Michael R. McKeown

Bio: Michael R. McKeown is an academic researcher from University of California, San Diego. The author has contributed to research in topics: Förster resonance energy transfer & Aequorea victoria. The author has an hindex of 5, co-authored 6 publications receiving 1853 citations. Previous affiliations of Michael R. McKeown include University of California.

Improving the photostability of bright monomeric orange and red fluorescent proteins.

Abstract: All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

Improving FRET dynamic range with bright green and red fluorescent proteins

Abstract: A variety of genetically encoded reporters use changes in fluorescence (or Forster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Forster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.

Evaluating and improving the photostability of fluorescent proteins

Abstract: Fluorescent proteins are the most common and versatile class of genetically encoded optical probes. While structure-guided rational design and directed evolution approaches have largely overcome early problems such as oligomerization, poor folding at physiological temperatures, and availability of wavelengths suitable for multi-color imaging, nearly all fluorescent proteins have yet to be fully optimized. We have developed novel methods for evaluating the current generation of fluorescent proteins and improving their remaining suboptimal properties. Little is yet known about the mechanisms responsible for photobleaching of fluorescent proteins, and inadequate photostability is a chief complaint among end users. In order to compare the performance of fluorescent proteins across the visual spectrum, we have standardized a method used to measure photostability in live cells under both widefield and confocal laser illumination. This method has allowed us to evaluate a large number of commonly used fluorescent proteins, and has uncovered surprisingly complex and unpredictable behaviors in many of these proteins. We have also developed novel methods for selecting explicitly for high photostability during the directed evolution process, leading to the development of highly improved monomeric orange and red fluorescent proteins. These proteins, most notably our photostable derivative of TagRFP, have remarkably high photostability and have proven useful as fusion tags for long-term imaging. Our methods should be applicable to any of the large number of fluorescent proteins still in need of improved photostability.

Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues

Abstract: Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

An Expanded Palette of Genetically Encoded Ca2+ Indicators

Abstract: Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca2+) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca2+ indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca2+ imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca2+ was imaged in three subcellular compartments, and, in conjunction with a cyan FP–yellow FP–based indicator, Ca2+ and adenosine 5′-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca2+ imaging.

A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum.

Abstract: We report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest monomeric green or yellow fluorescent protein yet described to our knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.

Direct stochastic optical reconstruction microscopy with standard fluorescent probes

Abstract: Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ∼20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.