A spotlight on Beamline B24

B24 is a dedicated biology beamline that bridges the resolution gap between electron microscopy and conventional light microscopy. It has been designed specifically to meet the rising demand for tomographic imaging of biological specimens under near physiological conditions.

The full-field X-ray microscope allows whole cells to be rapidly imaged in great detail ( to 25nm) as shown in the diagram below. Users can study all aspects of cellular structure, including gross changes resulting from the effects of, for example, drugs, disease, and parasites. The beamline is ideal for collecting data on cells, as well as bacteria and lipid structures.
 
 
This state of the art equipment uses cryo soft X-ray tomography and absorption contrast imaging, making it ideally suited for projects that require high-resolution 3-D imaging of the cellular ultra- structure, to provide context and allow the interpretation of existing heterologous data.
 

The benefits of using Cryo-Transmission X-ray Microscopy (Cryo-TXM)

  •  Enables users to study the ultrastructure of vitrified whole cells at near-native states (no fixation, staining or micro-sectioning required)
  • The wavelengths of the X-rays used allows longer exposures and serial imaging without significant radiation-enduced sample deterioration.
  • Integrates with complementary techniques such as cryo-EM and light microscopy modalities.
  • Provides the capability to capture images of cellular ultrastructure to a resolution of 25-40 nanometres.

Find out more about this technique.

About B24

The beamline is designed to operate at low (soft) X-ray energies in the region termed the “water window” although it has also the capability of operating at higher energies. The water window is the region between the K-absorption edges of carbon at 284 eV and oxygen at 543 eV. Between these two energies oxygen is virtually transparent to X-rays whereas carbon is highly absorbing. Thus there exists a natural contrast between the vitreous ice surrounding a frozen biological cell and the carbon containing structures making up the cell. Initially B24 will operate in absorption contrast mode within the water window. In time, phase contrast imaging will be developed and the beamline will be upgraded to allow the study of samples designated as requiring containment level 3 facilities.

       Data collection at ~500eV (water window) using absorption contrast mode
       Extensive field of view (16 x 16 microns with stitching capability)
       Limited angle tomography (140 degrees total tilt)
       Resolution: ~25-40nm in up to ~12um thick samples
       Samples vitrified, not sectioned, stained, dehydrated or otherwise processed.
       Automatic data processing in place
       Scheduled upgrades to phase contrast and Biosafety level 3
 
 
 
 

Process and workflow.

The diagram below outlines a typical workflow at B24.  Apart from the benefits of its close proximity to other techniques within the Diamond synchtrotron, this facility also houses two distinct microscopes alongside the beamline to enable the acquisition of complementary 3D imaging data on chemical localisation within samples under study at visible light super-resolution.

In specific, a conventional visible light microscope coupled to a cryo-stage allows the overview, mapping and evaluation of samples, while the custom-made on-site super resolution cryo-Structured Illumination Microscope (cryoSIM) allow the fluorescence imaging of biological determinants to clear 3D representations that can be directly correlated to X-ray tomograms of the same areas. 
 
 

Complementary Techniques

Cryo- X-ray microscopy can be used in correlation with other imaging techniques, such as electron tomography and fluorescence to further enhance findings.
 
 

Applications

Near Native Cell Imaging

• Generate 3D slices through the sample;
• Collect data on intact and unstained cells through entire cell thickness (up to 8 μm);
• Probe a wide range of sample types from bacteria to whole mammalian cells
 
Structural Cell Biology
• Study the effect of drugs on living cells;
• Follow the effects of biological processes on the cell e.g. starvation;
• Monitor cell regulation processes;
• Investigate impact of nanoparticles on cells
 
Cell Infection Process
• Study cell infection;
• Image viral particles within infected cells;
• Examine changes to cell organelles post infection;
• Explore host-pathogen interactions
 
Functional Studies of Cellular Processes
• Perform correlative studies with cryofluorescence microscopy;
• Reveal localisation of fluorescent proteins within complex organelles;
• Capture processes of dynamic membrane trafficking.

 

Meet the team

 

To find out more or to speak to one of the team, visit our team page.