Current Pharmaceutical Biotechnology - Volume 5, Issue 2, 2004

Fluorescence Correlation Spectroscopy (FCS) has developed into a routine method to quantitatively study diffusion of molecules and kinetic processes. FCS has recently become popular to complement live cell imaging with biophysical information. The enabling technology has been commercially realised by combining laser scanning microscopes and fluorescence correlation spectrometers in one integrated platform. Read More

Fluorescence correlation spectroscopy (FCS) is an important technique for studying low concentrations of analyte molecules in solution. The core molecular characteristic that can be addressed by FCS is the translational diffusion coefficient of the analyte molecules, which can be used for i.e. studying molecular binding and reactions, or conformational changes of macromolecules. The present paper discusses several possible Read More

Many theoretical models of molecular interactions, biochemical and chemical reactions are described on the single-molecule level, although our knowledge about the biochemical/chemical structure and dynamics primarily originates from the investigation of many-molecule systems. At present, there are four experimental platforms to observe the movement and the behavior of single fluorescent molecules: wide-field e Read More

The selection of specific binding molecules like peptides and proteins from biolibraries using, for instance, phage display methods can be quite time-consuming. It is therefore desirable to develop a strategy that is much faster in selection and sorting of potential binders out of a biolibrary. In this contribution we separately discuss the current achievements in generation of biolibraries, single-molecule detection techniques and Read More

Compounds can be screened for pharmaceutical activity either by detecting interactions with specified target molecules such as receptors or enzymes (molecular screening) or observing effects on the structure or physiological activities of cells or tissues (phenotypic screening). Screening at the molecular level has been greatly enhanced by fluorescence methods. Especially the combination of confocal detection with meas Read More

The direct analysis of single biological molecules is getting increasingly important in basic as well as pharmaceutical research (e.g. for gene expression analysis). In particular single-molecule fluorescence detection provides exciting new opportunities to probe biochemical processes in unprecedented detail. Currently several academic and industrial research groups work on the development of single molecule detection Read More

FCS and FCCS measurements provide two important analytical parameters, the average number of molecules in the detection area and the translational diffusion constant of the molecules at the single molecule level. Considering these properties, FCS and FCCS have been applied to analysis of the cellular environment and dynamic processes of molecules in the living cell. More recently, a systematic approach for the Read More

Receptor binding studies most often require the use of radioactively labeled ligands. In certain cases, the numbers of receptors are few per cell and no specific binding is detected because of a high background. Specific interactions between certain ligands (e.g. peptides, hormones, natural products) and their receptors are, therefore, often overlooked by the conventional binding technique. Fluorescence correlation spectros Read More

Cell-to-cell communication is mediated by molecular interactions at the surface of the cell by soluble ligands released from distant cells or by cell surface molecules on adjacent cells. These interactions lead to activation of intracellular signaling pathways that subsequently can lead to activation of specific genes. This signal transduction process controls cellular activities as diverse as proliferation, differentiation and apo Read More

During the last years confocal techniques have become increasingly popular for probing biological enzymatic reactions. In this paper we will summarize some of these methods, mainly focusing on measurement techniques suitable for analysis of freely diffusing molecules, where either the substrates or the enzymes are fluorescently labeled. The different approaches are classified according to their basic principles and a Read More

The past few years we have witnessed a tremendous surge of interest in so-called array-based miniaturised analytical systems due to their value as extremely powerful tools for high-throughput sequence analysis, drug discovery and development, and diagnostic tests in medicine (see articles in Issue I). Terminologies that have been used to describe these array-based bioscience systems include (but are not limited to): DNA-chip, Read More