Teaching Old Dyes New Tricks: Biological Probes Built from Fluoresceins and Rhodamines.

Small-molecule fluorophores, such as fluorescein and rhodamine derivatives, are critical tools in modern biochemical and biological research. The field of chemical dyes is old; colored molecules were first discovered in the 1800s, and the fluorescein and rhodamine scaffolds have been known for over a century. Nevertheless, there has been a renaissance in using these dyes to create tools for biochemistry and biology. The application of modern chemistry, biochemistry, molecular genetics, and optical physics to these old structures enables and drives the development of novel, sophisticated fluorescent dyes. This critical review focuses on an important example of chemical biology-the melding of old and new chemical knowledge-leading to useful molecules for advanced biochemical and biological experiments.

A technical review of optical mapping of intracellular calcium within myocardial tissue.

Optical mapping of Ca(2+)-sensitive fluorescence probes has become an extremely useful approach and adopted by many cardiovascular research laboratories to study a spectrum of myocardial physiology and disease conditions. Optical mapping data are often displayed as detailed pseudocolor images, providing unique insight for interpreting mechanisms of ectopic activity, action potential and Ca(2+) transient alternans, tachycardia, and fibrillation. Ca(2+)-sensitive fluorescent probes and optical mapping systems continue to evolve in the ongoing effort to improve therapies that ease the growing worldwide burden of cardiovascular disease. In this technical review we provide an updated overview of conventional approaches for optical mapping of Cai (2+) within intact myocardium. In doing so, a brief history of Cai (2+) probes is provided, and nonratiometric and ratiometric Ca(2+) probes are discussed, including probes for imaging sarcoplasmic reticulum Ca(2+) and probes compatible with potentiometric dyes for dual optical mapping. Typical measurements derived from optical Cai (2+) signals are explained, and the analytics used to compute them are presented. Last, recent studies using Cai (2+) optical mapping to study arrhythmias, heart failure, and metabolic perturbations are summarized.

The origin of in situ hybridization - A personal history.

In situ hybridization is the technique by which specific RNA or DNA molecules are detected in cytological preparations. Basically it involves formation of a hybrid molecule between an endogenous single-stranded RNA or DNA in the cell and a complementary single-stranded RNA or DNA probe. In its original form the probe was labeled with (3)H and the hybrid was detected by autoradiography. The first successful experiments in 1968 involved detection of the highly amplified ribosomal DNA in oocytes of the frog Xenopus, followed soon after by the reiterated "satellite DNA" in mouse and Drosophila chromosomes. Fluorescent probes were developed about ten years later.

Analytical ancestry: "firsts" in fluorescent labeling of nucleosides, nucleotides, and nucleic acids.

BACKGROUND: The inherent fluorescent properties of nucleosides, nucleotides, and nucleic acids are limited, and thus the need has arisen for fluorescent labeling of these molecules for a variety of analytical applications. CONTENT: This review traces the analytical ancestry of fluorescent labeling of nucleosides, nucleotides, and nucleic acids, with an emphasis on the first to publish or patent. The scope of labeling includes (a) direct labeling by covalent labeling of nucleic acids with a fluorescent label or noncovalent binding or intercalation of a fluorescent dye to nucleic acids and (b) indirect labeling via covalent attachment of a secondary label to a nucleic acid, and then binding this to a fluorescently labeled ligand binder. An alternative indirect strategy involves binding of a nucleic acid to a nucleic acid binder molecule (e.g., antibody, antibiotic, histone, antibody, nuclease) that is labeled with a fluorophore. Fluorescent labels for nucleic acids include organic fluorescent dyes, metal chelates, carbon nanotubes, quantum dots, gold particles, and fluorescent minerals. SUMMARY: Fluorescently labeled nucleosides, nucleotides, and nucleic acids are important types of reagents for biological assay methods and underpin current methods of chromosome analysis, gel staining, DNA sequencing and quantitative PCR. Although these methods use predominantly organic fluorophores, new types of particulate fluorophores in the form of nanoparticles, nanorods, and nanotubes may provide the basis of a new generation of fluorescent labels and nucleic acid detection methods.

Extrinsic fluorescent dyes as tools for protein characterization.

Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization.

Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present.

Understanding the role of bacteria in microbial food webs is intimately connected to the methods applied in the direct enumeration of bacteria. We have examined over 220 papers describing studies in which fluorochrome staining followed by epifluorescent microscopic direct counts was used to estimate total bacterial abundances. In this review, we summarize patterns in the use of 3,6-bis[dimethylamino]acridinium chloride (acridine orange) and 4',6-diamidino-2-phenylindole (DAPI), the two stains most frequently used in bacterial enumeration. The staining of samples with these fluorochromes, followed by filtration and direct counting of bacterial cells on filter surfaces, has become routine over the past 10 years. We examine trends in features of the standard direct count methods, such as sample preservation and preparation techniques, membrane filter types used, applied stain concentrations, duration of staining, and counting strategies, in relation to the types of samples being examined. The high variability in bacterial counts observed within similar sample types may be partially accounted for by differences in methods. Synthesizing review findings, we include a recommended method for the direct enumeration of bacteria in environmental samples.

Fluorescent voltage-sensitive dyes: applications for neurophysiology.

Voltage-sensitive dyes are a means to optically monitor changes in membrane potential. Their application in research has grown steadily over the last two decades as better dyes have been developed. The techniques presently in use are providing unique information about biologic systems from bacteria to the functional organization of primate occipital cortex. This review provides a history of the dyes, the data supporting their voltage sensitivity, and the techniques required for their use. The limitations in using and interpreting the voltage-sensitive dyes, as well as their diverse applications in all areas of research, especially neurophysiology, are comprehensively presented.