Fluorescent probes in membrane studies.

A number of spectroscopic techniques are suitable for studying biological membranes. Of these, fluorescence has the sensitivity and time resolution for following membrane events associated with nerve excitation. In this paper, the nature of the information derived from measurements of the fluorescence properties of externally introduced chromophores in membranes is examined. In particular, the locations of various probes are described on the basis of nuclear magnetic resonance (n.m.r.) experiments in model situations. Then the motional characteristics of the probe molecules (rotation and diffusion) are discussed. Finally experiments designed to relate the detailed observations that can be made in lipid bilayers using n.m.r. and fuorescence measurements to those (more limited in nature) that can be made in membranes are described.     

Fluorescent probes for living cells

The functional characteristics of fluorescent probes used for imaging and measuring dynamic processes in living cells are reviewed. Initial consideration is given to general design requirements for delivery, targeting, detectability and fluorescence readout, and current technologies for attaining them. Discussion then proceeds to the more application-specific properties of intracellurion indicators, membrane potential sensors, probes for proteins and lipids, and cell viability markers. 1998 © Chapman & Hall     

Review: Fluorescent probes for living cells

The functional characteristics of fluorescent probes used for imaging and measuring dynamic processes in living cells are reviewed. Initial consideration is given to general design requirements for delivery, targeting, detectability and fluorescence readout, and current technologies for attaining them. Discussion then proceeds to the more application-specific properties of intracellurion indicators, membrane potential sensors, probes for proteins and lipids, and cell viability markers. 1998 © Chapman & Hall     

Fluorescent probes for bioimaging applications

Fluorescent probes based on small organic molecules have become indispensable tools in modern biology because they provide dynamic information concerning the localization and quantity of the molecules of interest, without the need of genetic engineering of the sample. In this review, following a brief outline of the principle of fluorescence imaging, we recount some recent achievements in the field of small-molecular fluorescent probes. First, probes for metal cations, including those suitable for two-photon imaging, are introduced. Next, methodologies to visualize proteases are discussed, with special emphasis on activity-based probes for use in vivo. All these probes have been confirmed to be applicable to cellular or in vivo imaging.     

Fluorescent neoglycolipids. Improved probes for oligosaccharide ligand discovery.

A second generation of lipid-linked oligosaccharide probes, fluorescent neoglycolipids, has been designed and synthesized for ligand discovery within highly complex mixtures of oligosaccharides. The aminolipid 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE), which has been used extensively to generate neoglycolipids for biological and structural studies, has been modified to incorporate a fluorescent label, anthracene. This new lipid reagent, N-aminoacetyl-N-(9-anthracenylmethyl)-1, 2-dihexadecyl-sn-glycero-3-phosphoethanolamine (ADHP), synthesized from anthracenaldehyde and DHPE gives an intense fluorescence under UV light. Fluorescent neoglycolipids derived from a variety of neutral and acidic oligosaccharides by conjugation to ADHP, by reductive amination, can be detected and quantified by spectrophotometry and scanning densitometry, and resolved by TLC and HPLC with subpicomole detection. Antigenicities of the ADHP-neoglycolipids are well retained, and picomole levels can be detected using monoclonal carbohydrate sequence-specific antibodies. Among O-glycans from an ovarian cystadenoma mucin, isomeric oligosaccharide sequences, sialyl-Lea- and sialyl-Lex-active, could be resolved by HPLC as fluorescent neoglycolipids, and sequenced by liquid secondary-ion mass spectrometry. Thus the neoglycolipid technology now uniquely combines high sensitivity of immuno-detection with a comparable sensitivity of chemical detection. Principles are thus established for a streamlined technology whereby an oligosaccharide population is carried through ligand detection and ligand isolation steps, and sequence determination by mass spectrometry, enzymatic sequencing and other state-of-the-art technologies for carbohydrate analysis.     

Fluorescent DNA probes for flow cytometry. Considerations and prospects.

Techniques employing base specific deoxyribonucleic acid (DNA)-binding fluorochromes and flow cytometry (FCM) are potentially useful for obtaining information of the compositional features of chromatin or chromosomes of mammalian cells. Fluorescent compounds which form complexes preferentially at the A-T rich regions (i.e., DNA-reactive Hoechst dyes) or the G-C rich regions (i.e., mithramycin, chromomycin, olivomycin) in DNA are available and compatible with current FCM technology as are other compounds (i.e., ethidium bromide, propidium iodide) which show little or no base specificity and bind by intercalation in the double stranded regions of helical DNA. Energy transfer between appropriate DNA-bound dyes is a reflection of the quantity and proximity of regions containing the respective base pair segments. Since extrinsic fluorescent probes provide only a measure of available binding sites or regions unobstructed by chromatin-associated or chromosomal-associated proteins, interpretations of fluorescence measurements need to be substantiated by adequate control measures.     

Fluorescent probes for detection of protozoan parasites

Fluorescent probes generally provide a rapid and simple staining technique, valuable for the rapid diagnosis of protozoal infections. However, many of these staining techniques have disadvantages for clinical tests: (I) they require a fluorescence microscope which is not always available in clinical laboratories; (2) the preparations are not permanent because the fluorescent probes do not withstand dehydration; (3) variable quenching of the fluorescence may occur, unless proper preventive measures are taken. In this article, Fumihiko Kawamoto and Nobuo Kumodo explain some of the most widely used fluorescent probes, and discuss how problems in their use can be minimised.     

Fluorescent and photoaffinity labeling probes for retinoic acid receptors.

A fluorescent probe for retinoid receptors (RARs) was designed and prepared. The probe consists of a retinoid moiety and a dansyl moiety, i.e., 2-[3-(5-dimethylaminonaphthalene-1-sulfonyl)- aminopropyl-1-oxy]-4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)carboxamido]benzoic acid: DAM-3. DAM-3 specifically bound RARs. Additionally, a photoreactive RAR fluorescent probe was designed and prepared, i.e., 2-[3-(5-azidonaphthalene- 1-sulfonyl)aminopropyl-1-oxy]-4-[(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (ADAM-3). ADAM-3 irreversibly and specifically bound RARs using ultraviolet irradiation.     

Fluorescent probes for bacterial cytoplasmic membrane research

Fluorescent methods in biological and medical research are extremely useful at the cellular and molecular levels. This is due to sensitive and affordable detection equipment and a variety of specific and more general fluorescent probes, and analytical procedures. In this article, I examine the use of fluorescence membrane probes to study the fluidity (membrane polarization) of the bacterial cytoplasmic membrane, central to energy transduction, ion and nutrient transport and diffusion of water and gases.     

Fluorescent membrane probes incorporating dipyrrometheneboron difluoride fluorophores.

The spectroscopic properties of a new series of fatty acid analogs in which a dipyrrometheneboron difluoride fluorophore forms a segment of the acyl methylene chain are presented and their characteristics as fluorescent membrane probes are examined. When incorporated as a low mole fraction component in model phospholipid membranes, the probes retain the principal characteristics of the parent fluorophore: green fluorescence emission with high quantum yield, extensive spectral overlap, and low environmental sensitivity. The fluorescence quantum yield is typically two to three times that of comparable membrane probes based on the nitrobenzoxadiazole fluorophore. The spectral overlap results in a calculated F?rster energy transfer radius (Ro) of about 57 A. Consequently, increasing fluorescence depolarization and quenching are observed as the mole fraction of the probe species incorporated in the membrane is increased. Low environmental sensitivity is manifested by retention of high quantum yield emission in aqueous dispersions of fatty acids. Partition coefficient data derived from fluorescence anisotropy measurements and iodide quenching experiments indicate that in the presence of fluid phase phospholipid bilayers the aqueous fraction of fatty acid is very small. Fluorescence intensity and anisotropy responses to phospholipid phase transitions are examined and found to be indicative of nonrandom fluorophore distribution in the gel phase. It is concluded that the spectroscopic properties of the fatty acid probes and their phospholipid derivatives are particularly suited to applications in fluorescence imaging of cellular lipid distribution and membrane level studies of lateral lipid segregation.     

Fluorescent peptide probes for in vivo diagnostic imaging

Recently, many novel peptide-based near-infrared (NIR) fluorescent molecular probes have been developed for in vivo biomedical imaging. To report specific information of biological targets, the probes were individually designed according to the unique property or functions of their targets. These peptide-based probes can be classified into targeting, crosslinking, and enzyme-activatable probes. Several of them have been tested in various in vitro and in vivo models, and the obtained imaging information has been applied to disease detection, medical diagnosis, and drug evaluations.     

Fluorescent probes for investigating peptidoglycan biosynthesis in mycobacteria

Tuberculosis killed 1.5 million people in 2018. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the most deadly infectious bacteria in the world. A strength of mycobacterial pathogens — their formidable cell wall — could also be one of their greatest molecular vulnerabilities. As in other bacteria, peptidoglycan (PG) maintenance and integrity is essential to mycobacterial survival. But Mtb PG is unique, and a better understanding of its biosynthetic machinery could lead to new drugs or more effective treatment regimens. Such investigations are being accelerated by the application of fluorescent probes, including those based on vancomycin, β-lactams, PG stem mimics, d-amino acids, and reactive glycans. This review will describe how fluorescent probes are being used to uncover new information on the regulation and drug susceptibility of two classes of enzymes that fortify the Mtb PG: the penicillin-binding proteins and the L,D-transpeptidases.