Technical Beamline Information

B24 is the transmission cryo-soft X-ray tomography (cryo-SXT) beamline at Diamond Light Source aiming to satisfy the growing need for biologically relevant X-ray tomography on unstained biological samples under near-physiological conditions. The operational energy range for B24 is 200eV-2.6keV, which allows imaging via absorption contrast within the water window as well as phase contrast at higher energies.

B24 has also invested in the development of a bespoke super-resolution light microscope, cryo-structured illumination microscope (cryo-SIM), located at the beamline which allows correlative light/X-ray microscopy (CLXM) techniques to be explored on site. This cryo-SIM equipment is the first of its kind worldwide.

Representative cells and organisms that have been examined by cryo-SXT

Design Specification

• Beamline acceptance: 2x2mrad
• Energy range: 200eV-2600eV
• Single toroidal focusing 1:1 magnification
• Plane grating monochromator (2x VLS gratings, 1x plain mirror)
• Absorption- and Phase-contrast modes
• Resolving power > 1000
• Beam size at sample: 20um
• Photon flux: ~10⁸ photons/um²
• Resolution: 30-40nm
• Time to collect single tomographic series < 5 mins
• Detector: Direct detection CCD (Pixis) and back-illuminated sCMOS (PCO.edge)

Beamline Implementation

B24 uses bending magnet radiation and comprises a horizontal deflection toroidal mirror (located within the synchrotron machine vault) which focuses and directs the beam through a Variable Line Spacing - Plane Grating Monochromator (VLS-PGM) to a focal point at the PGM's exit slits. Diagnostics after the exit slits allow the beam to be characterised before it enters the Endstation (a commercial in-vacuum cryo X-ray microscope). There, the beam is focused onto the sample by either a single-bounce capillary condenser (for absorption contrast) or another wider single-bounce capillary condenser (for phase contrast). The resulting image is refocused by a Zone Plate (25nm or 40nm for absorption; 30nm for phase contrast) onto a direct detection CCD (for absorption contrast) or sCMOS (for phase contrast).

Endstation Characteristics

The Endstation, supplied by Xradia (now Zeiss), is designed to allow the collection of 3D tomographic data on vitrified biological samples mounted on electron microscopy grids. An automated sample loading system allows up to four samples to be loaded into the chamber at a single time. Samples are maintained at cryo-temperatures throughout data collection.

Ancillary Components

• Basic laboratory equipment 
• Zeiss Axioimager with Linkam cryostage for grid preliminary analysis and mapping (max resolution 50x, variable filters)
• Leica EM GP Plunge freezer
• Static and shaking CO₂ incubator 
• Laminar flow cabinet
• Leica EM HPM100 High pressure freezer
• Biodrop
• FisherBrand Sonicator
• Zeiss Stemi 2000 Stereo microscope
• Haier -80°C Freezer

Current Operating Parameters

• Absorption contrast at 500eV (water window) with a 25nm zone plate
• Zernike phase contrast at 2.5keV with a 30nm zone plate (under commissioning)
• Samples mounted/grown on 3.05mm finder grids (e.g. Quantifoil G200F1 Au)
• TXRM loading capacity: 4x grids
• Data collection strategy: tilt series, -70° to 70°, 0.5°/1° steps
• Data handling: automated backup and reconstruction In-line fluorescence detection capability