Microscopy & Microtechniques


The intracellular world in 3D and in colour: developments at the biological cryo-imaging beamline B24 at the UK’s national synchrotron, Diamond Light Source


Maria Harkiolaki and Chidinma Okolo.


Principal Beamline Scientist Maria Harkiolaki and Support Scientist Chidinma Okolo, explain how two powerful microscopy methods come together to deliver complementary high resolution cryo-imaging of cell structure and compositions at near-physiological conditions.


The biological cryo-imaging beamline B24 at the UK synchrotron Diamond Light Source (https://www.diamond.ac.uk/Instruments/Biological-Cryo-Imaging/B24.html) has developed a high resolution 3D cryo-imaging platform that delivers detailed 3D X-ray and visible light fl uorescence imaging of cells and cell populations at near- physiological conditions. The platform has been presented in a recent publication [1] in which, it was used in the study of reovirus vesicle egress in mammalian cells and allowed the tracking of viral capsids within endosomal vesicles across the cytoplasm in 3D in the early hours post infection.


Background


The success of biological research depends on understanding a living system in depth from its basic chemical composition to the structures that make up cells and the interactions within tissues and organisms. Microscopy is fundamental in this process allowing us to directly document the complex architecture of life, but as all research tools, it stands restricted by its subject matter. To see the macroscopic means that the observer gains a top level view of a system but misses the fi ne details, whereas when we look into the microscopic, context is often sacrifi ced. For example a CAT scan with a wide fi eld of view will document whole tissue arrangements within an organism but it takes an electron microscope with a fi eld of view of just a few microns to visualise the fi ne structures within individual cells from that same organism. And for each and every step we take towards ever increasing imaging resolution and detail capture, sacrifi ces need to be made. Cells need to be extracted from their natural environment, modifi cations need to be made to highlight them and their very chemical composition needs to be altered to immobilise them - an image can only be as clear as sample movement and vibrations within allow it to be, therefore, becomes imperative when we seek to gain information on cellular structures and fi ne chemical localisation to engage microscopy techniques with variable resolutions while retaining as much as possible physiological features and relative sizes. Moreover, when progressing the scales of image resolution, we ideally move across methods that overlap in their capability boundaries (resolution increases gradually as fi eld of view collapses) allowing the productive overlap of data from one scale to the next with high resolution imaging benefi ting from the wider context supplied by less powerful techniques.


The correlative cryo-imaging beamline B24 at the UK synchrotron Diamond Light Source endeavours to address all of the above and deliver directly correlatable 3D imaging data across scales and contrasting schemes on samples at near-physiological state. The imaging platform at B24 comprises of a novel super resolution fl uorescence structured illumination microscope (cryoSIM) and soft X-ray tomography end station (cryoSXT) and allows users to visualise biological samples such as cells and tissue slices.


It offers 3D cellular correlative imaging at cryogenic temperatures and features (a) 3D data capture, (b) same sample fl uorescence and X-ray imaging, (c) minimal sample processing requirements, (d) image resolution to 25nm, (e) rapid data collection and (f) easy to follow access procedures through peer-review processes (for academics). In addition, the beamline also provides access and support on accessory equipment to allow sample preparation and evaluation as well as data processing, analyses and correlation.


Beamline B24


Biological imaging across scales and B24 remit


You would benefit from B24 if you...


work with cells and want to understand/image their architecture


can track your protein of


interest intracellularly with fluorescence


are interested in the effects of a substance in cell structure/function


study a pathogen and


would like to better understand its biology


study the effect of vaccines or


pharmaceutics in cells


investigate intracellular or intercellular cell-to-cell signaling and communication


study microorganisms and their interactions


need to map size,


structure and distribution pf of organelles in cells


Apply for rapid access for preliminary results


an established research project with a real need for correlative cryo- imaging


Apply for standard access for full investigations


expertise in biological research or related area


Visit beamline B24 (in person or online) and research relevant literature


Contact the beamline


team to discuss suitability and requirements


Projects awarded time


get full support on sample handling and transfer, data collection and processing


Help is available on sample preparation and experiment design (protocols, access to facilities on site and hands-on assistance) after consultation with the beamline team in the form


pursue new and exciting insight into biological systems and processes


You need to have...


To access beamline B24:


What kind of support you can expect.


You as the user are expected to...


communicate and publish results as appropriate in a timely fashion (academic users only)


deposit and make Support for. data


processing and analyses is also available


publicly available project data (usually post- publication)


Suitability and requirements for use of the B24 facility. Current requirements for biological imaging


Diamond Light Source beamline B24 main cabin (top panel); the cryoSIM (cryogenic temperature-Structured Illumination Microscope) (bottom left) and cryo-SXT (cryogenic temperature-Soft X-ray Tomography) microscope (bottom right).


The requirements for biological/biomedical imaging at the cellular level continue to increase and become more complex as we probe further into biological systems.


INTERNATIONAL LABMATE - SEPTEMBER 2020


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80  |  Page 81  |  Page 82  |  Page 83  |  Page 84