No abstract

The advent of fluorescent proteins (FP) for genetic labeling of molecules and cells has revolutionized fluorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning the blue to red spectral regions. Common to autofluorescent FPs is their tight β-barrel structure, which provides the rigidity and chemical environment needed for effectual fluorescence. Despite the common structure, each FP has its own unique photophysical properties. Thus, there is no single “best” fluorescent protein for every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is right for any given application, we have characterized quantitatively over 40 different FPs for their brightness, photostability, pH stability, and monomeric properties, which permits easy apples-to-apples comparisons between these FPs. We report the values for all of the FPs measured, but focus the discussion on the more popular and/or best performing FPs in each spectral region.

Single gold-tagged epidermal growth factor (EGF) molecules bound to cellular EGF receptors of fixed fibroblast cells were imaged in liquid with a scanning transmission electron microscope (STEM). The cells were placed in buffer solution in a microfluidic device with electron transparent windows inside the vacuum of the electron microscope. A spatial resolution of 4 nm and a pixel dwell time of 20 s were obtained. The liquid layer was sufficiently thick to contain the cells with a thickness of 7 ؎ 1 m. The experimental findings are consistent with a theoretical calculation. Liquid STEM is a unique approach for imaging single molecules in whole cells with significantly improved resolution and imaging speed over existing methods. cellular imaging ͉ molecular labels U nderstanding cellular function at a molecular level requires imaging techniques capable of imaging whole cells with a resolution sufficient to image individually tagged proteins. Electron microscopy and X-ray diffraction are traditionally used to resolve the structures of individual proteins and to image proteins distributions in cells (1). Imaging with these techniques demands extensive sample preparation to obtain, e.g., proteins crystals, stained thin sections, or frozen samples. The cells are thus not in their native liquid state. Light microscopy is used to image protein distributions via fluorescent labels on fixed cells in liquid and in live cells to investigate cellular function (2). Superresolution techniques surpass the diffraction limit in optical microscopy (3-6), but despite recent advances, these methods are still restricted to spatial resolutions Ͼ10-20 nm. Further, their optimal performance requires extended imaging times, and significant data postprocessing. The speed can only be increased at the cost of resolution.Here, we describe a direct technique for imaging whole cells in liquid that offers nanometer spatial resolution and a high imaging speed. The principle is explained in Fig. 1. The eukaryotic cells in liquid are placed in a microfluidic flow cell with a thickness of up to 10 m contained between 2 ultrathin electron transparent windows. This flow cell is placed in the vacuum of a STEM, using a fluid specimen holder. The annular dark field (ADF) detector in the STEM is sensitive to scattered electrons, which are generated in proportion to the atomic number (Z) of the atoms in the specimen (7, 8), so-called Z contrast, where the contrast varies with ϷZ 2 . It is thus possible to image specific high-Z atoms, such as gold, inside a thick (several micrometer) layer of low-Z material, such as water, protein, or the embedding medium of a thin section (9). We used this approach to raster image single gold-tagged epidermal growth factor (EGF) molecules bound to cellular EGF receptors on fibroblast cells with a spatial resolution of 4 nm and a pixel dwell time of 20 s. ResultsCOS7 fibroblast cells were labeled with 10-nm gold nanoparticles conjugated with epidermal growth factor (EGF-Au). The cells were grown, labeled, and fixed directl...

Enhanced cyan and yellow fluorescent proteins are widely used for dual color imaging and protein-protein interaction studies based on fluorescence resonance energy transfer. Use of these fluorescent proteins can be limited by their thermosensitivity, dim fluorescence, and tendency for aggregation. Here we report the results of a site-directed mutagenesis approach to improve these fluorescent proteins. We created monomeric optimized variants of ECFP and EYFP, which fold faster and more efficiently at 37 degrees C and have superior solubility and brightness. Bacteria expressing SCFP3A were 9-fold brighter than those expressing ECFP and 1.2-fold brighter than bacteria expressing Cerulean. SCFP3A has an increased quantum yield (0.56) and fluorescence lifetime. Bacteria expressing SYFP2 were 12 times brighter than those expressing EYFP(Q69K) and almost 2-fold brighter than bacteria expressing Venus. In HeLa cells, the improvements were less pronounced; nonetheless, cells expressing SCFP3A and SYFP2 were both 1.5-fold brighter than cells expressing ECFP and EYFP(Q69K), respectively. The enhancements of SCFP3A and SYFP2 are most probably due to an increased intrinsic brightness (1.7-fold and 1.3-fold for purified recombinant proteins, compared to ECFP & EYFP(Q69K), respectively) and due to enhanced protein folding and maturation. The latter enhancements most significantly contribute to the increased fluorescent yield in bacteria whereas they appear less significant for mammalian cell systems. SCFP3A and SYFP2 make a superior donor-acceptor pair for fluorescence resonance energy transfer, because of the high quantum yield and increased lifetime of SCFP3A and the high extinction coefficient of SYFP2. Furthermore, SCFP1, a CFP variant with a short fluorescence lifetime but identical spectra compared to ECFP and SCFP3A, was characterized. Using the large lifetime difference between SCFP1 and SCFP3A enabled us to perform for the first time dual-lifetime imaging of spectrally identical fluorescent species in living cells.