Download the user pre-visit checklist prior to your beamtime and complete the necessary tasks.
User Pre-Visit Checklist
This checklist is designed to familiarise users with the workflow at B24 and to prepare them to gain the most out of their visit. If you are visiting the beamline for the first time, please complete these tasks.
STEP 1 Sample preparation
- Read the step-by-step guide for grid and fiducial preparation.
- Read the step-by-step guide for plunge freezing.
- OPTIONAL: For more detailed instructions, see this Nature Protocols paper
STEP 2 Cryo-structured illumination microscopy (cryoSIM)
To load samples and acquire data:
- Read the user manual for the cryoSIM. You may find this concise guide helpful on the day.
- Read the tutorial in the JoVE paper and watch its video demonstration.
- Consult the Setup menu in SpekCheck to determine which of the ‘cryoSIM’ laser/filter combinations would be most suitable for the fluorophores in your samples.
- Familiarise yourself with the concept of chromatic aberration, and be aware that this will cause fluorescence to focus at different Z depths in your cryoSIM sample.
- Download and install Chromagnon, a program you will use align cryoSIM data by correcting for chromatic aberration between data collected at different wavelengths.
To inspect data:
- Install Fiji and the SIMCheck plugin:
Go to ‘Help’ -> ‘Update…’. A window labelled ‘ImageJ Updater’ should appear. Select ‘Manage update sites’ within the ImageJ Updater window, then scroll down to SIMCheck and ensure it is ticked. Close that window and select ‘Apply changes’. Restart Fiji.
- Consult the SIMCheck website and publication to learn how to use the plugin.
- View Figure 1 of this publication to familiarise yourself with common artefacts in cryoSIM data, including ghosting, hammerstroke, hatching, honeycomb, lensing, and Z-wrapping.
- OPTIONAL: Download this Fiji macro to generate maximum Z projections of your data in batches as you may find these useful when choosing regions of interests on the X-ray microscope. SIMCheck must be installed to use this.
- OPTIONAL: Consult this guide and this presentation on how to write macros in Fiji.
STEP 3 Cryo-soft-X-ray tomography (cryoSXT)
To collect X-ray tomograms:
- Read the step-by-step guide
- To process X-ray tomograms:View this quick-start guide to using IMOD.
- Download the model datasets designed for cryo-electron tomography.
- Follow this tutorial to process the model datasets in IMOD.
- Once you receive your cryoSXT datasets, follow this tutorial to process them in IMOD.
STEP 4 Storage and transfer
To transfer data:
- Follow the steps here to use Globus or the file transfer protocol (FTP). It is best to do this as soon as possible after your visit because the data will only be available through this system for a limited time, after which it gets more difficult due to data archiving.
To organise data:
- Read the guide.
- Consult the directory structure system.
STEP 5 Correlative Light X-ray Tomography (CLXT)
- Beamline staff will show users how to perform a correlation on a beamline computer. In order to carry out correlation after your visit, you should download and install Icy, a platform for bioimage analysis, on a computer with at least 64-bit resolution and at least 16 GB RAM.
- Install the eC-CLEM v2 plugin in Icy. To do this, open Icy, then select the puzzle piece in the top-left corner of the window. This will open ‘Online Plugin’ in the setting menu. Select just ‘Plugin’, then tick ‘Allow beta version’ and press Apply. Return to ‘Online Plugin’, search for ‘ec-clemv2’, select it and click ‘Install’.
- Read this quick start guide to correlation.
- Read the full standard protocol in this publication while you watch the video tutorial.
STEP 6 Quantitation & Segmentation (OPTIONAL)
- Consult this decision tree to determine which segmentation tool is most suitable for your data.
- To segment dark thick features like mitochondria or lipid droplets, download Contour, read the publication and follow the protocol.
- To segment other features manually, use the built-in Segmentation Editor plugin in Fiji and follow the video tutorial.
- To segment other features using machine-learning, download SuRVoS, read the publication, and follow the protocol.
Congratulations on completing the checklist! We look forward to welcoming you to the beamline.