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Carnegie Mellon University
Aleksandra Klimas and Yongxin Zhao
ACS Nano, ISSN: 19360851, eISSN: 1936086X, Pages: 7689-7695, Published: 28 July 2020 American Chemical Society (ACS)
Expansion microscopy (ExM) has become a powerful imaging tool for visualizing the nanoscale organization of protein and nucleic acid targets in cells and tissues using only a conventional microscope. Until recently, current ExM approaches have had limited applicability to imaging other biomolecules, such as lipids and small molecules. With the new TRITON probes reported by Wen et al. in this issue of ACS Nano, ExM can now be used to perform nanoscale imaging of the cytoskeleton and lipid membranes. In this Perspective, we offer a brief overview of recent developments in ExM, with a focus on biomolecule anchoring and labeling strategies that target a wide range of biomolecules to the water-swellable polymer formed in situ, a key step that ensures biomolecules or labels of interest are separated in space and can be resolved on a conventional microscope. In addition to these new advancements, we discuss challenges and future directions in this exciting field.
Octavian Bucur, Feifei Fu, Mike Calderon, Geetha H. Mylvaganam, Ngoc L. Ly, Jimmy Day, Simon Watkin, Bruce D. Walker, Edward S. Boyden, and Yongxin Zhao
Nature Protocols, ISSN: 17542189, eISSN: 17502799, Pages: 1649-1672, Published: 1 May 2020 Springer Science and Business Media LLC
In pathology, microscopy is an important tool for the analysis of human tissues, both for the scientific study of disease states and for diagnosis. However, the microscopes commonly used in pathology are limited in resolution by diffraction. Recently, we discovered that it was possible, through a chemical process, to isotropically expand preserved cells and tissues by 4–5× in linear dimension. We call this process expansion microscopy (ExM). ExM enables nanoscale resolution imaging on conventional microscopes. Here we describe protocols for the simple and effective physical expansion of a variety of human tissues and clinical specimens, including paraffin-embedded, fresh frozen and chemically stained human tissues. These protocols require only inexpensive, commercially available reagents and hardware commonly found in a routine pathology laboratory. Our protocols are written for researchers and pathologists experienced in conventional fluorescence microscopy. The conventional protocol, expansion pathology, can be completed in ~1 d with immunostained tissue sections and 2 d with unstained specimens. We also include a new, fast variant, rapid expansion pathology, that can be performed on <5-µm-thick tissue sections, taking <4 h with immunostained tissue sections and <8 h with unstained specimens. Paraffin-embedded, fresh-frozen or chemically stained fixed human tissues are isotropically expanded by 4–5× in linear dimension to enable nanoscale-resolution imaging on conventional microscopes.
Aleksandra Klimas, Brendan Gallagher, and Yongxin Zhao
Current Protocols in Cytometry, ISSN: 19349297, eISSN: 19349300, Published: 1 December 2019 Wiley
Optical imaging techniques are often used in neuroscience to understand brain function and discern disease pathogenesis. However, the optical diffraction limit precludes conventional optical imaging approaches from resolving nanoscopic structures with feature sizes smaller than 300 nm. Expansion microscopy (ExM) circumvents this limit by physically expanding preserved tissues embedded in a swellable hydrogel. Biomolecules of interest are covalently linked to a polymer matrix, which is then isotropically expanded at least 100‐fold in size in pure water after mechanical homogenization of the tissue‐gel. The sample can then be investigated with nanoscale precision using a conventional diffraction‐limited microscope. The protocol described here is a variant of ExM that uses regents and equipment found in a typical biology laboratory and has been optimized for imaging proteins in expanded brain tissues. © 2019 by John Wiley & Sons, Inc.
Yufeng Zhao, Wei Zhang, Yongxin Zhao, Robert E. Campbell, and D. Jed Harrison
Lab on a Chip, ISSN: 14730197, eISSN: 14730189, Pages: 3880-3887, Published: 21 November 2019 Royal Society of Chemistry (RSC)
We introduce a single-phase flow microfluidic cell sorter with a two-point detection system capable of two-parameter screening to assist with directed evolution of a fluorescent protein based Ca2+ sensor expressed in bacterial cells.
Yoav Adam, Jeong J. Kim, Shan Lou, Yongxin Zhao, Michael E. Xie, Daan Brinks, Hao Wu, Mohammed A. Mostajo-Radji, Simon Kheifets, Vicente Parot, Selmaan Chettih, Katherine J. Williams, Benjamin Gmeiner, Samouil L. Farhi, Linda Madisen, E. Kelly Buchanan, Ian Kinsella, Ding Zhou, Liam Paninski, Christopher D. Harvey, Hongkui Zeng, Paola Arlotta, Robert E. Campbell, and Adam E. Cohen
Nature, ISSN: 00280836, eISSN: 14764687, Volume: 569, Issue: 7756, Pages: 413-417, Published: 16 May 2019 Springer Science and Business Media LLC
A technology that simultaneously records membrane potential from multiple neurons in behaving animals will have a transformative effect on neuroscience research1,2. Genetically encoded voltage indicators are a promising tool for these purposes; however, these have so far been limited to single-cell recordings with a marginal signal-to-noise ratio in vivo3–5. Here we developed improved near-infrared voltage indicators, high-speed microscopes and targeted gene expression schemes that enabled simultaneous in vivo recordings of supra- and subthreshold voltage dynamics in multiple neurons in the hippocampus of behaving mice. The reporters revealed subcellular details of back-propagating action potentials and correlations in subthreshold voltage between multiple cells. In combination with stimulation using optogenetics, the reporters revealed changes in neuronal excitability that were dependent on the behavioural state, reflecting the interplay of excitatory and inhibitory synaptic inputs. These tools open the possibility for detailed explorations of network dynamics in the context of behaviour.Fig. 1Photoactivated QuasAr3 (paQuasAr3) reports neuronal activity in vivo.a, Schematic of the paQuasAr3 construct. b, Photoactivation by blue light enhanced voltage signals excited by red light in cultured neurons that expressed paQuasAr3 (representative example of n = 4 cells). c, Model of the photocycle of paQuasAr3. d, Confocal images of sparsely expressed paQuasAr3 in brain slices. Scale bars, 50 μm. Representative images, experiments were repeated in n = 3 mice. e, Simultaneous fluorescence and patch-clamp recordings from a neuron expressing paQuasAr3 in acute brain slice. Top, magnification of boxed regions. Schematic shows brain slice, patch pipette and microscope objective. f, Simultaneous fluorescence and patch-clamp recordings of inhibitory post synaptic potentials in an L2–3 neuron induced by electrical stimulation of L5–6 in acute slice. g, Normalized change in fluorescence (ΔF/F) and SNR of optically recorded post-synaptic potentials (PSPs) as a function of the amplitude of the post-synaptic potentials. The voltage sensitivity was ΔF/F = 40 ± 1.7% per 100 mV. The SNR was 0.93 ± 0.07 per 1 mV in a 1-kHz bandwidth (n = 42 post-synaptic potentials from 5 cells, data are mean ± s.d.). Schematic shows brain slice, patch pipette, field stimulation electrodes and microscope objective. h, Optical measurements of paQuasAr3 fluorescence in the CA1 region of the hippocampus (top) and glomerular layer of the olfactory bulb (bottom) of anaesthetized mice (representative traces from n = 7 CA1 cells and n = 13 olfactory bulb cells, n = 3 mice). Schematics show microscope objective and the imaged brain region. i, STA fluorescence from 88 spikes in a CA1 oriens neuron. j, Frames from the STA video showing the delay in the back-propagating action potential in the dendrites relative to the soma. k, Sub-Nyquist fitting of the action potential delay and width shows electrical compartmentalization in the dendrites. Experiments in k–m were repeated in n = 2 cells from n = 2 mice.A combination of improved near-infrared voltage indicators, high-speed microscopes and targeted gene expression schemes enabled simultaneous in vivo optogenetic control and recording of voltage dynamics in multiple neurons in the hippocampus of behaving mice.
Ruixuan Gao, Shoh M. Asano, Srigokul Upadhyayula, Igor Pisarev, Daniel E. Milkie, Tsung-Li Liu, Ved Singh, Austin Graves, Grace H. Huynh, Yongxin Zhao, John Bogovic, Jennifer Colonell, Carolyn M. Ott, Christopher Zugates, Susan Tappan, Alfredo Rodriguez, Kishore R. Mosaliganti, Shu-Hsien Sheu, H. Amalia Pasolli, Song Pang, C. Shan Xu, Sean G. Megason, Harald Hess, Jennifer Lippincott-Schwartz, Adam Hantman, Gerald M. Rubin, Tom Kirchhausen, Stephan Saalfeld, Yoshinori Aso, Edward S. Boyden, and Eric Betzig
Science, ISSN: 00368075, eISSN: 10959203, Volume: 363, Issue: 6424, Published: 18 January 2019 American Association for the Advancement of Science (AAAS)
Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entireDrosophilabrain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large-scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.
Asmamaw T. Wassie, Yongxin Zhao, and Edward S. Boyden
Nature Methods, ISSN: 15487091, eISSN: 15487105, Pages: 33-41, Published: 1 January 2019 Springer Science and Business Media LLC
Many biological investigations require 3D imaging of cells or tissues with nanoscale spatial resolution. We recently discovered that preserved biological specimens can be physically expanded in an isotropic fashion through a chemical process. Expansion microscopy (ExM) allows nanoscale imaging of biological specimens with conventional microscopes, decrowds biomolecules in support of signal amplification and multiplexed readout chemistries, and makes specimens transparent. We review the principles of how ExM works, advances in the technology made by our group and others, and its applications throughout biology and medicine.Expansion microscopy allows super-resolution images of diverse samples to be acquired on conventional microscopes, thus democratizing super-resolution imaging. This Perspective reviews available methods and provides practical guidance for users.
Aleksandra Klimas, Octavian Bucur, Brigdet Njeri, and Yongxin Zhao
Journal of Visualized Experiments, ISSN: 1940087X, Volume: 2019, Issue: 151, Published: 2019 MyJove Corporation
In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, the fundamental physical diffraction limit prevents interrogation of nanoscale anatomy and subtle pathological changes when using conventional optical imaging approaches. Here, we describe a simple and inexpensive protocol, called expansion pathology (ExPath), for nanoscale optical imaging of common types of clinical primary tissue specimens, including both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. This method circumvents the optical diffraction limit by chemically transforming the tissue samples into tissue-hydrogel hybrid and physically expanding them isotropically across multiple scales in pure water. Due to expansion, previously unresolvable molecules are separated and thus can be observed using a conventional optical microscope.
Yufeng Zhao, Daniel Bushey, Yongxin Zhao, Eric R. Schreiter, D. Jed Harrison, Allan M. Wong, and Robert E. Campbell
Scientific Reports, eISSN: 20452322, Published: 1 December 2018 Springer Science and Business Media LLC
We have developed a series of yellow genetically encoded Ca2+ indicators for optical imaging (Y-GECOs) with inverted responses to Ca2+ and apparent dissociation constants (Kd′) ranging from 25 to 2400 nM. To demonstrate the utility of this affinity series of Ca2+ indicators, we expressed the four highest affinity variants (Kd′s = 25, 63, 121, and 190 nM) in the Drosophila medulla intrinsic neuron Mi1. Hyperpolarization of Mi1 by optogenetic stimulation of the laminar monopolar neuron L1 produced a decrease in intracellular Ca2+ in layers 8–10, and a corresponding increase in Y-GECO fluorescence. These experiments revealed that lower Kd′ was associated with greater increases in fluorescence, but longer delays to reach the maximum signal change due to slower off-rate kinetics.
Octavian Bucur and Yongxin Zhao
Frontiers in Medicine, eISSN: 2296858X, Issue: NOV, Published: 2018 Frontiers Media SA
Kidney glomerular diseases, such as the minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), and other nephrotic syndromes, are typically diagnosed or confirmed via electron microscopy. Although optical microscopy has been a vital tool to examine clinical specimens for diagnoses in pathology for decades, the optical resolution is constricted by the physical diffraction limit of the optical microscope, which prevents high-resolution investigation of subcellular anatomy, such as of the podocyte tertiary foot processes. Here, we describe a simple, fast, and inexpensive protocol for nanoscale optical imaging of kidney glomeruli. The protocol is based on Expansion Pathology (ExPath), a new principle of microscopy that overcomes optical diffraction limit by chemically embedding specimens into a swellable polymer and physically expanding it homogenously prior to imaging. Our method uses only commercially available reagents, a conventional fluorescence microscope and it can be applied to both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. It requires minimal operative experience in a wet lab, optical microscopy and imaging processing. Finally, we also discuss challenges, limitations and prospective applications for ExPath-based imaging of glomeruli.
Yongxin Zhao, Octavian Bucur, Humayun Irshad, Fei Chen, Astrid Weins, Andreea L Stancu, Eun-Young Oh, Marcello DiStasio, Vanda Torous, Benjamin Glass, Isaac E Stillman, Stuart J Schnitt, Andrew H Beck, and Edward S Boyden
Nature Biotechnology, ISSN: 10870156, eISSN: 15461696, Pages: 757-764, Published: 1 August 2017 Springer Science and Business Media LLC
Expansion microscopy (ExM), a method for improving the resolution of light microscopy by physically expanding a specimen, has not been applied to clinical tissue samples. Here we report a clinically optimized form of ExM that supports nanoscale imaging of human tissue specimens that have been fixed with formalin, embedded in paraffin, stained with hematoxylin and eosin, and/or fresh frozen. The method, which we call expansion pathology (ExPath), converts clinical samples into an ExM-compatible state, then applies an ExM protocol with protein anchoring and mechanical homogenization steps optimized for clinical samples. ExPath enables ∼70-nm-resolution imaging of diverse biomolecules in intact tissues using conventional diffraction-limited microscopes and standard antibody and fluorescent DNA in situ hybridization reagents. We use ExPath for optical diagnosis of kidney minimal-change disease, a process that previously required electron microscopy, and we demonstrate high-fidelity computational discrimination between early breast neoplastic lesions for which pathologists often disagree in classification. ExPath may enable the routine use of nanoscale imaging in pathology and clinical research.
Longteng Tang, Yanli Wang, Weimin Liu, Yongxin Zhao, Robert E. Campbell, and Chong Fang
Journal of Physical Chemistry B, ISSN: 15206106, eISSN: 15205207, Volume: 121, Pages: 3016-3023, Published: 13 April 2017 American Chemical Society (ACS)
Fluorescent protein (FP)-based biosensors have become an important and promising tool to track metal ion movement inside living systems. Their working principles after light irradiation, however, remain elusive. To facilitate the rational design of biosensors, we dissect the fluorescence modulation mechanism of a newly developed excitation ratiometric green FP-based Ca2+ biosensor, GEX-GECO1, using femtosecond stimulated Raman spectroscopy (FSRS) in the electronic excited state. Upon 400 nm photoexcitation, characteristic vibrational marker bands at ∼1180 and 1300 cm-1 show concomitant decay and rise dynamics, probing the progression of an ultrafast excited state proton transfer (ESPT) reaction. The Ca2+-bound biosensor exhibits two distinct populations that undergo ESPT with ∼6 and 80 ps time constants, in contrast to one dominant population with a 25 ps time constant in the Ca2+-free biosensor. This result is supported by key structural constraints from molecular dynamics simulations with and without Ca2+. The blueshift of the ∼1265 cm-1 C-O stretch mode unravels the vibrational cooling dynamics of the protonated chromophore regardless of Ca2+ binding events. This unique line of inquiry reveals the essential structural dynamics basis of fluorescence modulation inside an excitation ratiometric protein biosensor, correlating the uncovered chromophore structural heterogeneity with different H-bonding configurations and intrinsic proton transfer rate in the photoexcited state.
Paul W Tillberg, Fei Chen, Kiryl D Piatkevich, Yongxin Zhao, Chih-Chieh Yu, Brian P English, Linyi Gao, Anthony Martorell, Ho-Jun Suk, Fumiaki Yoshida, Ellen M DeGennaro, Douglas H Roossien, Guanyu Gong, Uthpala Seneviratne, Steven R Tannenbaum, Robert Desimone, Dawen Cai, and Edward S Boyden
Nature Biotechnology, ISSN: 10870156, eISSN: 15461696, Pages: 987-992, Published: 1 September 2016 Springer Science and Business Media LLC
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 ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (∼70 nm) imaging of cells and mammalian tissues on conventional microscopes.
Ahmed S. Abdelfattah, Samouil L. Farhi, Yongxin Zhao, Daan Brinks, Peng Zou, Araya Ruangkittisakul, Jelena Platisa, Vincent A. Pieribone, Klaus Ballanyi, Adam E. Cohen, and Robert E. Campbell
Journal of Neuroscience, ISSN: 02706474, eISSN: 15292401, Pages: 2458-2472, Published: 24 February 2016 Society for Neuroscience
Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (∼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation. SIGNIFICANCE STATEMENT Fluorescent-protein-based voltage indicators enable imaging of the electrical activity of many genetically targeted neurons with high spatial and temporal resolution. Here, we describe the engineering of a bright red fluorescent protein-based voltage indicator designated as FlicR1 (fluorescent indicator for voltage imaging red). FlicR1 has sufficient speed and sensitivity to report single action potentials and voltage fluctuations at frequencies up to 100 Hz in single-trial recordings with wide-field microscopy. Because it is excitable with yellow light, FlicR1 can be used in conjunction with blue-light-activated optogenetic actuators. However, spatially distinct patterns of optogenetic activation and voltage imaging are required to avoid fluorescence artifacts due to photoactivation of the FlicR1 chromophore.
Feras Hatahet, Jessica L. Blazyk, Eugenie Martineau, Eric Mandela, Yongxin Zhao, Robert E. Campbell, Jonathan Beckwith, and Dana Boyd
Proceedings of the National Academy of Sciences of the United States of America, ISSN: 00278424, eISSN: 10916490, Volume: 112, Pages: 15184-15189, Published: 8 December 2015 Proceedings of the National Academy of Sciences
Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.
Lin Zhuang, Yongxin Zhao, Huixiang Zhong, Jinhua Liang, Jianhua Zhou, and Hui Shen
Scientific Reports, eISSN: 20452322, Published: 23 November 2015 Springer Science and Business Media LLC
Hydrophilic Fe3O4 nanoparticles with controllable size and shape have been fabricated using a facile solvothermal approach followed by surface modification with polyacrylic acid (PAA). The nanoparticles form one-dimension photonic crystal structure under external magnetic field ranging from 29.6 to 459 G. The reflection peaks of formed photonic crystals cover the entire visible spectrum, which indicates a good magnetochromatic property and prospect of wide applications. The size and shape of Fe3O4 nanoparticles are controlled by changing the ratio between ethylene glycol and diethylene glycol. In the process of surface modification, PAA synthesized by free radical polymerization was chemisorbed onto the surface of Fe3O4 particles with the aid of Fe3+ cations, which renders the particles well dispersed in aqueous solution with high thermo-stability. The Fe3O4 particles exhibit ferrimagnetism with a high saturation magnetization value of 88.0 emu/g. Both the high magnetization and the wide reflection spectrum under magnetic field make the magnetochromatic nanoparticles a promising material for visualization of the distribution of magnetic field intensity on microfluidic chips.
Longteng Tang, Weimin Liu, Yanli Wang, Yongxin Zhao, Breland G. Oscar, Robert E. Campbell, and Chong Fang
Chemistry - A European Journal, ISSN: 09476539, eISSN: 15213765, Pages: 6481-6490, Published: 20 April 2015 Wiley
Imaging Ca(2+) dynamics in living systems holds great potential to advance neuroscience and cellular biology. G-GECO1.1 is an intensiometric fluorescent protein Ca(2+) biosensor with a Thr-Tyr-Gly chromophore. The protonated chromophore emits green upon photoexcitation via excited-state proton transfer (ESPT). Upon Ca(2+) binding, a significant population of the chromophores becomes deprotonated. It remains elusive how the chromophore structurally evolves prior to and during ESPT, and how it is affected by Ca(2+) . We use femtosecond stimulated Raman spectroscopy to dissect ESPT in both the Ca(2+) -free and bound states. The protein chromophores exhibit a sub-200 fs vibrational frequency shift due to coherent small-scale proton motions. After wavepackets move out of the Franck-Condon region, ESPT gets faster in the Ca(2+) -bound protein, indicative of the formation of a more hydrophilic environment. These results reveal the governing structure-function relationship of Ca(2+) -sensing protein biosensors.
Tobias Albrecht, Yongxin Zhao, Trang Hai Nguyen, Robert E. Campbell, and James D. Johnson
Cell Calcium, ISSN: 01434160, eISSN: 15321991, Pages: 263-274, Published: 1 April 2015 Elsevier BV
Live cell imaging has revealed that calcium ions (Ca(2+)) pass in and out of many cellular organelles. However, technical hurdles have limited measurements of Ca(2+) in acidic organelles, such as endosomes. Although evidence hints that endosomes play a role in Ca(2+) signaling, direct measurements within endosomal lumina represent one of the final frontiers in organelle imaging. To measure Ca(2+) in a TiVAMP-positive endosome sub-population, the pH-resistant ratiometric Ca(2+) biosensor GEM-GECO1 and the ratiometric pH biosensor mKeima were used. A positive correlation between acidic endosomal pH and higher Ca(2+) was observed within these Rab5a- and Rab7-positive compartments. Ca(2+) concentration in most endosomes was estimated to be below 2μM, lower than Ca(2+) levels in several other intracellular stores, indicating that endosomes may take up Ca(2+) during physiological stimulation. Indeed, endosomes accumulated Ca(2+) during glucose-stimulation, a condition where endosomal pH did not change. Our biosensors permitted the first measurements revealing a role for endosomes in cellular Ca(2+) homeostasis during physiological stimulation.
Lin Zhuang, Wei Zhang, Yongxin Zhao, Hui Shen, Han Lin, and Jinhua Liang
Scientific Reports, eISSN: 20452322, Published: 23 March 2015 Springer Science and Business Media LLC
Novel-morphological Fe3O4 nanosheets with magnetochromatic property have been prepared by a modified solvothermal method. Such nanosheets could form one-dimension photonic crystal under an external magnetic field. The Fe3O4 nanosheets suspension could strongly diffract visible light and display varied colors with changing the intensity of the magnetic field. The photonic response is rapid, fully reversible and widely tunable in the entire visible spectrum. Excellent magnetic properties of these Fe3O4 nanosheets are exhibited with a high saturation magnetization (82.1 emu/g), low remanence (13.85 emu/g) and low coercive force (75.95 Oe). The amount of the solvent diethylene glycol (DEG) plays a key role in the formation of the sheet-shaped morphology. When the ratio of the DEG reaches 100%, the growing of the crystal plane (111) of Fe3O4 is inhibited and the sheet-like Fe3O4 crystals are formed.
Yanli Wang, Longteng Tang, Weimin Liu, Yongxin Zhao, Breland G. Oscar, Robert E. Campbell, and Chong Fang
Journal of Physical Chemistry B, ISSN: 15206106, eISSN: 15205207, Volume: 119, Pages: 2204-2218, Published: 12 February 2015 American Chemical Society (ACS)
Fluorescent proteins (FPs) are luminescent biomolecules that emit characteristic hues upon irradiation. A group of calmodulin (CaM)-green FP (GFP) chimeras have been previously engineered to enable the optical detection of calcium ions (Ca(2+)). We investigate one of these genetically encoded Ca(2+) biosensors for optical imaging (GECOs), GEM-GECO1, which fluoresces green without Ca(2+) but blue with Ca(2+), using femtosecond stimulated Raman spectroscopy (FSRS). The time-resolved FSRS data (<800 cm(-1)) reveal that initial structural evolution following 400 nm photoexcitation involves small-scale coherent proton motions on both ends of the chromophore two-ring system with a <250 fs time constant. Upon Ca(2+) binding, the chromophore adopts a more twisted conformation in the protein pocket with increased hydrophobicity, which inhibits excited-state proton transfer (ESPT) by effectively trapping the protonated chromophore in S1. Both the chromophore photoacidity and local environment form the ultrafast structural dynamics basis for the dual-emission properties of GEM-GECO1. Its photochemical transformations along multidimensional reaction coordinates are evinced by distinct stages of FSRS spectral evolution, particularly related to the ∼460 and 504 cm(-1) modes. The direct observation of lower frequency modes provides crucial information about the nuclear motions preceding ESPT, which enriches our understanding of photochemistry and enables the rational design of new biosensors.
Peng Zou, Yongxin Zhao, Adam D. Douglass, Daniel R. Hochbaum, Daan Brinks, Christopher A. Werley, D. Jed Harrison, Robert E. Campbell, and Adam E. Cohen
Nature Communications, eISSN: 20411723, Published: 13 August 2014 Springer Science and Business Media LLC
Genetically encoded fluorescent reporters of membrane potential promise to reveal aspects of neural function not detectable by other means. We present a palette of multi-colored brightly fluorescent genetically encoded voltage indicators with sensitivities from 8 – 13% ΔF/F per 100 mV, and half-maximal response times from 4 – 7 ms. A fluorescent protein is fused to an Archaerhodopsin-derived voltage sensor. Voltage-induced shifts in the absorption spectrum of the rhodopsin lead to voltage-dependent nonradiative quenching of the appended fluorescent protein. Through a library screen, we identify linkers and fluorescent protein combinations which report neuronal action potentials in cultured rat hippocampal neurons with a single-trial signal-to-noise ratio from 7 to 9 in a 1 kHz imaging bandwidth at modest illumination intensity. The freedom to choose a voltage indicator from an array of colors facilitates multicolor voltage imaging, as well as combination with other optical reporters and optogenetic actuators.
B. G. Oscar, W. Liu, Y. Zhao, L. Tang, Y. Wang, R. E. Campbell, and C. Fang
Proceedings of the National Academy of Sciences of the United States of America, ISSN: 00278424, eISSN: 10916490, Volume: 111, Pages: 10191-10196, Published: 15 July 2014 Proceedings of the National Academy of Sciences
Significance Fluorescent proteins (FPs) started their incredible, colorful journey in bioimaging and biomedicine with the extraction and purification of GFP from the Pacific jellyfish Aequorea victoria more than 50 years ago. Recently, an expanded palette of genetically encodable Ca2+-sensing FPs have paved the way to image neural activities and important biological processes where Ca2+ is the ubiquitous messenger. To unravel the molecular choreography of FPs engineered for visualizing Ca2+ movement, we study the embedded chromophore upon photoexcitation and monitor its subsequent excited-state structural evolution with femtosecond Raman spectroscopy. The vivid insights on H-bonding network and functional roles played by strategic mutations provide a deep understanding of excited-state processes in biology and will guide future bioengineering efforts toward better biosensors. Fluorescent proteins (FPs) have played a pivotal role in bioimaging and advancing biomedicine. The versatile fluorescence from engineered, genetically encodable FP variants greatly enhances cellular imaging capabilities, which are dictated by excited-state structural dynamics of the embedded chromophore inside the protein pocket. Visualization of the molecular choreography of the photoexcited chromophore requires a spectroscopic technique capable of resolving atomic motions on the intrinsic timescale of femtosecond to picosecond. We use femtosecond stimulated Raman spectroscopy to study the excited-state conformational dynamics of a recently developed FP-calmodulin biosensor, GEM-GECO1, for calcium ion (Ca2+) sensing. This study reveals that, in the absence of Ca2+, the dominant skeletal motion is a ∼170 cm−1 phenol-ring in-plane rocking that facilitates excited-state proton transfer (ESPT) with a time constant of ∼30 ps (6 times slower than wild-type GFP) to reach the green fluorescent state. The functional relevance of the motion is corroborated by molecular dynamics simulations. Upon Ca2+ binding, this in-plane rocking motion diminishes, and blue emission from a trapped photoexcited neutral chromophore dominates because ESPT is inhibited. Fluorescence properties of site-specific protein mutants lend further support to functional roles of key residues including proline 377 in modulating the H-bonding network and fluorescence outcome. These crucial structural dynamics insights will aid rational design in bioengineering to generate versatile, robust, and more sensitive optical sensors to detect Ca2+ in physiologically relevant environments.
Paul Tewson, Mara Westenberg, Yongxin Zhao, Robert E. Campbell, Anne Marie Quinn, and Thomas E. Hughes
PLoS ONE, eISSN: 19326203, Published: 3 January 2014 Public Library of Science (PLoS)
The graph shown in Figure 3C is mislabeled. The drug that followed the ionomycin was PDBu (not carbachol). The description in the figure legend is correct.
Yongxin Zhao, Ahmed S. Abdelfattah, Yufeng Zhao, Araya Ruangkittisakul, Klaus Ballanyi, Robert E. Campbell, and D. Jed Harrison
Integrative Biology (United Kingdom), ISSN: 17579694, eISSN: 17579708, Pages: 714-725, Published: July 2014 Oxford University Press (OUP)
We describe the use of μFACS to aid the directed evolution of a genetically encoded yellow fluorescent Ca2+indicator.
Daniel R Hochbaum, Yongxin Zhao, Samouil L Farhi, Nathan Klapoetke, Christopher A Werley, Vikrant Kapoor, Peng Zou, Joel M Kralj, Dougal Maclaurin, Niklas Smedemark-Margulies, Jessica L Saulnier, Gabriella L Boulting, Christoph Straub, Yong Ku Cho, Michael Melkonian, Gane Ka-Shu Wong, D Jed Harrison, Venkatesh N Murthy, Bernardo L Sabatini, Edward S Boyden, Robert E Campbell, and Adam E Cohen
Nature Methods, ISSN: 15487091, eISSN: 15487105, Pages: 825-833, Published: August 2014 Springer Science and Business Media LLC
All-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk–free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell–derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.