Before you apply, there are a few steps to take:
- Please take the time to look through our publications (here and here) to help you make the best use of our techniques.
- Make sure you can prepare samples correctly - this should be discussed with us before applying.
- Please get in touch! We are keen to discuss potential projects with you.
Routes of Application
This route is for proof-of-principle or seed/feasibility studies. It is allocated based on availability and after consultation with the Principal Beamline Scientist.
This access route is for projects that have preliminary data or which have corroborating evidence that the B24 scheme will most probably produce good imaging which will contribute towards publication or further experiments.
Brief Instructions for Submission of Proposals
- Please register or login to the Diamond UAS and 'Create New Proposal'.
- You will need to identify it as standard call or rapid access when prompted and a proposal number will then be allocated and you will be directed to a proposal setup page.
- At the Science Overview tab please pick up the template and fill in accordingly.
- It is important to have a strong biological/biomedical science case and present previous results from X-ray or other imaging techniques. Please include images (e.g. fluorescence in tissue culture) if available. We will evaluate the proposal on its scientific merit but also on the availability of samples and the know-how of the home laboratory on sample handling and sample preparation for our beamline.
- If we have discussed working together on sample preparation then that needs to be mentioned here with an estimate of the time/effort that you will require.
- Please refer to the beamline webpages and in specific the call for proposals tab to see what we can offer.
- Through absorption contrast you are expecting to get a clear three dimensional map of cellular compartments/organelles (membranes absorb really well!) as well as potentially highly clustered or structured protein-containing molecular modules. The nuclear area has high background and although we can identify nucleoli the level of detail obtained from there is not great at present.
- The technique will allow you to see structural features of your cells (which can be anything from tissue mammalian to larger bacteria but only cat 1 & 2 for now) but will also provide context for any fluorescence marking that you have observed or engineered already.
- If you want to use the in house cryo-SIM before X-ray data collection, please include this in your proposal but keep in mind that the equipment is not accessed via the UAS as it is still being developed. However, we would be happy to organise access if there is a scientific case and it is at the time in full working order.
- We would expect you not to need funding for consumables but please do let me know if you think you might.
- During your proposal preparation, please select 'B24 - Cryo Soft X-ray Tomography' at the beamline tab.
- When prompted to declare the number of shifts please keep in mind that we will allocate user time in 8hr lots (9am-5pm = 1 shift) and you will need at least two days per sample (which includes sample transfers and data collection but not sample preparation) so a three day request for a specific project is reasonable for a start given delays we might encounter at the beamline. If however you have several projects you will need to scale up accordingly.
- You will require no access to the Research Complex at Harwell (RCaH). Please refer to Beamline Summary for ancillary components at B24.
- All highlighted sections need to have some info before you can submit your proposal.
Typical Workflow for One Experiment
Before TXM allocated time:
Month -1: Grid mapping (conventional cryo-fluorescence)
Month -2: Cryo-SIM (equal shifts to TXM required)
Activity per 3-day allocation - 4 samples loaded:
Day 1: sample loading, preliminary mapping, strategy
Day 2: Data collection
Day 3: Data collection
After TXM allocated time:
Month +1: Data processing, segmentation and correlation
B24 X-ray Tomography Workflow
- Users are strongly advised to use 3.05mm finder grids (e.g. Quantifoil G200F1 Au).
- If B24 team members have been consulted, sample prep is likely to be successful.
- Users will be asked to provide standard microscopy evidence that samples arriving at the beamline are suitable for both X-ray and SIM data collection.
- Image acquisition based on absorption with 40nm and 25nm zoneplates will be offered on alternate runs.
- Phase contrast mode will be under commissioning – no time offered yet.
Scheduling Pre- and Post-Beamtime
Setup time is often required prior to beamtime. UAS now has the functionality to book pre and post beamtime providing that it is contiguous with the beamtime. The benefit of scheduling time in this way means that users are able to request additional nights for accommodation and can clearly indicate when they are arriving, making all information much more joined up.
NOTE: This time can only be scheduled by the scheduler if no arrangements have been submitted by the team. If arrangements have already been booked then any updates must be done manually via the User Office. For that reason, pre and post time should be scheduled as part of the AP scheduling process.
- In the scheduling screen unlock the session (right mouse click to unlock).
- Use the right mouse click again to get the menu and select "Edit".
- A pop-up box will appear where you can add Pre Time/Post Time or both. Time can be added in days/hours/minutes or a combination of all three. Add scheduling notes and reason for the session change. Press OK button at bottom of page.
For up-to-date manuals of B24, refer to beamline manuals.
Two 8-hour shifts are available each day (except Machine Days).
Representitive Shift Structure
For single shifts
09:00 – 10:00
Arrival of users
10:00 – 12:00
Sample mapping and testing
12:00 – 13:00
13:00 – 16:30
Cryo-soft X-ray Tomography (2 grids per shift are likely to be imaged)
16:30 – 17:00
End of day activities
For consecutive shifts
09:00 – 10:00
Arrival of users
10:00 – 12:00
Sample mapping and testing
12:00 – 13:00
13:00 – 17:00
end of shift 1
17:00 – 00:00
00:00 – 01:00 (next day)
end of shift 2
End of day activities
Health and Safety
Cryo Soft X-ray Tomography (cryo-SXT)
The full field transmission microscope at beamline B24 is a Zeiss UltraXRM-S220C, and delivers imaging by absorption contrast within a defined spectral area known as the ‘water window’ (between the k absorption edges of carbon at 284eV and oxygen at 543eV). Within this region, carbon-rich biological structures absorb X-rays more than the surrounding oxygen-rich medium and the resulting contrast allows the delineation of cellular features in recorded projections.
Illumination at the B24 transmission X-ray microscope (TXM) is supplied by a bending magnet, focused by a toroidal mirror and conditioned with a plane grating monochromator and exit slit module that can deliver highly monochromated beam between 200-2600eV. This focused beam (now 25.6m away from the bending magnet source) is the real source point for our imaging and it is further focused by a glass capillary condenser lens on the sample. A zone plate objective focuses the resulting projections onto a highly sensitive photon detector (Pixis 1024B CCD, Princeton Instruments). Samples are placed into the X-ray microscope using a transfer chamber that facilitates transition from liquid nitrogen storage under atmospheric pressure to cooling via conduction in high vacuum (soft X-rays have poor penetration in atmosphere and successful propagation relies on good vacuum conditions). Samples at the imaging position are in close proximity to the microscope optics and this results in limited tilt potential. The TXM has in-line visible light microscopy capacity and samples are examined using a broad-spectrum LED and a conventional 20x objective located within the sample chamber for a pre-data collection inspection to locate and confirm regions of interest; previously identified through by any number of visible cryo-light microscopies (samples are vitrified before exposure to X-ray radiation to preserve them against radiation and heat damage) conventional or super resolution. 25nm and 40nm objectives (depth of focus of circa 1 and 3.5 microns respectively) are available for use depending on the imaging requirements of samples at hand. The magnification of the system is up to 1,300x with a field of view of between 10-16 microns allowing for 2.5 pixel sampling at the resolution cut off with the detector.
3D imaging data is collected as a series of projections at defined regular angular steps (tilt series up to 140°; projections collected typically at 0.5-1s exposures) and is subsequently processed into tomograms using IMOD through a custom, fully automated, in-silico pipeline. When a larger field of view is required to capture substantial cellular content extending to several tens of microns with the aim of capturing vesicles and substructures of interest as well as the overall cellular landscape in which they reside, multiple tomograms can be collected at adjacent overlapping areas.
3D cryo-Structured Illumination Microscope (cryo-SIM)
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 www.micron.ox.ac.uk/software/spekcheck/ 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 didmensions, 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 proteins, (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
Experimental workflow of correlative cryo-SXT and cryo-SIM
Cells that are to be imaged are typically grown as adherent monolayers, multilayers or in suspesion. The basic steps of the experimental workflow includes:
• Adherent cells are cultured on carbon films on gold transmission electron microscopy grids while fluorescent markers cand be endogenously expressed or added to cell culture media for uptake.
• Conventional light and flurescence microscopy can be used to evaluate cell attachement, growth, confluency and distribution of live cells in media.
• Sample cryo-preservation by following plunge freezing technique.
• 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.
• Cryo-SIM for high-resolution fluresence imaging.
• 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.
• 3D X-ray data reconstruction to tomograms using IMOD's weighted back projection (WBP), serial iterative reconstruction technique (SIRT) and automatic patch traching methods.
• In silico 3D correlation of the proceesed data from both high-end microscopes using eC-CLEM plug in the open-source software, Icy.