B22-Multimode InfraRed imaging And Microspectroscopy
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Open Access
Abstract: Measuring live cells by FTIR spectroscopy is challenging due to their small size and the absorbance of water in the mid-IR region. However, measuring cells in their live state is important to observe changes in the biological processes of cells that are unaffected by fixation and drying processes. Recently, ZnS hemispheres were used to sandwich live cells in a 6 µm layer of cell medium, which simultaneously limit the absorbance of water and increase the spatial resolution by x2.25, thereby enabling high quality spectra to be acquired from living cells. So far, this method has been used as an imaging technique to showcase the distribution of biomolecules within a single cell. In this work, we present an alternative use of these ZnS hemispheres as a high throughput screening tool. We obtained high quality spectra of a single cell at a measurement rate of ∼1 min/cell. We’ve applied this technique to observe the biochemical effects of various polystyrene microplastics on two mammalian cell lines (J774A.1 and A549), however the method can easily be expanded to other cell lines, microplastics, and alternative xenobiotics.
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Jun 2026
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Serial synchrotron crystallography (SSX) enables structure determination from microcrystals under near-physiological, room-temperature conditions but is limited in part due to the inevitable onset of radiation damage. The ability to reduce the absorbed dose while retaining, or even improving, data quality is an attractive means of mitigating this limitation. Advances in detector technology have made the use of high-energy X-rays a routine approach in MX, improving diffraction efficiency and enhancing overall data quality. Here, we systematically evaluate low-dose SSX data collected at five different X-ray energies from 12.4 to 25 keV using a CdTe Eiger2 detector while maintaining a constant dose. Higher photon energies increased the mean diffracted intensity and signal-to-noise ratio per unit dose, and facilitated higher-resolution structure determination, even with limited crystal numbers. These findings highlight the advantages of high-energy X-rays and provide practical guidance for optimizing SSX experiments in probing protein dynamics.
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Mar 2026
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I14-Hard X-ray Nanoprobe
I20-Scanning-X-ray spectroscopy (XAS/XES)
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Diamond Proposal Number(s):
[34837, 38234, 38452]
Open Access
Abstract: Metals play an essential role in cellular homeostasis and are key components of several formulations currently used in the clinic. Synchrotron-based X-ray microscopy at submicron resolution is a powerful approach to map intracellular elemental distributions and to monitor how these patterns change upon genetic or pharmacological perturbations. However, existing sample-preparation protocols often rely on costly and highly specialized equipment for vitrification and dehydration, limiting their widespread adoption. Here, we present an adapted plunge-freezing and freeze-drying workflow that enables the preparation of mammalian cell samples for X-ray fluorescence (XRF) and X-ray absorption spectroscopy (XAS) studies with submicron resolution in a cost-effective and versatile manner. Furthermore, we define acquisition parameters optimized for the reliable detection of low-abundance metals, such as endogenous iron. We anticipate that this accessible protocol will facilitate the broader implementation of synchrotron-based inner-shell spectromicroscopy in cell biology.
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Mar 2026
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Open Access
Abstract: Serial crystallography relies on the reproducible production of high-density suspensions of microcrystals, yet sample optimization remains a resource-intensive bottleneck. While phase diagrams provide a theoretical framework for controlling crystal size and number, experimental mapping is traditionally hindered by relatively high sample consumption. We present an automated microbatch-under-oil crystallization approach that rapidly maps phase boundaries using only 15–60 µl (∼0.15–3.8 mg) of protein. While this workflow is ideally suited for refining existing hits, it serves as a standalone platform for characterizing the crystallization landscape of new protein targets. The power of this approach lies in the integration of three distinct strategies that exploit the stable chemical environment of microbatch-under-oil. Firstly, we utilize an ingenious diagonal sampling strategy that traverses the phase boundary parallel to the solubility curve by systematically varying protein-to-precipitant ratios, identifying primary nucleation zones with far greater efficiency than traditional orthogonal grids. Secondly, we employ a linked variation of multiple precipitants to reveal morphology-specific regions, such as the rod versus plate transitions crucial for time-resolved experiments. Finally, we incorporate automated seed-stock titration to precisely define the metastable zone, enabling the predictive rescue of nucleation-limited systems. The synergy of these three strategies enables the systematic decoupling of nucleation from growth, providing a rational route to optimize microcrystal density, size and lattice order. Crucially, by eliminating the evaporation-related variables inherent in vapor diffusion, this method ensures that the chemical coordinates identified during screening remain constant during scale-up to larger volumes. This workflow transforms empirical serial crystallography sample preparation into a rational, reproducible and highly efficient process applicable to both the optimization of known conditions and the de novo development of microcrystal suspensions, tailored to the rigorous demands of modern serial diffraction experiments.
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Mar 2026
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I14-Hard X-ray Nanoprobe
I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[39618, 39938, 302085, 31588, 5116, 36811]
Open Access
Abstract: Correlative microscopy linking synchrotron X-ray fluorescence (SXRF) with optical imaging is valuable for contextualizing chemical element distributions in biology. The spatial correlation necessary to achieve this presents fundamental challenges and can be a significant constraint on accuracy and data interpretation. We present a technical solution based on a finder grid concept, optimized for SXRF correlative studies of metals in biological tissues, with scope for wider adaptation and application. A hierarchically patterned fiducial system was directly etched onto spectroscopically clean quartz substrates via femtosecond laser ablation. This design enables improved correlation among SXRF, optical imaging, and histological staining over a greater range of length scales than conventional registration methods such as the use of tissue architecture from serial sections and the use of electron-microscopy-resolution finder grids and applied fiduciary markers that can introduce XRF-signal-dominating levels of elements such as copper, nickel, gold, and titanium. We present two quartz finder grid formats: a microgrid and a nanogrid design. We demonstrate their utility for rapid ROI relocalization and same-section correlative workflows using human brain tissue. The etched quartz finder grid approach facilitates rapid and reproducible ROI relocalization and alignment across instruments, particularly where integral fiducial markers are sparse or ambiguous.
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Mar 2026
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[33984]
Abstract: In situ tomography enables non-destructive, time-lapse imaging of biological tissues under load, offering insights into structural and mechanical changes. However, repeated scans can expose samples to high radiation doses, potentially altering tissue properties. This study evaluated the feasibility of low-dose synchrotron computed tomography (sCT) for high-resolution, in situ imaging of intact bovine intervertebral discs (IVDs), and assessed the effects of repeated x-ray exposure on mechanical, microstructural, and molecular integrity. Intact oxtail IVD segments were imaged using propagation-based phase contrast sCT at 54 keV. Scan parameters were optimised to achieve high image quality within 66 seconds per scan, resulting in a total absorbed dose of ∼30 kGy over six scans. Mechanical properties were assessed under cyclic loading, microstructural changes via digital volume correlation (DVC), and molecular alterations using Raman spectroscopy. High-resolution imaging of soft and calcified tissues was achieved. Changes in sample stiffness, hysteresis, or stress recovery between irradiated and control were not identified. DVC revealed no microstructural damage or strain accumulation in the calcified endplate. Raman spectroscopy indicated minimal changes in soft tissues, with bone showing slightly increased collagen crosslinking and reduced mineralisation. Overall, this study demonstrates that high-energy, low-dose sCT enables repeated imaging of musculoskeletal tissues without compromising integrity, supporting its application in dynamic, time-lapse imaging studies. Importantly, larger, intact samples, such as whole bovine IVDs, were imaged overcoming limitations of previous studies that relied on small animal models. This approach supports more physiologically relevant investigations of tissue mechanics and degeneration in complex systems.
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Feb 2026
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Ruihai
Wang
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Qianhao
Zhao
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Julia
Quinn
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Liming
Yang
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Yuhui
Zhu
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Feifei
Huang
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Chengfei
Guo
,
Tianbo
Wang
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Pengming
Song
,
Michael
Murphy
,
Thanh D.
Nguyen
,
Andrew
Maiden
,
Francisco E.
Robles
,
Guoan
Zheng
Open Access
Abstract: The mesoscale characterization of biological specimens has traditionally required compromises between resolution, field-of-view, depth-of-field, and molecular specificity, with most approaches relying on external labels. Here we present the Deep-ultrAviolet ptychogRaphic pockeT-scope (DART), a handheld platform that transforms label-free molecular imaging through intrinsic deep-ultraviolet spectroscopic contrast. By leveraging biomolecules’ natural absorption fingerprints and combining them with lensless ptychographic microscopy, DART resolves down to 308-nm linewidths across centimeter-scale areas while maintaining millimeter-scale depth-of-field. The system’s virtual error-bin methodology effectively eliminates artifacts from limited temporal coherence and other optical imperfections, enabling high-fidelity molecular imaging without lenses. Through differential spectroscopic imaging at deep-ultraviolet wavelengths, DART quantitatively maps nucleic acid and protein distributions with femtogram sensitivity, providing an intrinsic basis for explainable virtual staining. We demonstrate DART’s capabilities through imaging of tissue sections, cytopathology specimens, blood cells, and neural populations, revealing detailed molecular contrast without external labels. The combination of high-resolution molecular mapping and broad mesoscale imaging in a portable platform opens new possibilities from rapid clinical diagnostics, tissue analysis, to biological characterization in space exploration.
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Jan 2026
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B22-Multimode InfraRed imaging And Microspectroscopy
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Lewis
Dowling
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Charlotte
Evans
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Paul
Roach
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Lisa
Vaccari
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Gianfelice
Cinque
,
Chiara Maria
Stani
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Giovanni
Birarda
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Vishnu Anand
Muruganandan
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Srinivas
Pillai
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Daniel Gey
Van Pittius
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Apurna
Jegannathen
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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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Jingjing
Zhao
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Chen
Huang
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Ali
Mostaed
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Amirafshar
Moshtaghpour
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James M.
Parkhurst
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Ivan
Lobato
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Marcus
Gallagher-Jones
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Judy S.
Kim
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Mark
Boyce
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David
Stuart
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Elena A.
Andreeva
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Jacques-Philippe
Colletier
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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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I24-Microfocus Macromolecular Crystallography
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Peter
Smyth
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Sofia
Jaho
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Lewis J.
Williams
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Gabriel
Karras
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Ann
Fitzpatrick
,
Amy J.
Thompson
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Sinan
Battah
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Danny
Axford
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Sam
Horrell
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Marina
Lucic
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Kotone
Ishihara
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Machika
Kataoka
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Hiroaki
Matsuura
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Kanji
Shimba
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Kensuke
Tono
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Takehiko
Tosha
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Hiroshi
Sugimoto
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Shigeki
Owada
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Michael A.
Hough
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Jonathan A. R.
Worrall
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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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