Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first expansion microscopy method was unable to retain native proteins in the gel and used custom made reagents not widely available. Here, we describe protein retention ExM (proExM), a variant of ExM that anchors proteins to the swellable gel allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validate and demonstrate utility of proExM for multi-color super-resolution (~70 nm) imaging of cells and mammalian tissues on conventional microscopes.

The ability of non-invasive monitoring of deep-tissue developmental, metabolic, and pathogenic processes will advance modern biotechnology. Imaging of live mammals using fluorescent probes is more feasible within a “near-infrared optical window” (NIRW)1. Here we report a phytochrome-based near infra-red fluorescent protein (iRFP) with the excitation/emission maxima at 690/713 nm. Bright fluorescence in a living mouse proved iRFP to be a superior probe for non-invasive imaging of internal mammalian tissues. Its high intracellular stability, low cytotoxicity, and lack of the requirement to add external biliverdin-chromophore makes iRFP as easy to use as conventional GFP-like proteins. Compared to earlier phytochrome-derived fluorescent probes, the iRFP protein has better in vitro characteristics and performs well in cells and in vivo, having greater effective brightness and photostability. Compared to the far-red GFP-like proteins, iRFP has substantially higher signal to background ratio in a mouse model owing to its infra-red shifted spectra.

We here present a new way to engineer complex proteins toward multidimensional specifications, through a simple yet scalable directed evolution strategy. By robotically picking mammalian cells that are identified, under a microscope, to express proteins that simultaneously exhibit several specific properties, we can screen through hundreds of thousands of proteins in a library in a matter of a few hours, evaluating each along multiple performance axes. We demonstrate the power of this approach by identifying a novel genetically encoded fluorescent voltage indicator, simultaneously optimizing brightness and membrane localization of the protein using our microscopy-guided cell picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1, and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices as well as in larval zebrafish in vivo. We also demonstrate measurement of postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.

Optogenetic control of individual neurons with high temporal precision, within intact mammalian brain circuitry, would enable powerful explorations of how neural circuits operate. Two-photon computer generated holography enables precise sculpting of light, and could in principle enable simultaneous illumination of many neurons in a network, with the requisite temporal precision to simulate accurate neural codes. We designed a high efficacy soma-targeted opsin, finding that fusing the N-terminal 150 residues of kainate receptor subunit 2 (KA2) to the recently discovered high-photocurrent channelrhodopsin CoChR restricted expression of this opsin primarily to the cell body of mammalian cortical neurons. In combination with two-photon holographic stimulation, we found that this somatic CoChR (soCoChR) enabled photostimulation of individual cells in intact cortical circuits with single cell resolution and <1 millisecond temporal precision, and use soCoChR to perform connectivity mapping on intact cortical circuits.

Based on the mechanism for chromophore formation in red fluorescent proteins, we developed three mCherry-derived monomeric variants, called fluorescent timers (FTs), that change their fluorescence from the blue to red over time. These variants exhibit distinctive fast, medium and slow blue-to-red chromophore maturation rates that depend on the temperature. At 37 °C, the maxima of the blue fluorescence are observed at 0.25, 1.2 and 9.8 h for the purified fast-FT, medium-FT and slow-FT, respectively. The half-maxima of the red fluorescence are reached at 7.1, 3.9 and 28 h, respectively. The FTs show similar timing behavior in bacteria, insect and mammalian cells. Medium-FT allowed for tracking of the intracellular dynamics of the lysosome-associated membrane protein type 2A (LAMP-2A) and determination of its age in the targeted compartments. The results indicate that LAMP-2A transport through the plasma membrane and early or recycling endosomes to lysosomes is a major pathway for LAMP-2A trafficking.Monomeric fluorescent proteins of various emission wavelengths have become invaluable tools for studying the spatial behavior of intracellular molecules, including their localization and interaction 1 . To visualize temporal and spatial molecular events, FTs 2 , which change their emission wavelengths over time, could be especially valuable. The only currently available FT is DsRed-Timer FT (also known as DsRed-E5) 3 ; however, it is a tetramer, which prevents its application as a protein fusion tag. Nevertheless, the tetrameric state of the DsRed-Timer does not limit its use to study gene activities 4 , relative age of organelles 5 and cell differentiation 3 .It has been suggested that a red DsRed-like chromophore in the red fluorescent proteins (RFPs) results from an oxidation of a protonated blue form of the GFP-like chromophore, not from the green anionic form, which is a dead-end product 6 . This suggested scheme for red chromophore maturation provides a basis for developing monomeric FTs that change their color from blue to red. The most suitable templates for this appear to be the monomeric variants of DsRed 7 . One of these variants, mCherry, was chosen for a directed molecular evolution to develop three monomeric FTs with different maturation rates between the protonated blue GFP-like and the anionic red DsRed-like chromophore states.FTs can be used as molecular genetically encoded tools to study trafficking of different cellular proteins and to provide accurate insight into the timing of intracellular processes. The sequence NIH Public Access Author ManuscriptNat Chem Biol. Author manuscript; available in PMC 2010 February 1. Published in final edited form as:Nat Chem Biol. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript of events during trafficking of different cellular proteins before they reach their final compartment has often been the subject of contradictory investigations. An example of a longstanding dilemma is the contribution of different pathways to trafficking a...

A longstanding goal in neuroscience has been to image membrane voltage across a population of individual neurons in an awake, behaving mammal. Here, we report a genetically encoded fluorescent voltage indicator, SomArchon, which exhibits millisecond response times and compatibility with optogenetic control, and which increases the sensitivity, signal-to-noise ratio, Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Two-photon microscopy has advanced fluorescence imaging of cellular processes in living animals. Fluorescent proteins in the blue-green wavelength range are widely used in two-photon microscopy; however, the use of red fluorescent proteins is limited by the low power output of Ti-Sapphire lasers above 1,000 nm. To overcome this limitation we have developed two red fluorescent proteins, LSS-mKate1 and LSS-mKate2, which possess large Stokes shifts with excitation/emission maxima at 463∕624 and 460∕605 nm, respectively. These LSS-mKates are characterized by high pH stability, photostability, rapid chromophore maturation, and monomeric behavior. They lack absorbance in the green region, providing an additional red color to the commonly used red fluorescent proteins. Substantial overlap between the two-photon excitation spectra of the LSS-mKates and blue-green fluorophores enables multicolor imaging using a single laser. We applied this approach to a mouse xenograft model of breast cancer to intravitally study the motility and Golgi-nucleus alignment of tumor cells as a function of their distance from blood vessels. Our data indicate that within 40 μm the breast cancer cells show significant polarization towards vessels in living mice.cell polarity | intravital imaging | Keima | two-photon microscopy | mKate

We report an intensiometric, near-infrared (NIR) fluorescent, genetically encoded calcium ion (Ca 2+ ) indicator (GECI) with excitation and emission maxima at 678 nm and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca 2+ transients in cultured mammalian cells and brain tissue with sensitivity comparable to currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca 2+ imaging in combination with other optogenetic indicators and actuators.