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Instruments / Technical Area	Publication Details	Published
B24-Cryo Soft X-ray Tomography	Sample preparation strategies for efficient correlation of 3D SIM and soft X-ray tomography data at cryogenic temperatures Chidinma Okolo , Ilias Kounatidis , Johannes Groen , Kamal Nahas , Stefan Balint , Thomas M. Fish , Mohamed A. Koronfel , Aitziber L. Cortajarena , Ian M. Dobbie , Eva Pereiro , Maria Harkiolaki Nature Protocols 11 DOI: 10.1038/s41596-021-00522-4 Diamond Proposal Number(s): [18737, 20321, 22274, 23046, 25162] Show Abstract ▼ Abstract: 3D correlative microscopy methods have revolutionized biomedical research, allowing the acquisition of multidimensional information to gain an in-depth understanding of biological systems. With the advent of relevant cryo-preservation methods, correlative imaging of cryogenically preserved samples has led to nanometer resolution imaging (2–50 nm) under harsh imaging regimes such as electron and soft X-ray tomography. These methods have now been combined with conventional and super-resolution fluorescence imaging at cryogenic temperatures to augment information content from a given sample, resulting in the immediate requirement for protocols that facilitate hassle-free, unambiguous cross-correlation between microscopes. We present here sample preparation strategies and a direct comparison of different working fiducialization regimes that facilitate 3D correlation of cryo-structured illumination microscopy and cryo-soft X-ray tomography. Our protocol has been tested at two synchrotron beamlines (B24 at Diamond Light Source in the UK and BL09 Mistral at ALBA in Spain) and has led to the development of a decision aid that facilitates experimental design with the strategic use of markers based on project requirements. This protocol takes between 1.5 h and 3.5 d to complete, depending on the cell populations used (adherent cells may require several days to grow on sample carriers).	May 2021
I24-Microfocus Macromolecular Crystallography	The cubicon method for concentrating membrane proteins in the cubic mesophase Pikyee Ma , Dietmar Weichert , Luba A. Aleksandrov , Timothy J. Jensen , John R. Riordan , Xiangyu Liu , Brian K. Kobilka , Martin Caffrey Nature Protocols 12, 1745 - 1762 DOI: 10.1038/nprot.2017.057 Diamond Proposal Number(s): [11890, 10357, 9406] Show Abstract ▼ Abstract: The lipid cubic phase (in meso) method is an important approach for generating crystals and high-resolution X-ray structures of integral membrane proteins. However, as a consequence of instability, it can be impossible—using traditional methods—to concentrate certain membrane proteins and complexes to values suitable for in meso crystallization and structure determination. The cubicon method described here exploits the amphiphilic nature of membrane proteins and their natural tendency to partition preferentially into lipid bilayers from aqueous solution. Using several rounds of reconstitution, the protein concentration in the bilayer of the cubic mesophase can be ramped up stepwise from less than a milligram per milliliter to tens of milligrams per milliliter for crystallogenesis. The general applicability of the method is demonstrated with five integral membrane proteins: the β2-adrenergic G protein-coupled receptor (β2AR), the peptide transporter (PepTSt), diacylglycerol kinase (DgkA), the alginate transporter (AlgE) and the cystic fibrosis transmembrane conductance regulator (CFTR). In the cases of β2AR, PepTSt, DgkA and AlgE, an effective 20- to 45-fold concentration was realized, resulting in a protein-laden mesophase that allowed the formation of crystals using the in meso method and structure determination to resolutions ranging from 2.4 Å to 3.2 Å. In addition to opening up in meso crystallization to a broader range of integral membrane protein targets, the cubicon method should find application in situations that require membrane protein reconstitution in a lipid bilayer at high concentrations. These applications include functional and biophysical characterization studies for ligand screening, drug delivery, antibody production and protein complex formation. A typical cubicon experiment can be completed in 3–5 h.	Aug 2017
B22-Multimode InfraRed imaging And Microspectroscopy	Using Raman spectroscopy to characterize biological materials Holly J. Butler , Lorna Ashton , Benjamin Bird , Gianfelice Cinque , Kelly Curtis , Jennifer Dorney , Karen Esmonde-white , Nigel J Fullwood , Benjamin Gardner , Pierre L Martin-hirsch , Michael J Walsh , Martin R Mcainsh , Nicholas Stone , Francis L. Martin Nature Protocols 11, 664 - 687 DOI: 10.1038/nprot.2016.036 Show Abstract ▼ Abstract: Raman spectroscopy can be used to measure the chemical composition of a sample, which can in turn be used to extract biological information. Many materials have characteristic Raman spectra, which means that Raman spectroscopy has proven to be an effective analytical approach in geology, semiconductor, materials and polymer science fields. The application of Raman spectroscopy and microscopy within biology is rapidly increasing because it can provide chemical and compositional information, but it does not typically suffer from interference from water molecules. Analysis does not conventionally require extensive sample preparation; biochemical and structural information can usually be obtained without labeling. In this protocol, we aim to standardize and bring together multiple experimental approaches from key leaders in the field for obtaining Raman spectra using a microspectrometer. As examples of the range of biological samples that can be analyzed, we provide instructions for acquiring Raman spectra, maps and images for fresh plant tissue, formalin-fixed and fresh frozen mammalian tissue, fixed cells and biofluids. We explore a robust approach for sample preparation, instrumentation, acquisition parameters and data processing. By using this approach, we expect that a typical Raman experiment can be performed by a nonspecialist user to generate high-quality data for biological materials analysis.	Mar 2016

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