A5052613436- Advanced Fluorescence Microscopy Techniques
- Click Chemistry and Applications
- Genomics and Chromatin Dynamics
- Advanced biosensing and bioanalysis techniques
- RNA and protein synthesis mechanisms
- RNA Research and Splicing
- Cell Image Analysis Techniques
- Photoreceptor and optogenetics research
- Single-cell and spatial transcriptomics
- Synthetic Organic Chemistry Methods
- Neural dynamics and brain function
- Luminescence and Fluorescent Materials
- Photochromic and Fluorescence Chemistry
- Advanced Electron Microscopy Techniques and Applications
- Neuroscience and Neuropharmacology Research
- Receptor Mechanisms and Signaling
- Diffusion and Search Dynamics
- Chemical Synthesis and Analysis
- Advanced Biosensing Techniques and Applications
- Nanoplatforms for cancer theranostics
- Cellular transport and secretion
- Molecular Sensors and Ion Detection
- Particle Dynamics in Fluid Flows
- T-cell and B-cell Immunology
- Photodynamic Therapy Research Studies
Howard Hughes Medical Institute
2016-2025
Janelia Research Campus
2016-2025
Helix (United States)
2011-2024
MRC Laboratory of Molecular Biology
2018
Medical Research Council
2018
Merck & Co., Inc., Rahway, NJ, USA (United States)
2007-2009
University of Chicago
2008
University of Wisconsin–Madison
2004-2007
University of Virginia
2003
Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility existing GEVIs has been limited by brightness photostability fluorescent proteins rhodopsins. We engineered a GEVI, called Voltron, that uses bright photostable synthetic dyes instead protein-based fluorophores, thereby extending number neurons imaged simultaneously in vivo factor 10 enabling imaging for significantly longer durations relative to...
Although messenger RNA (mRNA) translation is a fundamental biological process, it has never been imaged in real time vivo with single-molecule precision. To achieve this, we developed nascent chain tracking (NCT), technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify protein synthesis dynamics at the single-mRNA level. NCT reveals an elongation rate of ~10 amino acids per second, initiation occurring stochastically every ~30 seconds. Polysomes contain ~1...
Single-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due biases both data analysis experimental design. To address bias, we introduce 'Spot-On', intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving...
Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate with functional consequences vivo. We developed live-cell super-resolution approach uncover correlation between mRNA synthesis and dynamics RNA Polymerase II (Pol II) clusters at gene locus. For endogenous β-actin genes mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol basal output. During serum stimulation, stereotyped...
Combinatorial cis-regulatory networks encoded in animal genomes represent the foundational gene expression mechanism for directing cell-fate commitment and maintenance of cell identity by transcription factors (TFs). However, 3D spatial organization cis-elements how such sub-nuclear structures influence TF activity remain poorly understood. Here, we combine lattice light-sheet imaging, single-molecule tracking, numerical simulations, ChIP-exo mapping to localize functionally probe Sox2...
Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and nonfluorescent lactone. Tuning this toward the lactone form can improve cell-permeability allow creation of "fluorogenic" compounds-ligands that shift to upon binding biomolecular target. An archetype fluorogenic dye is far-red tetramethyl-Si-rhodamine (SiR), which has been used create exceptionally useful labels for advanced microscopy. Here, we develop quantitative framework development new dyes, determining...
Fluorogenic molecules are important tools for advanced biochemical and biological experiments. The extant collection of fluorogenic probes is incomplete, however, leaving regions the electromagnetic spectrum unutilized. Here, we synthesize green-excited fluorescent analogues classic fluorescein rhodamine 110 fluorophores by replacement xanthene oxygen with a quaternary carbon. These anthracenyl "carbofluorescein" "carborhodamine 110" exhibit excellent properties can be masked enzyme-...
Fluorescence microscopy relies on dyes that absorb and then emit photons. In addition to fluorescence, fluorophores can undergo photochemical processes decrease quantum yield or result in spectral shifts irreversible photobleaching. Chemical strategies suppress these undesirable pathways—thereby increasing the brightness photostability of fluorophores—are crucial for advancing frontier bioimaging. Here, we describe a general method improve small-molecule by incorporating deuterium into...
Significance Local translation in dendrites of neurons has been shown to be important for neuronal function and synaptic biology. We imaged changes the localization β-actin mRNA protein dendritic spines. Our results showed that activating specific synapses can drive endogenous reporter RNA hippocampal neurons. Enhancing our understanding spatial temporal kinetics informs local synthesis These provide direct evidence away from soma allow us determine how could influence physiology plasticity.
mRNA translation is a key step in decoding genetic information. Genetic surprisingly heterogeneous because multiple distinct polypeptides can be synthesized from single sequence. To study translational heterogeneity, we developed the MoonTag, fluorescence labeling system to visualize of mRNAs. When combined with orthogonal SunTag system, MoonTag enables dual readouts translation, greatly expanding possibilities interrogate complex heterogeneity. By placing and sequences different reading...
Rhodamine dyes are excellent scaffolds for developing a broad range of fluorescent probes. A key property rhodamines is their equilibrium between colorless lactone and zwitterion. Tuning the lactone–zwitterion constant (KL–Z) can optimize dye properties specific biological applications. Here, we use known novel organic chemistry to prepare comprehensive collection rhodamine elucidate structure–activity relationships that govern KL–Z. We discovered auxochrome substituent strongly affects...
The century-old fluoresceins and rhodamines persist as flexible scaffolds for fluorescent fluorogenic compounds. Extensive exploration of these xanthene dyes has yielded general structure-activity relationships where the development new probes is limited only by imagination organic chemistry. In particular, replacement oxygen with silicon resulted in red-shifted Si-fluoresceins Si-rhodamines, whose high brightness photostability enable advanced imaging experiments. Nevertheless, efforts to...
Abstract The rhodamine system is a flexible framework for building small‐molecule fluorescent probes. Changing N ‐substitution patterns and replacing the xanthene oxygen with dimethylsilicon moiety can shift absorption fluorescence emission maxima of dyes to longer wavelengths. Acylation nitrogen atoms forces molecule adopt nonfluorescent lactone form, providing convenient method make fluorogenic compounds. Herein, we take advantage all these structural manipulations describe novel...
We demonstrate stimulated emission depletion (STED) microscopy of whole bacterial and eukaryotic cells using fluorogenic labels that reversibly bind to their target structure. A constant exchange guarantees the removal photobleached fluorophores replacement by intact fluorophores, thereby circumventing bleaching-related limitations STED super-resolution imaging. achieve a labeling density fluorescence signal for long theoretically unlimited acquisition times. Using this concept, we...
Abstract Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known novel 1,2,4,5-tetrazine-dye conjugates spanning entire visible wavelength range. A hallmark this is its fluorogenicity - tetrazine moiety can elicit substantial quenching dye. We discovered that photoinduced electron transfer (PET) from...
Despite the apparent simplicity of xanthene fluorophores, preparation caged derivatives with free carboxy groups remains a synthetic challenge. A straightforward and flexible strategy for preparing rhodamine fluorescein was developed using reduced, "leuco" intermediates.