Fluorescence detection fluorescent molecules

This teclnhque can be used both to pennit the spectroscopic detection of molecules, such as H2 and HCl, whose first electronic transition lies in the vacuum ultraviolet spectral region, for which laser excitation is possible but inconvenient [ ], or molecules such as CH that do not fluoresce. With 2-photon excitation, the required wavelengdis are in the ultraviolet, conveniently generated by frequency-doubled dye lasers, rather than 1-photon excitation in the vacuum ultraviolet. Figure B2.3.17 displays 2 + 1 REMPI spectra of the HCl and DCl products, both in their v = 0 vibrational levels, from the Cl + (CHg) CD reaction [ ]. For some electronic states of HCl/DCl, both parent and fragment ions are produced, and the spectrum in figure B2.3.17 for the DCl product was recorded by monitoring mass 2 (D ions. In this case, both isotopomers (D Cl and D Cl) are detected. 

A laser beam highly focused by a microscope into a solution of fluorescent molecules defines the open illuminated sample volume in a typical FCS experiment. The microscope collects the fluorescence emitted by the molecules in the small illuminated region and transmits it to a sensitive detector such as a photomultiplier or an avalanche photodiode. The detected intensity fluctuates as molecules diffuse into or out of the illuminated volume or as the molecules within the volume undergo chemical reactions that enhance or diminish their fluorescence (Fig. 1). The measured fluorescence at time t,F(t), is proportional to the number of molecules in the illuminated volume weighted by the... 

Abstract Fluorescent molecules have been widely used as biomolecular labels, enzyme substrates, environmental indicators, and cellular stains and thus constitute indispensable tools in chemistry, physics, biology, and medicinal sciences. The large variation in the photophysics of the available fluorophores connected with chemical alterations give fluorescent probe techniques an almost unlimited scope for the detection of specific molecules and the investigation of intermolecular interactions on a molecular scale. 

It is necessary to note that fluorescence characteristics demonstrate remarkable sensitivity to variations of physicochemical parameters of the environment. Therefore, such parameters as polarity, viscosity, temperature, electric potential, local electric field, pressure, pH, etc., can be registered successfully using the modem sensitive apparatus for fluorescence detection [1, 4—12]. As a consequence, fluorescent molecules are used successfully as molecular probes to study the local characteristics of physicochemical, biochemical and biological systems. 

Fluorescence molecules are analogous to roaming molecular spies with radio transmitters, radiating information to the experimenter about the state of affairs on the molecular scale, and informing us where the spies are located and how many there are. A feature unique to FRET is the capability to inform us whenever two or more molecules (usually biological macromolecules) are close to one another on a molecular scale (<80 A), and whether they are moving relative to each other (Section 1.3.1.1). It is even sometimes possible to detect how the D and A transition moments are oriented... 

Fig. 6.11. Two types of FRET probes. (A) Ratiometric probes are formed by two fluorescent molecules that allow determination of emission ratio. (B) Quenched probes feature a donor fluorophore and a quencher. The emission increase of the donor after release of the acceptor is detected. Both types are frequently used to build proteinases probes.

Use of sulfo-NHS-LC-SPDP or other heterobifunctional crosslinkers to modify PAMAM dendrimers may be done along with the use of a secondary conjugation reaction to couple a detectable label or another protein to the dendrimer surface. Patri et al. (2004) used the SPDP activation method along with amine-reactive fluorescent labels (FITC or 6-carboxytetramethylrhodamine succinimidyl ester) to create an antibody conjugate, which also was detectable by fluorescent imaging. Thomas et al. (2004) used a similar procedure and the same crosslinker to thiolate dendrimers for conjugation with sulfo-SMCC-activated antibodies. In this case, the dendrimers were labeled with FITC at a level of 5 fluorescent molecules per G-5 PAMAM molecule. 

The original polymeric latex particles still are widely used for separation and detection. Polymers provide a matrix that can be swollen for embedding other molecules in their core, such as organic dyes or fluorescent molecules. Even nanoparticle quantum dots can be incorporated into larger latex particles to form highly fluorescent composite microparticles. 

Covalent attachment of antibody molecules to liposomes can provide a targeting capacity to the vesicle that can modulate its binding to specific antigenic determinants on cells or to molecules in solution. Antibody-bearing liposomes may possess encapsulated components that can be used for detection or therapy (Figure 22.17). For instance, fluorescent molecules encapsulated within antibody-targeted vesicles can be used as imaging tools or in flow cytometry... 

Figure 22.17 Antibody-liposome conjugates may be used as targeting reagents for detection or therapeutic applications. The liposome may be constructed to contain fluorescent molecules for detection purposes or bioactive agents for therapy. The antibody component targets the complex for binding to specific antigenic determinants.

Figure 22.18 Biotinylated liposomes may be used in immunoassay systems to enhance the signal for detection or measurement of specific analytes. The liposome components may be constructed to include fluorescent molecules to facilitate detection of antigens within tissue sections.

Oligonucleotide probes may be labeled with small fluorescent molecules for detection of hybridization by luminescence. Fluorescent probes are widely used in assay systems involving bio-specific interactions (Chapter 9). Receptors for ligands may be localized in tissues or cells by modification of the ligand with the appropriate fluorophore. Targeted molecules may be... 

The point is now to estimate the maximum number of photons that can be detected from a burst. The maximum rate at which a molecule can emit is roughly the reciprocal of the excited-state lifetime. Therefore, the maximum number of photons emitted in a burst is approximately equal to the transit time divided by the excited-state lifetime. For a transit time of 1 ms and a lifetime of 1 ns, the maximum number is 106. However, photobleaching limits this number to about 105 photons for the most stable fluorescent molecules. The detection efficiency of specially designed optical systems with high numerical aperture being about 1%, we cannot expect to detect more than 1000 photons per burst. The background can be minimized by careful dean-up of the solvent and by using small excitation volumes ( 1 pL in hydrodynamically focused sample streams, 1 fL in confocal exdtation and detection with one- and two-photon excitation, and even smaller volumes with near-field excitation). 

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