Manuals and Software

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.

Manuals for B24 instruments, techniques and workflow:

• Fiducialisation and grid preparation - this is a quick guide on how to prepare grids prior to tissue culture as well as the current standard at B24 for fiducialisation.
• Plunge freezing - step-by-step protocol for vitrification of biological samples at B24 on TEM grids.
• Cryo-SIM -
  • User manual (new version of cockpit)
  • concise protocol (old version of cockpit)
  • expanded user manual: (old version of cockpit) 
  • JoVE protocol and video outlining steps for acquiring super-resolution fluorescence images using the cryo-SIM.
• Batch proecssing of raw structured illumination (SIM) data to pseudo wide-field using this custom-written Fiji macro. Please note that the SIMCheck plugin needs to be installed in Fiji before this macro will work.
• Batch intensity projection of processes structured illumination (SIM) data using this custom-written Fiji macro.
• TXRM - step-by-step protocol for mosaic and tilt series acquisition using B24's transmission X-ray microscope (TXRM).
• Tomogram reconstruction 
  • Start here: Quick start guide for tomogram reconstruction at B24
  • Online tutorial on how to use etomo in IMOD for tomogram reconstruction
  • Resource files for practicing tomogram reconstruction with IMOD
  • A PowerPoint presentation from a B24 workshop on how to use IMOD for tilt series reconstruction into a tomogram. 
• Project data organisation - a concise guide on directory structure during data sorting.
• Correlation -
  • Start here: Quick start guide for correlation of B24 data
  • a STAR protocols paper on using eC-CLEM for 2D and 3D correlation of fluorescence and X-ray images.
  • Video tutorial showing correlation usig eC-CLEM and chromatic shift correction using Chromagnon
• Image analysis - a concise guide on how to write Macros for automated image analysis with Fiji (ImageJ).
• Segmentation-​​​
  • Start here: Quick start guide for SXT segmentation
  • Contour Instruction guide
  • Quick guide to using Fiji segmentation editor

In-house software and scripts:

• StitchM: a Python 3 package with a command line interface. StitchM creates a flat mosaic image from the cryoSIM brighfield data. Regions of interest can be added with the OME-TIFF standard, viewable with Bioformats in ImageJ/Fiji.
• txrm2tiff: a Python 3 package with a command line interface. txrm2tiff converts the raw data from the SXM into tiff files, a widely-supported lossless format. This is has been developed for B24 as part of our automatic processing pipeline.
• GridSNAP: currently undergoing commissioning, this software offers a web-based GUI for semi-automatically alignment 2D brightfield images from the cryoSIM and SXT. This enables a coordinate transform between microscopes, which ensures that the regions of interest from the cryoSIM can be quickly identified and imaged on the SXM. GridSNAP also ouputs transform data that can later be used as a starting point when correlating the 3D data sets.
• omexml-dls: a Python 2/3 module for creating and interacting with OME xml metadata. This was created for lightweight access to OME metadata by B24's software.
• Fluorescence Merging Macro: for merging and flattening SIM fluorescence images in Fiji. To download, right click this link and select "Save as...".
• Batch proecssing of raw structured illumination (SIM) data to pseudo wide-field using this custom-written Fiji macro. Please note that the SIMCheck plugin needs to be installed in Fiji before this macro will work.
• Batch intensity projection of processes structured illumination (SIM) data using this custom-written Fiji macro.
• SuRVoS: Super Region Volume Segmentation.

Recommendations:

• Chromagnon: Open source software for chromatic shift correction of fluorescence images
• IMOD: a set of image processing, modeling and display programs used for tomographic reconstruction. IMOD's batchruntomo is used by our automatic processing pipeline and we recommend IMOD's etomo for manual reconstruction.
• eC-CLEM: a plugin of Icy for multidimensional image alignment in 2D or 3D.
• Fiji (Fiji is just ImageJ): an image processing package, ImageJ, bundled with commonly used plugins. Our commonly used plugins include:
  • SIMCheck: a suite for super-resolution structured illumination microscopy data quality control.
  • Bioformats: for reading and writing life sciences image file formats (including OME metadata).

Reference Library:

B24 has a continually updated Endnote Library list with relevant references for TXM, SIM and correlation. We are happy to share this with you; please contact any of the B24 team, tell us your email address and we will send you a link to the library.