluorescence excitation and detection bands. Samples contained per ml 67 pg protein and 1.7 nmoles 9-AA in the case of cyanobacteria, and 70 pg protein and 20 pmoles 9-AA in the case of spinach thylakoids. The background monovalent cation concentration was approx. 0.8 mM. Fluorescence quenching was released by adding MgCl and KCl. 

Figure C 1.5.5. Time-dependent fluorescence signals observed from liquid solutions of rhodamine 6G by confocal fluorescence microscopy. Data were obtained with 514.5 mn excitation and detected tlirough a 540-580 nm...

Fisz, J. J. (2007). Fluorescence polarization spectroscopy at combined high-aperture excitation and detection Application to one-photon-excitation fluorescence microscopy. J. Phys. Chem. A 111, 8606-21. 

Figure 11.10. Phase-locked detection of fluorescence lifetime using single reference signal. FID = fluorescence inducing and detecting devices LPF = low-pass electronic filter VCO = voltage-controlled oscillator v, = signal to modulate the output intensity of the excitation light source v/= the fluorescence signal.

T. P. Burghardt and N. L. Thompson, Effect of planar dielectric interfaces on fluorescence emission and detection Evanescent excitation with high aperture collection, Biophys. J. 46, 729-737 (1984). 

The integrated fluorescence signal //was collected with a g-in. glass light pipe and detected through a combination of dielectric and colored glass filters with a photomultiplier tube. Fluorescence excitation and elastic scattering spectra were recorded simultaneously, in order to identify the type (TM or TE) of resonance responsible for the peaks seen in the excitation spectrum. 

Fluorescence excitation and emission spectra of the two sodium D lines in an air-acetylene flame, (a) In the excitation spectrum, the laser was scanned, (to) In the emission spectrum, the monochromator was scanned. The monochromator slit width was the same for both spectra. [From s. J. Weeks, H. Haraguchl, and J. D. Wlnefordner, Improvement of Detection Limits in Laser-Excited Atomic Fluorescence Flame Spectrometry," Anal. Chem. 1976t 50,360.]... 

A simple, rapid, sensitive, and selective spectrofluorimetric method (2ex/ lem = 345/455nm) has been developed for the determination of zaleplon. Tang et al. have studied the influence of micellar medium on the absorption, fluorescent excitation, and emission spectra character of zaleplon The nonionic surfactant of Triton X-100 showed a strong sensitizing effect for the fluorescence of zaleplon in a pH 5.0 buffer. The possible enhancement mechanism was discussed. Based on the optimum conditions, the linear range was 1.32 x 10 8-1.00 x 10 mol/1. The detection limit was 4.0 x 10 mol/1 with a relative standard deviation (RSD) of 0.06%. The proposed method was successfully applied to the determination of zaleplon in tablets, serum, and urine. 

Experimentally, commercial steady-state fluorescence spectrometers can be equipped with polarizer attachments, either sheet or Glan-Thompson polarizers. Alternatively, sheet polarizers are usually easily incorporated into the sample cavity in the excitation and emission pathways. Likewise, for time-resolved spectroscopy, polarizers may simply be introduced into the excitation and detection paths. Frequently, the excitation source in time-resolved experiments is a laser which will be inherently polarized. 

The experimental conditions for the excitation and detection of all the species are listed in Table I along with the radiative lifetimes of the excited states. Under the narrow detection bandwidth conditions for these measurements the quench term is much greater than T 1 for the species studied and the fluorescence efficiency varies as x T1/2. Thus with fixed geometry, laser excitation wavelength, and detection parameters, the fluorescence intensity in Equation (1) simplifies to... 

Some fluorophores are used to detect the presence of sodium or potassium in cells. For example, the maxima of the fluorescence excitation and emission spectra of SBFI increase, and its emission maximum shifts toward short wavelengths in the presence of sodium (Figure 7.17). 

Fluorescent dyes, especially the cyanine dyes Cy3 and Cy5, are the most popular choice for dual colour microarray analysis. The main benefit of using Cy dye fluorophors in particular is that the two dyes can be excited and detected from the same slide. Fluorescent dyes can be directly incorporated into nucleic acid by either enzymatic or chemical methods. Basically there are two fluorescent dye-labeling strategies ... 

The monobromobimane derivatives of cysteine, y-glutamylcysteine, and glutathione were separated on a Merck Supersphere RP-18 column (4.0 mm X 250 mm). The mobile phase was methanol-water (18 82) containing 0.25% acetic acid adjusted to pH 3.8 with NaOH. The flow rate was 1.0 mL/min. Detection was by fluorescence, excitation and emission wavelengths 370 and 485 nm, respectively. 

The o-phthaldialdehyde derivatives of glutamate, y-aminobutyric acid (GABA), and S-aminovaleric acid (DAVA internal standard) were separated on an Altex Ultrasphere ODS column (4.6 mm x 250 mm, 5 /tin). The mobile phase contained 0.2 M sodium acetate (pH 3.8), 100 mg/mL EDTA, and 40% acetonitrile (v/v). Detection was by fluorescence, with excitation and detection wavelengths of 330 and 440 nm, respectively. 

Isoxanthopterin was separated from 2-amino-4-hydroxypteridine by chromatography on a Unisil, ODS column (4.6 mm x 50 mm, 3 /u.m). The mobile phase was 0.1 M phosphate buffer (pH 2.0). A fluorescence detector was used, with excitation and detection wavelengths 340 and 410 nm, respectively. 

The fiber optic evanescent wave sensor (FO-EWS) belongs to a sensor in which the fiber core interacts with the analyte. This interaction occurs through the attenuated total reflection (ATR) and the evanescent wave excitation in a dielectric medium of smaller refractive index in the vicinity of fiber core. If the surrounding medium is fluorescent, then the fluorescence signal in the reaction region of evanescent wave field is excited and detected. This is illustrated in Figure 8.2. 

---