Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and malachite green. When displayed on yeast or mammalian cell surfaces, these FAPs bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. Spectral variation can be generated by combining different FAPs and fluorogen derivatives. Visualization of FAPs on the cell surface or within the secretory apparatus of mammalian cells can be achieved by choosing membrane permeant or impermeant fluorogens. The FAP technique is extensible to a wide variety of nonfluorescent dyes.

A theoretical treatment has been developed for the optical properties of a layered structure which absorbs and scatters light. This theory predicts that the logarithm of the inverse of reflectance (LIR) of the surface should be a useful parameter for the examination of that structure. This approach has been applied to a study of skin in vivo. An instrument was constructed for use in clinical situations to measure the LIR spectrum of skin over the visible region of the spectrum (450-760 nm). The contributions to the observed spectra made by pigments and the skin structure were deduced by reference to the theoretical model. Numerical indices were used to quantify the changes in skin haemoglobin content following the application of vasoconstricting preparations. The indices also provided a means of measuring erythema and melanin pigmentation induced in the skin by exposure to ultraviolet radiation. The assessments made using this instrument were more reproducible and sensitive than judgments made by eye.

Abstract. Fluorescence ratio imaging microscopy (Tanasugarn, L., P. McNeil, G. Reynolds, and D. L. Taylor, 1984, J. Cell Biol., 98:717-724) has been used to measure the spatial variations in cytoplasmic pH of individual quiescent and nonquiescent Swiss 3T3 cells. Fundamental issues of ratio imaging that permit precise and accurate temporal and spatial measurements have been addressed including: excitation light levels, lamp operation, intracellular probe concentrations, methods of threshold selection, photobleaching, and spatial signal-to-noise ratio measurements. SubceUular measurements can be measured accurately (<3 % coefficient of variation) in an area of 3.65 lxm 2 with the present imaging system. Quiescent Swiss 3T3 cells have a measured cytoplasmic pH of 7.09 (0.01 SEM), whereas nonquiescent cells have a pH of 7.35 (0.01 SEM) in the presence of bicarbonate buffer. A unimodal distribution of mean cytoplasmic pH in both quiescent and nonquiescent cells was identified from populations of cells measured on a cell by cell basis. Therefore, unlike earlier studies based on cell population averages, it can be stated that cells in each population exhibit a narrow range of cytoplasmic pH. However, the mean cytoplasmic pH can change based on the physiological state of the cells. In addition, there appears to be little, if any, spatial variation in cytoplasmic pH in either quiescent or nonquiescent Swiss 3T3 cells. The pH within the nucleus was always the same as the surrounding cytoplasm. These values will serve as a reference point for investigating the role of temporal and spatial variations in cytoplasmic pH in a variety of cellular processes including growth control and cell movement.T hE cytoplasm of living cells is a highly organized and dynamic system of molecular interactions and chemical reactions (13,32,38,71,72). The precise spatial orchestration of these reactions leads to such cellular events as exocytosis, cell movement, cell division, chemotaxis, phagocytosis, and directed intracellular transport. Without some spatial component to the control and performance of these reactions, it is difficult to understand how such directed events could occur. Thus, an understanding of cell biology at the molecular level requires an understanding of how ceils control and perform this spatial orchestration.Quantitative fluorescence imaging microscopy has become a powerful tool in cell and molecular biology because it permits the measurement of both spatial and temporal dynamics of molecules and organelles in living cells (2,69,(70)(71)(72). The roots of this technology extend to the early use of microspectrophotometry on both living and fixed cells (9, 55). The superior sensitivity, specificity, and spectroscopic capabilities of fluorescence microscopy encouraged the development of advanced detection systems (6,14,31,53,59,63,73,83,85), fluorescent probes and biologicals (see 82), and physical optical methods (see 36). The developments in fluorescence imaging microscopy have been rapid in the last few years (1--4, 6...

Incorporation of a guanidine functional group into the PNA backbone facilitates cellular uptake of PNA into mammalian cells with efficiency comparable to that of the TAT transduction domain. The modified PNA recognizes and binds to the complementary DNA strand in accordance with Watson-Crick recognition rules. However, unlike polypyrimidine PNA which binds to DNA in 2:1 stoichiometry, the modified PNA binds to complementary DNA in a 1:1 ratio to form a highly stable duplex.