Iain Johnson

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

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 Förster 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.

Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP.

Green fluorescent protein (GFP) technology is rapidly advancing the study of morphogenesis, by allowing researchers to specifically focus on a subset of labeled cells within the living embryo. However, when imaging GFP‐labeled cells using confocal microscopy, it is often essential to simultaneously visualize all of the cells in the embryo using dual‐channel fluorescence to provide an embryological context for the cells expressing GFP. Although various counterstains are available, part of their fluorescence overlaps with the GFP emission spectra, making it difficult to clearly identify the cells expressing GFP. In this study, we report that a new fluorophore, BODIPY TR methyl ester dye, serves as a versatile vital counterstain for visualizing the cellular dynamics of morphogenesis within living GFP transgenic zebrafish embryos. The fluorescence of this photostable synthetic dye is spectrally separate from GFP fluorescence, allowing dual‐channel, three‐dimensional (3D) and four‐dimensional (4D) confocal image data sets of living specimens to be easily acquired. These image data sets can be rendered subsequently into uniquely informative 3D and 4D visualizations using computer‐assisted visualization software. We discuss a variety of immediate and potential applications of BODIPY TR methyl ester dye as a vital visualization counterstain for GFP in transgenic zebrafish embryos. Developmental Dynamics 232:359–368, 2005.

Practical Considerations in the Selection and Application of Fluorescent Probes

Due to its sensitivity, multiplexing capacity, and applicability to live specimens, fluorescence is the dominant contrast mechanism used in three-dimensional (3D) biological microscopy. Use of fluorescence detection generally requires specimens to be labeled with extrinsic probes. This is because most biological molecules and structures of interest are not intrinsically fluorescent in spectral ranges that are useful for detection, and even those that are cannot usually be discriminated from each other on the basis of their intrinsic fluorescence.

Detecting enzymes in living cells using fluorogenic substrates.

Characteristics of fluorogenic substrates designed for detection of enzyme activity in living cells are reviewed. Improved retention of the fluorescent products in the cell of origin can be achieved by structural modifications to the substrate that result in association with membrane lipids or conjugation to intracellular glutathione. Newly-developed substrates that yield fluorescent precipitates provide the additional advantage of allowing subcellular localization of sites of enzymatic activity. Improved detection sensitivity can also be achieved by targeted delivery of substrates for processing by specific organelles. Substrates designed for monitoring oxidative activity and lipid metabolism provide examples of this approach.

A Fluorescent High-Throughput Assay for Double-Stranded DNA: the RediPlate PicoGreen Assay

The fluorescent PicoGreen reagent for detection and quantitation of double-stranded DNA has been adapted for high-throughput screening: the RediPlate PicoGreen double-stranded DNA assay format. In the RediPlate PicoGreen assay format, the PicoGreen reagent is predistributed and co-dried into either 96- or 384-well microplates with the excipient trehalose. The user resuspends the dried reagents upon adding DNA, and measures the resulting fluorescence after a five minute incubation. Replicate fluorescence measurements on nominally identical wells have less than a 5% coefficient of variation. The assay is linear from 5 to 500 ng/ml DNA in a 200 micro l volume. The RediPlate PicoGreen assay format retains the advantages of the original PicoGreen reagent - sensitivity, speed, and specificity - but in a high-throughput format.

Multiphoton fluorescence spectroscopy of flourescent bioprobes and biomolecules

Multi-photon fluorescence spectra of a number of commonly used biological probes were measured in this study. Significant spectral variation has been detected between single and multi- photon excitation. The result is important for the proper selection of spectral setting/dichroic beam splitter in the set- up of a multi-photon fluorescence microscope. The information can also be useful in the detection of multi-photon fluorescence in bio-chip technology. In addition, we have investigated a few highly fluorescent bio-molecules commonly found in plant cells.

Cellular Function Probes

This unit covers many of the fluorescent probes used in cell biology, such as green fluorescent protein, esterase and peptidase substrates, intracellular thiols, lipid probes, and probes for intracellular ions. It brings to light the chemical relationships between certain probes and relates these properties to their functional application. Correct use of these probes requires an understanding of how they are taken up and distributed within the cell and where appropriate, what factors will affect the fluorescence emission from the probe. This is particularly important for probes of pH and calcium flux.