About the Beamline

Please refer to our recent publication for an overview of our capabilities.

The full-field transmission X-ray microscope (TXM) at beamline B24 is designed specifically to meet the rising demand for tomographic imaging of biological specimens under near physiological conditions. The technique bridges the resolution gap that exists between electron microscopy and conventional light microscopy and allows acquisition of tomographic data from both native and fluorescently-labelled samples.

At present B24 is operating at 500eV (in the “water window”) where the image contrast is generated by the preferential absorption of the X-rays by carbon with the surrounding water/ice remaining relatively transparent. Currently, operation of the beamline has extended outside the water window particularly to higher energies (although this is under commissioning). This should allow data to be collected either side of an absorption edge so that certain elements within a sample (e.g. Fe, P, Mg, Ca, Mn) can be highlighted. In addition it is planned to offer Zernike phase contrast at 2.5keV.

It is expected that, in the longer term, we will also upgrade the beamline to permit the study of samples which require Containment Level 3 facilities. The precise details of how this will be achieved will be established in due course. The timescale for this upgrade is at present unknown.

We have a novel CryoSIM (structured illumination microscope) which allows 3D super-resolution fluorescence cryo-imaging.

We have custom built our microscope (cryoSIM) around a commercial cryo-stage (by Linkam) with a long working distance air objective (100X, 0.9NA, 2mm working stage by Nikon) that delivers images with a maximum lateral resolution of circa 360nm at 525nm (green light). We currently have in place 4 illumination wavelengths (405, 488, 561 and 642nm) while the system's optical features are incorporated under the label cryoSIM in the available online SPEKcheck tool where the efficiency and performance of different fluorophores can be assessed in silico with respect to specific features in our setup, thereby enabling the intelligent design of fluroscence-dependent experiments. 

Our novel 3D-SIM optical setup with an optical design that overcomes many technical difficulties of imaging fluorescence at super-resolution under cryogenic conditions and doubles the achievable resolution in all threee dimensions, thus, giving us an 8-fold increase in volumetric resolution as compared to the diffraction limit. 3DSIM has distinct advantages over other suer-resolution techniques as

• (a) it is easily applied to multi-channel imaging using a range of common dyes or fluorescent protein
• (b) it uses short image acquisition times (20-100ms per single exposure) and relatively low light doses (10-100 W/cm² laser power) which in turns minimise sample heating and reducing the posibility of ice crystal formation
• (c) it is capable of imaging relatively thick samples and
• (d) it it has good out of focus light supression leading to high contrast images even in samples with thickness of 10μm or more. 

At beamline B24 the cryoSIM (first of its kind) has now been extensively tested and the data collected on a number of projects has been proved both robust and efficient. Raw 3D-SIM data is reconstructed and further analysed in itself or in the context of cryo-correlative imaging as described in the next step.

The cryoSIM has been developed by our collaborators at Micron Oxford. See  the latest publication giving more details on the construction of the microscope.

Cells that are to be imaged are typically grown as adherent monolayers, multilayers or in suspesion. The basic steps of the experimental workflow includes:

1. Adherent cells are cultured on carbon films on gold transmission electron microscopy grids while fluorescent markers can be endogenously expressed or added to cell culture media for uptake.  
2. Conventional light and flurescence microscopy can be used to evaluate cell attachement, growth, confluency and distribution of live cells in media.
3. Sample cryo-preservation by following plunge freezing technique.
4. Pre-mapping of the samples using a cryostage-equipped light microscope with fluoresence detection capabilites to examine cell morphology, to confirm absence of non-vitreous contaminants and mapped at low resolution.
5. Cryo-SIM for high-resolution fluresence imaging.
6. Development and application of TXRM data collection strategy including a) mapping with visible light in order to produce a new mosaic at the sample orientation followed by b) 2D X-ray mosaics at all ROIs and c) 3D X-ray data as a tilt series. 
7. 3D X-ray data reconstruction to tomograms using IMOD's weighted back projection (WBP), serial iterative reconstruction technique (SIRT) and automatic patch traching methods.
8. In silico 3D correlation of the proceesed data from both high-end microscopes using eC-CLEM plug in the open-source software, Icy.