Manuals and Software

Download the B24 User Pre-visit Checklist prior to your beamtime and complete the necessary tasks.

If you have any questions after reading the checklist, contact details for B24 staff members can be found in the Staff Profiles section.

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: this software offers semi-automatic alignment 2D of brightfield images from the cryoSIM and SXT to easily transfer regions of interest (ROIs) from the cryoSIM can to the the SXM.
• XMBatch: user-friendly software for queueing up batches of scans on the TXM.
• 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.