Publications by authors named "Philipp Kukura"

64 Publications

Mass Photometry of Membrane Proteins.

Chem 2021 Jan 14;7(1):224-236. Epub 2021 Jan 14.

Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.

Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane-mimetic systems at the single-particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterizing the particle size, sample purity, and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane-protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers-in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation.
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http://dx.doi.org/10.1016/j.chempr.2020.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815066PMC
January 2021

Quantifying the Monomer-Dimer Equilibrium of Tubulin with Mass Photometry.

J Mol Biol 2020 11 15;432(23):6168-6172. Epub 2020 Oct 15.

Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3QZ, UK. Electronic address:

The αβ-tubulin heterodimer is the fundamental building block of microtubules, making it central to several cellular processes. Despite the apparent simplicity of heterodimerisation, the associated energetics and kinetics remain disputed, largely due to experimental challenges associated with quantifying affinities in the <µM range. We use mass photometry to observe tubulin monomers and heterodimers in solution simultaneously, thereby quantifying the αβ-tubulin dissociation constant (8.48 ± 1.22 nM) and its tightening in the presence of GTP (3.69 ± 0.65 nM), at a dissociation rate >10 s. Our results demonstrate the capabilities of mass photometry for quantifying protein-protein interactions and clarify the energetics and kinetics of tubulin heterodimerisation.
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http://dx.doi.org/10.1016/j.jmb.2020.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763485PMC
November 2020

Emergence and Rearrangement of Dynamic Supramolecular Aggregates Visualized by Interferometric Scattering Microscopy.

ACS Nano 2020 09 18;14(9):11160-11168. Epub 2020 Aug 18.

Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom OX1 3TA.

Studying dynamic self-assembling systems in their native environment is essential for understanding the mechanisms of self-assembly and thereby exerting full control over these processes. Traditional ensemble-based analysis methods often struggle to reveal critical features of the self-assembly that occur at the single particle level. Here, we describe a label-free single-particle assay to visualize real-time self-assembly in aqueous solutions by interferometric scattering microscopy. We demonstrate how the assay can be applied to biphasic reactions yielding micellar or vesicular aggregates, detecting the onset of aggregate formation, quantifying the kinetics at the single particle level, and distinguishing sigmoidal and exponential growth of aggregate populations. Furthermore, we can follow the evolution in aggregate size in real time, visualizing the nucleation stages of the self-assembly processes and record phenomena such as incorporation of oily components into the micelle or vesicle lumen.
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http://dx.doi.org/10.1021/acsnano.0c02414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513470PMC
September 2020

Single molecule mass photometry of nucleic acids.

Nucleic Acids Res 2020 09;48(17):e97

Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.

Mass photometry is a recently developed methodology capable of measuring the mass of individual proteins under solution conditions. Here, we show that this approach is equally applicable to nucleic acids, enabling their facile, rapid and accurate detection and quantification using sub-picomoles of sample. The ability to count individual molecules directly measures relative concentrations in complex mixtures without need for separation. Using a dsDNA ladder, we find a linear relationship between the number of bases per molecule and the associated imaging contrast for up to 1200 bp, enabling us to quantify dsDNA length with up to 2 bp accuracy. These results introduce mass photometry as an accurate, rapid and label-free single molecule method complementary to existing DNA characterization techniques.
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http://dx.doi.org/10.1093/nar/gkaa632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515692PMC
September 2020

Coupled Metabolic Cycles Allow Out-of-Equilibrium Autopoietic Vesicle Replication.

Angew Chem Int Ed Engl 2020 11 3;59(46):20361-20366. Epub 2020 Sep 3.

Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, UK.

We report chemically fuelled out-of-equilibrium self-replicating vesicles based on surfactant formation. We studied the vesicles' autocatalytic formation using UPLC to determine monomer concentration and interferometric scattering microscopy at the nanoparticle level. Unlike related reports of chemically fuelled self-replicating micelles, our vesicular system was too stable to surfactant degradation to be maintained out of equilibrium. The introduction of a catalyst, which introduces a second catalytic cycle into the metabolic network, was used to close the first cycle. This shows how coupled catalytic cycles can create a metabolic network that allows the creation and perseverance of fuel-driven, out-of-equilibrium self-replicating vesicles.
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http://dx.doi.org/10.1002/anie.202007302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692917PMC
November 2020

Quantifying the heterogeneity of macromolecular machines by mass photometry.

Nat Commun 2020 04 14;11(1):1772. Epub 2020 Apr 14.

Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford, OX1 3QZ, UK.

Sample purity is central to in vitro studies of protein function and regulation, and to the efficiency and success of structural studies using techniques such as x-ray crystallography and cryo-electron microscopy (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize the heterogeneity of a sample using minimal material with high resolution within a matter of minutes. To benchmark our approach, we use negative stain electron microscopy (nsEM), a popular method for EM sample screening. We include typical workflows developed for structure determination that involve multi-step purification of a multi-subunit ubiquitin ligase and chemical cross-linking steps. When assessing the integrity and stability of large molecular complexes such as the proteasome, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP over current methods for rapid sample characterization, prioritization and workflow optimization.
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http://dx.doi.org/10.1038/s41467-020-15642-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156492PMC
April 2020

Myosin II Filament Dynamics in Actin Networks Revealed with Interferometric Scattering Microscopy.

Biophys J 2020 04 4;118(8):1946-1957. Epub 2020 Mar 4.

Centre for Mechanochemical Cell Biology, University of Warwick, Coventry, United Kingdom; Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, United Kingdom. Electronic address:

The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for a variety of cellular processes. Recent work has shown that the remodeling acto-myosin network modifies local membrane organization, but the molecular details are only partly understood because of difficulties with experimentally accessing the relevant time and length scales. Here, we use interferometric scattering microscopy to investigate a minimal acto-myosin network linked to a supported lipid bilayer membrane. Using the magnitude of the interferometric contrast, which is proportional to molecular mass, and fast acquisition rates, we detect and image individual membrane-attached actin filaments diffusing within the acto-myosin network and follow individual myosin II filament dynamics. We quantify myosin II filament dwell times and processivity as functions of ATP concentration, providing experimental evidence for the predicted ensemble behavior of myosin head domains. Our results show how decreasing ATP concentrations lead to both increasing dwell times of individual myosin II filaments and a global change from a remodeling to a contractile state of the acto-myosin network.
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http://dx.doi.org/10.1016/j.bpj.2020.02.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175421PMC
April 2020

Quantifying Protein-Protein Interactions by Molecular Counting with Mass Photometry.

Angew Chem Int Ed Engl 2020 06 2;59(27):10774-10779. Epub 2020 Apr 2.

Physical and Theoretical Chemistry, Department of Chemistry, University of Oxford, South Parks Road, Oxford, OX1 3TA, UK.

Interactions between biomolecules control the processes of life in health and their malfunction in disease, making their characterization and quantification essential. Immobilization- and label-free analytical techniques are desirable because of their simplicity and minimal invasiveness, but they struggle with quantifying tight interactions. Here, we show that mass photometry can accurately count, distinguish by molecular mass, and thereby reveal the relative abundances of different unlabelled biomolecules and their complexes in mixtures at the single-molecule level. These measurements determine binding affinities over four orders of magnitude at equilibrium for both simple and complex stoichiometries within minutes, as well as the associated kinetics. These results introduce mass photometry as a rapid, simple and label-free method for studying sub-micromolar binding affinities, with potential for extension towards a universal approach for characterizing complex biomolecular interactions.
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http://dx.doi.org/10.1002/anie.202001578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318626PMC
June 2020

Femtosecond Transient Absorption Microscopy of Singlet Exciton Motion in Side-Chain Engineered Perylene-Diimide Thin Films.

J Phys Chem A 2020 Apr 18;124(13):2721-2730. Epub 2020 Mar 18.

Department of Physics, Cavendish Laboratory, University of Cambridge, JJ Thomson Avenue, Cambridge CB3 0HE, U.K.

We present a statistical analysis of femtosecond transient absorption microscopy applied to four different organic semiconductor thin films based on perylene-diimide (PDI). By achieving a temporal resolution of 12 fs with simultaneous sub-10 nm spatial precision, we directly probe the underlying exciton transport characteristics within 3 ps after photoexcitation free of model assumptions. Our study reveals sub-picosecond coherent exciton transport (12-45 cm s) followed by a diffusive phase of exciton transport (3-17 cm s). A comparison between the different films suggests that the exciton transport in the studied materials is intricately linked to their nanoscale morphology, with PDI films that form large crystalline domains exhibiting the largest diffusion coefficients and transport lengths. Our study demonstrates the advantages of directly studying ultrafast transport properties at the nanometer length scale and highlights the need to examine nanoscale morphology when investigating exciton transport in organic as well as inorganic semiconductors.
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http://dx.doi.org/10.1021/acs.jpca.0c00346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7132576PMC
April 2020

Ultrafast Tracking of Exciton and Charge Carrier Transport in Optoelectronic Materials on the Nanometer Scale.

J Phys Chem Lett 2019 Nov 17;10(21):6727-6733. Epub 2019 Oct 17.

Department of Physics, Cavendish Laboratory , University of Cambridge , JJ Thompson Avenue , Cambridge CB3 0HE , United Kingdom.

We present a novel optical transient absorption and reflection microscope based on a diffraction-limited pump pulse in combination with a wide-field probe pulse, for the spatiotemporal investigation of ultrafast population transport in thin films. The microscope achieves a temporal resolution down to 12 fs and simultaneously provides sub-10 nm spatial accuracy. We demonstrate the capabilities of the microscope by revealing an ultrafast excited-state exciton population transport of up to 32 nm in a thin film of pentacene and by tracking the carrier motion in p-doped silicon. The use of few-cycle optical excitation pulses enables impulsive stimulated Raman microspectroscopy, which is used for in situ verification of the chemical identity in the 100-2000 cm spectral window. Our methodology bridges the gap between optical microscopy and spectroscopy, allowing for the study of ultrafast transport properties down to the nanometer length scale.
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http://dx.doi.org/10.1021/acs.jpclett.9b02437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844127PMC
November 2019

A molecular movie of ultrafast singlet fission.

Nat Commun 2019 09 16;10(1):4207. Epub 2019 Sep 16.

Department of Physics and Astronomy, University of Sheffield, Hounsfield Road, Sheffield, S3 7RH, UK.

The complex dynamics of ultrafast photoinduced reactions are governed by their evolution along vibronically coupled potential energy surfaces. It is now often possible to identify such processes, but a detailed depiction of the crucial nuclear degrees of freedom involved typically remains elusive. Here, combining excited-state time-domain Raman spectroscopy and tree-tensor network state simulations, we construct the full 108-atom molecular movie of ultrafast singlet fission in a pentacene dimer, explicitly treating 252 vibrational modes on 5 electronic states. We assign the tuning and coupling modes, quantifying their relative intensities and contributions, and demonstrate how these modes coherently synchronise to drive the reaction. Our combined experimental and theoretical approach reveals the atomic-scale singlet fission mechanism and can be generalized to other ultrafast photoinduced reactions in complex systems. This will enable mechanistic insight on a detailed structural level, with the ultimate aim to rationally design molecules to maximise the efficiency of photoinduced reactions.
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http://dx.doi.org/10.1038/s41467-019-12220-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746807PMC
September 2019

Editorial overview: Advances and future prospects of molecular imaging for studying and quantifying biological processes.

Curr Opin Chem Biol 2019 08 13;51:A4-A5. Epub 2019 Jul 13.

Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA. Electronic address:

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http://dx.doi.org/10.1016/j.cbpa.2019.06.014DOI Listing
August 2019

An ultra-stable gold-coordinated protein cage displaying reversible assembly.

Nature 2019 05 8;569(7756):438-442. Epub 2019 May 8.

Heddle Initiative Research Unit, RIKEN, Saitama, Japan.

Symmetrical protein cages have evolved to fulfil diverse roles in nature, including compartmentalization and cargo delivery, and have inspired synthetic biologists to create novel protein assemblies via the precise manipulation of protein-protein interfaces. Despite the impressive array of protein cages produced in the laboratory, the design of inducible assemblies remains challenging. Here we demonstrate an ultra-stable artificial protein cage, the assembly and disassembly of which can be controlled by metal coordination at the protein-protein interfaces. The addition of a gold (I)-triphenylphosphine compound to a cysteine-substituted, 11-mer protein ring triggers supramolecular self-assembly, which generates monodisperse cage structures with masses greater than 2 MDa. The geometry of these structures is based on the Archimedean snub cube and is, to our knowledge, unprecedented. Cryo-electron microscopy confirms that the assemblies are held together by 120 S-Au-S staples between the protein oligomers, and exist in two chiral forms. The cage shows extreme chemical and thermal stability, yet it readily disassembles upon exposure to reducing agents. As well as gold, mercury(II) is also found to enable formation of the protein cage. This work establishes an approach for linking protein components into robust, higher-order structures, and expands the design space available for supramolecular assemblies to include previously unexplored geometries.
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http://dx.doi.org/10.1038/s41586-019-1185-4DOI Listing
May 2019

Interferometric Scattering Microscopy.

Annu Rev Phys Chem 2019 06 12;70:301-322. Epub 2019 Apr 12.

Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3QZ, United Kingdom; email: ,

Interferometric scattering microscopy (iSCAT) is an extremely sensitive imaging method based on the efficient detection of light scattered by nanoscopic objects. The ability to, at least in principle, maintain high imaging contrast independent of the exposure time or the scattering cross section of the object allows for unique applications in single-particle tracking, label-free imaging of nanoscopic (dis)assembly, and quantitative single-molecule characterization. We illustrate these capabilities in areas as diverse as mechanistic studies of motor protein function, viral capsid assembly, and single-molecule mass measurement in solution. We anticipate that iSCAT will become a widely used approach to unravel previously hidden details of biomolecular dynamics and interactions.
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http://dx.doi.org/10.1146/annurev-physchem-050317-021247DOI Listing
June 2019

Shaky lattices for light-matter interactions.

Nat Mater 2019 04;18(4):307-308

Department of Chemistry, Physical and Theoretical Chemistry Laboratory, Oxford, UK.

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http://dx.doi.org/10.1038/s41563-019-0299-2DOI Listing
April 2019

Dissecting FOXP2 Oligomerization and DNA Binding.

Angew Chem Int Ed Engl 2019 06 29;58(23):7662-7667. Epub 2019 Apr 29.

Physik Department & Munich School of Bioengineering, Technische Universität München, Am Coulombwall 4a, 85784, Garching, Germany.

Protein-protein and protein-substrate interactions are critical to function and often depend on factors that are difficult to disentangle. Herein, a combined biochemical and biophysical approach, based on electrically switchable DNA biochips and single-molecule mass analysis, was used to characterize the DNA binding and protein oligomerization of the transcription factor, forkhead box protein P2 (FOXP2). FOXP2 contains domains commonly involved in nucleic-acid binding and protein oligomerization, such as a C H -zinc finger (ZF), and a leucine zipper (LZ), whose roles in FOXP2 remain largely unknown. We found that the LZ mediates FOXP2 dimerization via coiled-coil formation but also contributes to DNA binding. The ZF contributes to protein dimerization when the LZ coiled-coil is intact, but it is not involved in DNA binding. The forkhead domain (FHD) is the key driver of DNA binding. Our data contributes to understanding the mechanisms behind the transcriptional activity of FOXP2.
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http://dx.doi.org/10.1002/anie.201901734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986896PMC
June 2019

Pseudomonas aeruginosa orchestrates twitching motility by sequential control of type IV pili movements.

Nat Microbiol 2019 05 25;4(5):774-780. Epub 2019 Feb 25.

Institute of Bioengineering and Global Health Institute, School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

Prokaryotes have the ability to walk on surfaces using type IV pili (TFP), a motility mechanism known as twitching. Molecular motors drive TFP extension and retraction, but whether and how these movements are coordinated is unknown. Here, we reveal how the pathogen Pseudomonas aeruginosa coordinates the motorized activity of TFP to power efficient surface motility. To do this, we dynamically visualized TFP extension, attachment and retraction events at high resolution in four dimensions using label-free interferometric scattering microscopy (iSCAT). By measuring TFP dynamics, we found that the retraction motor PilT was sufficient to generate tension and power motility in free solution, while its partner ATPase PilU may improve retraction only in high-friction environments. Using precise timing of successive attachment and retraction, we show that P. aeruginosa engages PilT motors very rapidly and almost only when TFP encounter the surface, suggesting contact sensing. Finally, measurements of TFP dwell times on surfaces show that tension reinforced the adhesion strength to the surface of individual pili, thereby increasing effective pulling time during retraction. The successive control of TFP extension, attachment, retraction and detachment suggests that sequential control of motility machinery is a conserved strategy for optimized locomotion across domains of life.
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http://dx.doi.org/10.1038/s41564-019-0378-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522360PMC
May 2019

Revealing Compartmentalized Diffusion in Living Cells with Interferometric Scattering Microscopy.

Biophys J 2018 06;114(12):2945-2950

Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, United Kingdom. Electronic address:

The spatiotemporal organization and dynamics of the plasma membrane and its constituents are central to cellular function. Fluorescence-based single-particle tracking has emerged as a powerful approach for studying the single molecule behavior of plasma-membrane-associated events because of its excellent background suppression, at the expense of imaging speed and observation time. Here, we show that interferometric scattering microscopy combined with 40 nm gold nanoparticle labeling can be used to follow the motion of membrane proteins in the plasma membrane of live cultured mammalian cell lines and hippocampal neurons with up to 3 nm precision and 25 μs temporal resolution. The achievable spatiotemporal precision enabled us to reveal signatures of compartmentalization in neurons likely caused by the actin cytoskeleton.
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http://dx.doi.org/10.1016/j.bpj.2018.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026387PMC
June 2018

Complementary studies of lipid membrane dynamics using iSCAT and super-resolved fluorescence correlation spectroscopy.

J Phys D Appl Phys 2018 Jun 16;51(23):235401. Epub 2018 May 16.

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.

Observation techniques with high spatial and temporal resolution, such as single-particle tracking based on interferometric scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. In this work, we have used lipid analogues tagged with a hybrid fluorescent tag-gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous supported lipid bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50  ⩽  t  ⩽  100 ms) scales probed, the diffussion coefficient was reduced by 20- to 60-fold when tagging with 20 and 40 nm large gold particles as compared to when using dye tagged lipid analogues. These FCS measurements of hybrid fluorescent tag-gold nanoparticle labeled lipids also revealed that commercially supplied streptavidin-coated gold nanoparticles contain large quantities of free streptavidin. Finally, the values of apparent diffusion coefficients obtained by STED-FCS and iSCAT differed by a factor of 2-3 across the techniques, while relative differences in mobility between different species of lipid analogues considered were identical in both approaches. In conclusion, our experiments reveal that large and potentially cross-linking scattering tags introduce a significant slow-down in diffusion on SLBs but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.
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http://dx.doi.org/10.1088/1361-6463/aac04fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964363PMC
June 2018

Quantitative mass imaging of single biological macromolecules.

Science 2018 04;360(6387):423-427

Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.

The cellular processes underpinning life are orchestrated by proteins and their interactions. The associated structural and dynamic heterogeneity, despite being key to function, poses a fundamental challenge to existing analytical and structural methodologies. We used interferometric scattering microscopy to quantify the mass of single biomolecules in solution with 2% sequence mass accuracy, up to 19-kilodalton resolution, and 1-kilodalton precision. We resolved oligomeric distributions at high dynamic range, detected small-molecule binding, and mass-imaged proteins with associated lipids and sugars. These capabilities enabled us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomolecular interactions, one molecule at a time.
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http://dx.doi.org/10.1126/science.aar5839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6103225PMC
April 2018

Label-Free Single-Molecule Imaging with Numerical-Aperture-Shaped Interferometric Scattering Microscopy.

ACS Photonics 2017 Feb 18;4(2):211-216. Epub 2017 Jan 18.

Physical and Theoretical Chemistry Laboratory, University of Oxford , South Parks Road, OX1 3QZ Oxford, U.K.

Our ability to optically interrogate nanoscopic objects is controlled by the difference between their extinction cross sections and the diffraction-limited area to which light can be confined in the far field. We show that a partially transmissive spatial mask placed near the back focal plane of a high numerical aperture microscope objective enhances the extinction contrast of a scatterer near an interface by approximately , where is the transmissivity of the mask. Numerical-aperture-based differentiation of background from scattered light represents a general approach to increasing extinction contrast and enables routine label-free imaging down to the single-molecule level.
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http://dx.doi.org/10.1021/acsphotonics.6b00912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5323080PMC
February 2017

Sub-10 fs Time-Resolved Vibronic Optical Microscopy.

J Phys Chem Lett 2016 Dec 15;7(23):4854-4859. Epub 2016 Nov 15.

Physical and Theoretical Chemistry Laboratory, University of Oxford , South Parks Road, Oxford OX1 3QZ, United Kingdom.

We introduce femtosecond wide-field transient absorption microscopy combining sub-10 fs pump and probe pulses covering the complete visible (500-650 nm) and near-infrared (650-950 nm) spectrum with diffraction-limited optical resolution. We demonstrate the capabilities of our system by reporting the spatially- and spectrally-resolved transient electronic response of MAPbICl perovskite films and reveal significant quenching of the transient bleach signal at grain boundaries. The unprecedented temporal resolution enables us to directly observe the formation of band-gap renormalization, completed in 25 fs after photoexcitation. In addition, we acquire hyperspectral Raman maps of TIPS pentacene films with sub-400 nm spatial and sub-15 cm spectral resolution covering the 100-2000 cm window. Our approach opens up the possibility of studying ultrafast dynamics on nanometer length and femtosecond time scales in a variety of two-dimensional and nanoscopic systems.
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http://dx.doi.org/10.1021/acs.jpclett.6b02387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5684689PMC
December 2016

Wide-Field Detected Fourier Transform CARS Microscopy.

Sci Rep 2016 11 24;6:37516. Epub 2016 Nov 24.

Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.

We present a wide-field imaging implementation of Fourier transform coherent anti-Stokes Raman scattering (wide-field detected FT-CARS) microscopy capable of acquiring high-contrast label-free but chemically specific images over the full vibrational 'fingerprint' region, suitable for a large field of view. Rapid resonant mechanical scanning of the illumination beam coupled with highly sensitive, camera-based detection of the CARS signal allows for fast and direct hyperspectral wide-field image acquisition, while minimizing sample damage. Intrinsic to FT-CARS microscopy, the ability to control the range of time-delays between pump and probe pulses allows for fine tuning of spectral resolution, bandwidth and imaging speed while maintaining full duty cycle. We outline the basic principles of wide-field detected FT-CARS microscopy and demonstrate how it can be used as a sensitive optical probe for chemically specific Raman imaging.
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http://dx.doi.org/10.1038/srep37516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5121585PMC
November 2016

Mechanism for rapid growth of organic-inorganic halide perovskite crystals.

Nat Commun 2016 11 10;7:13303. Epub 2016 Nov 10.

Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1 3PU, UK.

Optoelectronic devices based on hybrid halide perovskites have shown remarkable progress to high performance. However, despite their apparent success, there remain many open questions about their intrinsic properties. Single crystals are often seen as the ideal platform for understanding the limits of crystalline materials, and recent reports of rapid, high-temperature crystallization of single crystals should enable a variety of studies. Here we explore the mechanism of this crystallization and find that it is due to reversible changes in the solution where breaking up of colloids, and a change in the solvent strength, leads to supersaturation and subsequent crystallization. We use this knowledge to demonstrate a broader range of processing parameters and show that these can lead to improved crystal quality. Our findings are therefore of central importance to enable the continued advancement of perovskite optoelectronics and to the improved reproducibility through a better understanding of factors influencing and controlling crystallization.
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http://dx.doi.org/10.1038/ncomms13303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5109546PMC
November 2016

Visualization of the spontaneous emergence of a complex, dynamic, and autocatalytic system.

Proc Natl Acad Sci U S A 2016 10 16;113(40):11122-11126. Epub 2016 Sep 16.

Chemistry Research Laboratory, University of Oxford, Oxford OX1 3TA, United Kingdom

Autocatalytic chemical reactions are widely studied as models of biological processes and to better understand the origins of life on Earth. Minimal self-reproducing amphiphiles have been developed in this context and as an approach to de novo "bottom-up" synthetic protocells. How chemicals come together to produce living systems, however, remains poorly understood, despite much experimentation and speculation. Here, we use ultrasensitive label-free optical microscopy to visualize the spontaneous emergence of an autocatalytic system from an aqueous mixture of two chemicals. Quantitative, in situ nanoscale imaging reveals heterogeneous self-reproducing aggregates and enables the real-time visualization of the synthesis of new aggregates at the reactive interface. The aggregates and reactivity patterns observed vary together with differences in the respective environment. This work demonstrates how imaging of chemistry at the nanoscale can provide direct insight into the dynamic evolution of nonequilibrium systems across molecular to microscopic length scales.
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http://dx.doi.org/10.1073/pnas.1602363113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056079PMC
October 2016

Vibronic Dynamics of the Ultrafast all-trans to 13-cis Photoisomerization of Retinal in Channelrhodopsin-1.

J Am Chem Soc 2016 Apr 30;138(14):4757-62. Epub 2016 Mar 30.

Physical and Theoretical Chemistry Laboratory, University of Oxford , South Parks Road, Oxford OX1 3QZ, United Kingdom.

Channelrhodopsins are light-gated ion channels with extensive applications in optogenetics. Channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) exhibits a red-shifted absorption spectrum as compared to Channelrhodopsin-2, which is highly beneficial for optogenetic application. The primary event in the photocycle of CaChR1 involves an isomerization of the protein-bound retinal chromophore. Here, we apply highly time-resolved vibronic spectroscopy to reveal the electronic and structural dynamics associated with the first step of the photocycle of CaChR1. We observe vibrationally coherent formation of the P1 intermediate exhibiting a twisted 13-cis retinal with a 110 ± 7 fs time constant. Comparison with low-temperature resonance Raman spectroscopy of the corresponding trapped photoproduct demonstrates that this rapidly formed P1 intermediate is stable for several hundreds of nanoseconds.
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http://dx.doi.org/10.1021/jacs.5b12251DOI Listing
April 2016

Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging.

Nat Protoc 2016 Apr 3;11(4):617-33. Epub 2016 Mar 3.

Physical and Theoretical Chemistry Laboratory, Oxford, UK.

Interferometric scattering microscopy (iSCAT) is a light scattering-based imaging modality that offers a unique combination of imaging speed and precision for tracking nanoscopic labels and enables label-free optical sensing down to the single-molecule level. In contrast to fluorescence, iSCAT does not suffer from limitations associated with dye photochemistry and photophysics, or the requirement for fluorescent labeling. Here we present a protocol for constructing an iSCAT microscope from commercially available optical components and demonstrate its compatibility with simultaneously operating single-molecule, objective-type, total internal reflection fluorescence microscopy. Given an intermediate level of experience with optics and microscopy, for instance graduate-level familiarity with laser beam steering and optical components, this protocol can be completed in a time frame of 2 weeks.
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http://dx.doi.org/10.1038/nprot.2016.022DOI Listing
April 2016

Label-free Imaging of Microtubules with Sub-nm Precision Using Interferometric Scattering Microscopy.

Biophys J 2016 Jan;110(1):214-7

Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom. Electronic address:

Current in vitro optical studies of microtubule dynamics tend to rely on fluorescent labeling of tubulin, with tracking accuracy thereby limited by the quantum yield of fluorophores and by photobleaching. Here, we demonstrate label-free tracking of microtubules with nanometer precision at kilohertz frame rates using interferometric scattering microscopy (iSCAT). With microtubules tethered to a glass substrate using low-density kinesin, we readily detect sequential 8 nm steps in the microtubule center of mass, characteristic of a single kinesin molecule moving a microtubule. iSCAT also permits dynamic changes in filament length to be measured with <5 nm precision. Using the arbitrarily long observation time enabled by label-free iSCAT imaging, we demonstrate continuous monitoring of microtubule disassembly over a 30 min period. The ability of iSCAT to track microtubules with nm precision together with its potential for label-free single protein detection and simultaneous single molecule fluorescence imaging represent a unique platform for novel approaches to studying microtubule dynamics.
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http://dx.doi.org/10.1016/j.bpj.2015.10.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806212PMC
January 2016