Image analysis for single molecule localization microscopy
Siân Culley (@SuperResoluSian)
In this session, we will look at Fiji workflows for single molecule localization microscopy (SMLM) data. The main points that we will cover are:
Analysis of sparse blinking datasets: the ‘classic’ SMLM dataset
- Use of QuickPALM and ThunderSTORM to localize individual molecules
- Interpretation of particle tables
- Drift correction
- Rendering and visualisation of images
3D SMLM data sets
- Different types of 3D calibration data
- Localization and visualisation in ThunderSTORM
Dense blinking datasets: the ‘tricky’ SMLM dataset
- Use of HAWK for pre-processing dense data
- Options in ThunderSTORM for fitting dense data
- Using SRRF for live-cell data
Multicolour datasets
- Chromatic aberration correction using NanoJ
Assessing data quality
- Using SQUIRREL to measure the resolution of images and to assess the quality of images.
Homework: To prepare for this session, students are asked to install Fiji (https://imagej.net/Fiji/Downloads). We will be using the following plugins:
- QuickPALM (pre-packaged into Fiji)
- ThunderSTORM (installation instructions at: https://github.com/zitmen/thunderstorm)
- HAWK (https://www.coxphysics.com/ - copy the version 1.1 .jar file into the plugins folder in your Fiji install)
- NanoJ-Core, NanoJ-SRRF, NanoJ-SQUIRREL (In Fiji, go to Help>Update…>Manage Update Sites and check the boxes for NanoJ-Core, NanoJ-SQUIRREL and NanoJ-SRRF. Close this window, then press ‘Apply changes’ We will also be using various test data sets – please download these from https://www.dropbox.com/sh/soz472wr42kvy4x/AABskdDlq4wixzTH91ESsCHxa?dl=0 (sorry the files are a bit big, this is an occupational hazard of SMLM!). There are two .zip files; one contains some freely available example data that I’ve taken from various sources (listed in .rtf file) and the other contains some of my own data. Please download both!
In case of any issues, please contact Siân: s.culley@ucl.ac.uk