About the Beamline

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.

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 cryo-SIM which allows 3D super-resolution fluorescence cryo-imaging.

We have custom built our microscope 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 647nm). The system's optical features, such as filters, can be found online in the SPEKcheck tool. This program allows the efficiency and performance of different fluorophores to be assessed in silico with respect to specific features in our setup, thereby enabling the intelligent design of fluorescence-dependent experiments. 

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

• (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 possibility of ice crystal formation
• (c) it can image relatively thick samples and
• (d) it has good out of focus light suppression leading to high contrast images even in samples with thickness of 10μm or more. 

At beamline B24, the cryo-SIM 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 cryo-SIM has been developed by our collaborators at Micron Oxford. See the 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 suspension. The basic steps of the experimental workflow includes:

1. Cells are cultured on carbon films attached to gold electron microscopy grids while fluorescent markers can be endogenously expressed or added to cell-culture media for staining.  
2. Conventional light and fluorescence 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 grid samples using a cryostage-equipped light microscope (Zeiss AxioImager2) with fluorescence detection capabilities to examine cell morphology and bright field imaging to confirm absence of non-vitreous ice or signs of damage, such as cracks.
5. Cryo-SIM for high-resolution fluoresence imaging.
6. Cryo-SXT data is then collected on the same grids in the same regions of interest.
7. 3D X-ray data reconstruction to tomograms using IMOD's weighted back projection (WBP), serial iterative reconstruction technique (SIRT) and automatic patch tracking methods.
8. In silico 3D correlation of the processed data from both high-end microscopes using eC-CLEM plug in the open-source software, Icy.