Excited molecular probes

BODIPY is a short for 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, the basic structure of this type of fluorophore (see Fig. 6.5). Derivatives of this dye have been created by modification of positions 1, 3, 5, 7, and 8, generating an array of fluorophores with very distinct excitation and emission properties [38]. Molecular Probes has synthesized a wide number of BODIPY dyes whose excitation... 

The fluorescence spectra of the monomeric cyanine nucleic acid stains family (PO-PRO-1, BO-PRO-1 and YO-PRO-1) introduced by Molecular Probes (http // probes.invitrogen.com) cover the entire visible wavelength range. These dyes may also be used with ultraviolet trans- or epi-illuminator excitation sources. The monomeric cyanine nucleic acid stains exhibit large degrees of fluorescence enhancement upon binding to DNA (or RNA) up to 1,800-fold. Consequently,... 

Dramatic advances in modem fluorophore technology have been achieved with the introduction of Alexa Fluor dyes by Molecular Probes (Alexa Fluor is a registered trademark of Molecular Probes). Alexa Fluor dyes are available in a broad range of fluorescence excitation and emission wavelength maxima, ranging from the ultraviolet and deep blue to the near-infrared regions. Because of the large... 

E Sacksteder, M. Lee, J. N. Demas, and B. A. DeGraff, Long lived, highly luminescent rhenium(I) complexes as molecular probes Intra- and intermolecular excited state interactions, J Am. Chem. Soc. 115, 8230-8238 (1993). 

Figure 5.22. Structures and electronic spectra of Fura-2 and Indo-l (a) excitation spectra of Fura-2 (b) emission spectra of Indo-l, in increasing concentrations of free calcium (from molecular probes).

In the literature, fluorescence spectroscopy in OFD has been limited to the use of ultraviolet (UV) or visible dyes as molecular probes.(1) The most common fluorescent dye used in OFD is fluorescein and its derivatives/21 23) Fluorescein possesses a good fluorescence quantum yield and is commercially available with an isothiocyanate functionality for linking to the polymeric support/24-26 Additionally, selective laser excitation can be performed because the absorbance maximum of fluorescein coincides with the 499-nm laser line emitted from an argon laser. Unfortunately, argon lasers are costly and bulky, thus limiting the practicality of their use. Similar difficulties exist with other popular commercial dyes. 

Excitation and emission spectra of molecules for donor-acceptor pairs can be found at one of the following Web sites Becton-Dickinson Fluorescence Spectrum Viewer (http //www. bdbiosciences.com/spectra), Invitrogen-Molecular Probes Fluorescence Spectra Viewer (http //www.probes.invitrogen. com/servlets/spectraviewer). 

Spontaneously Detected Photon Echoes in Excited Molecular Ensembles A Probe Pulse Laser Technique for the Detection of Optical Coherence of Inhomogeneously Broadened Electronic Transitions, A. H. Zewail, T. E. Orlowski, K. E. Jones, and D. E. Godar, Chem. Phys. Lett. 4S, 256 (1977). 

Another hydrazine derivative of fluorescein, 5-(((2-(Carbohydrazino)methyl)thio)-acetyl)-aminofluorescein, contains a longer spacer arm off its No. 5 carbon atom of its lower ring than fluorescein-5-thiosemicarbazide, described previously (Molecular Probes). The reagent can be used to react spontaneously with aldehyde- or ketone-containing molecules forming a hydrazone linkage (Fig. 209). It also can be used to label cytosine residues in DNA or RNA by use of the bisulfite activation procedure (Chapter 17, Section 2.1). The resulting fluorescent derivative exhibits a maximal excitation at 490 nm and a maximal luminescence emission peak at 516 nm when dissolved in buffer at pH 8. In the same buffered environment, the compound has an extinction coefficient of approximately 75,000 M-1cm 1 at 490 nm. 

Diethylamino-3-[(4 -(iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA) is a derivative of the basic aminomethylcoumarin structure that contains a sulfhydryl-reactive iodoacetyl group and diethyl substitutions on its amine. This particular cou-marin derivative is among the most fluorescent UV-excitable iodoacetamide probes available (Sippel, 1981) (Molecular Probes). 

BODIPY fluorophores are a relatively new class of probes based on the fused, multiring structure 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (Fig. 225) (note BODIPY is a registered trademark of Molecular Probes U.S. Patent 4,774,339). This fundamental molecule can be modified, particularly at its 1,3,5,7 and 8 carbon positions, to produce new fluorophores with different characteristics. The modifications cause spectral shifts in its excitation and emission wavelengths and can provide sites for chemical coupling to label other molecules. 

BODIPY 530/550 C3 is 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (Molecular Probes). This derivative of the basic BODIPY structure contains two phenyl rings off the No. 5 and 7 carbon atoms and a propionic acid group on the No. 3 carbon atom. The carboxylate group may be used to attach the fluo-rophore to amine-containing molecules via a carbodiimide reaction to create an amide bond. The substituents on this BODIPY fluorophore result in alterations to its spectral properties, pushing its excitation and emission maximums up to higher wavelengths. 

The absorption and emission maxima from this table will provide clues to the spectral ranges that are useful for excitation and for fluorescence detection with a particular fluorochrome. However, the absorption and emission spectra have breadth, with slopes and shoulders and secondary peaks (see Fig. 5.6). With efficient fluorochromes, excitation and fluorescence detection at wavelengths distant from the maxima may be possible. Therefore, inspection of the full, detailed spectra is necessary to get the full story. In addition, spectra may shift in different chemical environments (this will explain why maxima vary in different reference tables from different sources). Values in this table are derived primarily from the Molecular Probes Handbook and the article by Alan Waggoner (Chapter 12) in Melamed et al. 

Fluorescence of Excited Singlet-State Acids in Certain Organized Media Applications as Molecular Probes... 

Direct labeling of a biomolecule involves the introduction of a covalently linked fluorophore in the nucleic acid sequence or in the amino acid sequence of a protein or antibody. Fluorescein, rhodamine derivatives, the Alexa, and BODIPY dyes (Molecular Probes [92]) as well as the cyanine dyes (Amersham Biosciences [134]) are widely used labels. These probe families show different absorption and emission wavelengths and span the whole visible spectrum (e.g., Alexa Fluor dyes show UV excitation at 350 nm to far red excitation at 633 nm). Furthermore, for differential expression analysis, probe families with similar chemical structures but different spectroscopic properties are desirable, for example the cyanine dyes Cy3 and Cy5 (excitation at 548 and 646 nm, respectively). The design of fluorescent labels is still an active area of research, and various new dyes have been reported that differ in terms of decay times, wavelength, conjugatibility, and quantum yields before and after conjugation [135]. New ruthenium markers have been reported as well [136]. 

Recently, BOCILLIN FL, a fluorescent penicillin, was synthesized for the detection and characterization of PBPs [34]. BOCILLIN FL, a derivative of penicillin V, is an orange solid with extinction coefficient of 68,000 M 1 cm1 and a maximal absorption at 504 nm (Molecular Probes, Inc.). It fluoresces at 511 nm upon excitation at 504 nm. BOCILLIN FL has been used to detect PBPs from the membrane preparations of several bacterial species, including S. pneumoniae, E. coli, and P. aeruginosa (Fig. 3). A typical procedure of using BOCILLIN FL for the detection of PBPs involves preparation of the bacterial cytoplasmic membranes, incubation of the membrane preparations with BOCILLIN FL, and visualization of PBPs by a UV transilluminator or with the aid of a... 

Emission and excitation wavelengths are solute- and solvent dependent. A number of the excitation wavelengths correspond to the laser lines (325, 442, and 488 nm) and do not correspond to the actual excitation maxima. In addition, the excitation wavelength is frequently selected to avoid the Raman band and may not correspond to the actual emission maximum. c 3-(4-Carboxybenzoyl)-2-quinolinecarboxaldehyde, available from Molecular Probes. d 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate, available from Waters Chromatography. 

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