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Jingjing
Zhao
,
Chen
Huang
,
Ali
Mostaed
,
Amirafshar
Moshtaghpour
,
James M.
Parkhurst
,
Ivan
Lobato
,
Marcus
Gallagher-Jones
,
Judy S.
Kim
,
Mark
Boyce
,
David
Stuart
,
Elena A.
Andreeva
,
Jacques-Philippe
Colletier
,
Angus I.
Kirkland
Open Access
Abstract: Exit wavefunction reconstruction is important in transmission electron microscopy for structural studies. We describe electron Fourier ptychography and its application to phase reconstruction of both radiation-resistant and beam-sensitive materials. We demonstrate that the phase of the exit wave can be reconstructed to high resolution using a modified iterative phase retrieval algorithm from data collected in an alternative optical geometry. This method achieves a spatial resolution of 0.63 nm at a fluence of 4.5 × 102 e−/nm2, as validated on Cry11Aa protein crystals under cryogenic conditions. Notably, this method requires no instrumental modifications, is straightforward to implement, and can be seamlessly integrated with existing data collection software, providing a broadly accessible alternative approach for structural studies.
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Oct 2025
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B22-Multimode InfraRed imaging And Microspectroscopy
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Lewis
Dowling
,
Charlotte
Evans
,
Paul
Roach
,
Lisa
Vaccari
,
Gianfelice
Cinque
,
Chiara Maria
Stani
,
Giovanni
Birarda
,
Vishnu Anand
Muruganandan
,
Srinivas
Pillai
,
Daniel Gey
Van Pittius
,
Apurna
Jegannathen
,
Josep
Sulé-Suso
Diamond Proposal Number(s):
[36088]
Abstract: Liquid biopsy is revolutionizing cancer management, with circulating tumor cells (CTCs), offering a transformative approach to screening, diagnosis, and treatment monitoring. However, existing CTC isolation methods relying on antigen expression or physical properties lack robustness, are operator-dependent, and suffer from automation challenges, leading to inconsistent and time-intensive analyses. A universal, unbiased methodology for CTC detection across tumor types is critically needed. Here, we present the first proof-of-concept study demonstrating the use of Fourier transform infrared (FT-IR) microspectroscopy to study cytospun blood samples coupled with a random forest (RF) classifier, for the detection of a single CTC in the blood of a lung cancer patient as confirmed via immunohistochemistry. Notably, our method utilizes glass coverslips as substrates, routinely employed in pathology departments, enabling seamless integration with histopathological analyses (e.g., staining, immunohistochemistry). Using FT-IR spectral data from in vitro growing lung cancer cells as a training model, we achieved precise CTC identification based on biochemical composition, specifically within the Fingerprint region (1800 cm–1 to 1350 cm–1). This study introduces FT-IR microspectroscopy as a novel, label-free approach for CTCs detection in liquid biopsies, with the potential to redefine cancer diagnostics. By enhancing precision and accessibility in CTC identification, the clinical implementation of this methodology may represent a significant advancement in personalized oncology, offering a clinically viable tool for real-time cancer monitoring and improved patient stratification.
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Oct 2025
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I24-Microfocus Macromolecular Crystallography
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Peter
Smyth
,
Sofia
Jaho
,
Lewis J.
Williams
,
Gabriel
Karras
,
Ann
Fitzpatrick
,
Amy J.
Thompson
,
Sinan
Battah
,
Danny
Axford
,
Sam
Horrell
,
Marina
Lucic
,
Kotone
Ishihara
,
Machika
Kataoka
,
Hiroaki
Matsuura
,
Kanji
Shimba
,
Kensuke
Tono
,
Takehiko
Tosha
,
Hiroshi
Sugimoto
,
Shigeki
Owada
,
Michael A.
Hough
,
Jonathan A. R.
Worrall
,
Robin L.
Owen
Diamond Proposal Number(s):
[18565, 28583, 27313]
Open Access
Abstract: Time-resolved X-ray crystallography is undergoing a renaissance due to the development of serial crystallography at synchrotron and XFEL beamlines. Crucial to such experiments are efficient and effective methods for uniformly initiating time-dependent processes within microcrystals, such as ligand binding, enzymatic reactions or signalling. A widely applicable approach is the use of photocaged substrates, where the photocage is soaked into the crystal in advance and then activated using a laser pulse to provide uniform initiation of the reaction throughout the crystal. This work characterizes photocage release of nitric oxide and binding of this ligand to two heme protein systems, cytochrome c′-β and dye-decolourizing peroxidase B using a fixed target sample delivery system. Laser parameters for photoactivation are systematically explored, and time-resolved structures over timescales ranging from 100 µs to 1.4 s using synchrotron and XFEL beamlines are described. The effective use of this photocage for time-resolved crystallography is demonstrated and appropriate illumination conditions for such experiments are determined.
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Sep 2025
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Krios I-Titan Krios I at Diamond
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Gangshun
Yi
,
Dimitrios
Mamalis
,
Mingda
Ye
,
Loic
Carrique
,
Michael
Fairhead
,
Huanyu
Li
,
Katharina L.
Duerr
,
Peijun
Zhang
,
David B.
Sauer
,
Frank
Von Delft
,
Benjamin G.
Davis
,
Robert J. C.
Gilbert
Diamond Proposal Number(s):
[20223, 21004]
Open Access
Abstract: Whilst cryo-electron microscopy(cryo-EM) has become a routine methodology in structural biology, obtaining high-resolution cryo-EM structures of small proteins (<100 kDa) and increasing overall throughput remain challenging. One approach to augment protein size and improve particle alignment involves the use of binding proteins or protein-based scaffolds. However, a given imaging scaffold or linking module may prove inadequate for structure solution and availability of such scaffolds remains limited. Here, we describe a strategy that exploits covalent dimerization of nanobodies to trap an engineered, predisposed nanobody-to-nanobody interface, giving Di-Gembodies as modular constructs created in homomeric and heteromeric forms. By exploiting side-chain-to-side-chain assembly, they can simultaneously display two copies of the same or two distinct proteins through a subunit interface that provides sufficient constraint required for cryo-EM structure determination. We validate this method with multiple soluble and membrane structural targets, down to 14 kDa, demonstrating a flexible and scalable platform for expanded protein structure determination.
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Aug 2025
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B21-High Throughput SAXS
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Open Access
Abstract: Small angle X-ray scattering (SAXS) of particles in solution informs on the conformational states and assemblies of biological macromolecules (bioSAXS) outside of cryo- and solid-state conditions. In bioSAXS, the SAXS measurement under dilute conditions, is resolution-limited and through an inverse Fourier transform, the measured SAXS intensities directly relates to the physical space occupied by the particles via the P(r)-distribution. Yet, this inverse transform of SAXS data has been historically cast as an ill-posed, ill-conditioned problem requiring an indirect approach. Here, we show that through the applications of matrix and information theories, the inverse transform of SAXS intensity data is a well-conditioned problem. The so-called ill-conditioning of the inverse problem is directly related to the Shannon number. By exploiting the oversampling enabled by modern detectors, a direct inverse Fourier transform of the SAXS data is possible provided the recovered information does not exceed the Shannon number. The Shannon limit corresponds to the maximum number of significant singular values that can be recovered in a SAXS experiment suggesting this relationship is a fundamental property of band-limited inverse integral transform problems. This correspondence reduces the complexity of the inverse problem to the Shannon limit and maximum dimension. We propose a hybrid scoring function using an information theory framework that assesses both the quality of the model-data fit as well as the quality of the recovered P(r)-distribution. The hybrid score utilizes the Akaike Information Criteria and Durbin-Watson (DW) statistic that considers parameter-model complexity, i.e., degrees-of-freedom, and randomness of the model-data residuals. The described tests and findings extend the boundaries for bioSAXS by completing the information theory formalism initiated by Peter B. Moore to enable a quantitative measure of resolution in SAXS, robustly determine maximum dimension, and more precisely define the best paramater-model appropriately representing the observed scattering data.
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Jun 2025
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I13-1-Coherence
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Michela
Fratini
,
Lorenzo
Massimi
,
Francesco
Brun
,
Darren
Batey
,
Inna
Bukreeva
,
Alberto
Mittone
,
Alberto
Bravin
,
Elena
Longo
,
Giuliana
Tromba
,
Federico
Giove
,
Silvia
Cipiccia
,
Alejandra
Sierra
Diamond Proposal Number(s):
[32221]
Open Access
Abstract: The choice of fixative is critical in X-ray phase-contrast tomography (XPCT) because it affects tissue preservation, contrast enhancement and compatibility with other imaging techniques. A careful selection and optimization of fixatives can lead to significant improvements in the quality and accuracy of imaging results, which is especially important when studying complex biological systems such as those involved in neurodegeneration, where it is crucial to maintain the fine details of the Grey Matter (GM) and White Matter (WM) structures. Dehydration with ethanol and xylene is commonly used as it effectively removes water while minimising structural alterations. Using perfusion in ethanol and dehydration in xylene as a secondary fixative can increase the contrast, thereby improving the visibility of myelinated fibers without using a contrast agent. In this paper we discuss an optimised fixation method to significantly enhance the contrast and boost the signal to noise ratio (SNR) in XPCT images of WM in the central nervous system (CNS).
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Jun 2025
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B23-Circular Dichroism
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: A Capillary Zone Electrophoresis (CZE) fragment screening methodology was developed and applied to the human plasma protein Transthyretin (TTR), normally soluble, but could misfold and aggregate, causing amyloidosis. Termed Free Probe Peak Height Restoration (FPPHR), it monitors changes in the level of free ligand known to bind TTR (the Probe Ligand) in the presence of competing fragments. 129 fragments were screened, 12 of the 16 initial hits (12.4% hit rate) were co-crystallised with TTR, 11 were found at the binding site (92% confirmation rate). Subsequent analogue screens have identified a novel TTR-binding scaffold 4-(3H-pyrazol-4-yl)quinoline and its derived compounds were further studied by crystallography, circular dichroism (CD), isothermal titration calorimetry (ITC) and radiolabelled 125I-Thyroxine displacement assay in neat plasma. Two lead molecules had similar ITC Kd and 125I-Thyroxine displacement IC50 values to that of Tafamidis, adding another potential pipeline for transthyretin amyloidosis. The methodology is reproducible, procedurally simple, automatable, label-free without target immobilisation, non-fluorescence based and site-specific with low false positive rate, which could be applicable to fragment screening of many drug targets.
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May 2025
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Open Access
Abstract: Advancements in macromolecular crystallography, driven by improved sources and cryocooling techniques, have enabled the use of increasingly smaller crystals for structure determination, with microfocus beamlines now widely accessible. Initially developed for challenging samples, these techniques have culminated in advanced beamlines such as VMXm. Here, an in vacuo sample environment improves the signal-to-noise ratio in X-ray diffraction experiments, and thus enables the use of submicrometre crystals. The advancement of techniques such as microcrystal electron diffraction (MicroED) for atomic-level insights into charged states and hydrogen positions, along with room-temperature crystallography to observe physiological states via serial crystallography, has driven a resurgence in the use of microcrystals. Reproducibly preparing small crystals, especially from samples that typically yield larger crystals, requires considerable effort, as no one singular approach guarantees optimal crystals for every technique. This review discusses methods for generating such small crystals, including mechanical crushing and batch crystallization with seeding, and evaluates their compatibility with microcrystal data-collection modalities. Additionally, we examine sample-delivery methods, which are crucial for selecting appropriate crystallization strategies. Establishing reliable protocols for sample preparation and delivery opens new avenues for macromolecular crystallography, particularly in the rapidly progressing field of time-resolved crystallography.
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May 2025
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NONE-No attached Diamond beamline
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Open Access
Abstract: We have developed sample-efficient delivery and reaction initiation strategies that use room temperature microcrystal slurries and serial crystallography methods for time-resolved studies [1-3]. However, interpreting electron density maps from reaction cycle intermediates can be challenging when mixtures of species are present in the data. Therefore, to help reduce ambiguity we and our collaborators have also pioneered strategies to simultaneously collect time-resolved serial crystallography (tr- SSX/tr-SFX) diffraction data in the forward direction, and X-ray emission spectroscopy (tr-XES) data at ∼ 90°, using either XFEL (tr-SFX) or synchrotron (tr-SSX) sources. The resulting atomic and electronic structures are fully correlated and have been applied to a range of enzymes [1, 2, 4-8]. For instance, isopenicillin N synthase (IPNS) uses nonheme iron to catalyse the O2- dependent conversion of its tripeptide substrate delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) into isopenicillin N (IPN, the precursor of all penicillin/cephalosporin beta-lactam antibiotics). The unique four electron oxidation reaction leading to the beta-lactam bicyclic ring proceeds via two high-valent iron species, an Fe(III)-superoxo and a high-spin Fe(IV)=O oxyferryl species. These enable two sequential C-H bond cleavage steps that each exhibit large kinetic isotope effects (KIE). Our recent tr- SFX and tr-XES studies have characterised the Fe(III)-superoxo species and revealed unexpected, correlated motions throughout the whole protein caused by O2 binding [4].
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Mar 2025
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[17971]
Open Access
Abstract: We propose an imaging system and methodology for mapping soft-tissue samples in three dimensions, with micron-scale and isotropic spatial resolution, with low-concentrations as well as in the absence of heavy metal staining. We used hard x-ray phase-contrast imaging for the x-ray ability to nondestructively probe the internal structure of opaque specimens and for enhanced contrast obtained by exploiting phase effects, even in cases with reduced or absent staining agents. To demonstrate its applicability to soft-tissue specimens, we built a compact system that is easily deployable in a laboratory setting. The imaging system is based on a conventional rotating anode x-ray tube and a state-of-the-art custom-made radiation detector. The systems performance is quantitatively assessed on a calibration standard. Its potential for soft-tissue microscopy is demonstrated on two biological specimens and benchmarked against gold-standard synchrotron data. We believe that the approach proposed here can be valuable as a bridging imaging modality for intravital correlative light and electron microscopy and be applied across disciplines where the three-dimensional morphology of pristine-condition soft tissues is a key element of the investigation.
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Jan 2025
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