Poslední úprava: Mgr. Zuzana Burdíková, Ph.D. (19.01.2023)
The course is an introduction to superresolution microscopy techniques STORM and SIM. The theoretical background will be complemented with many practical presentations. Experts and scientists from the field teach the lectures and practical sessions. The two-day theoretical course with practical demonstrations and exercises is intensely devoted
to modern methodologies of super-resolution light microscopy (SIM, STORM). The priority is the practical demonstration of the image analysis software by the
authors themselves. After completing the course, the participants will be able to determine what is appropriate microscopy technique used to answer the research questions, including sample preparation and data processing for publication. The course will be taught in English.
Information about the course
Title – Image analysis and data processing in superresolution microscopy: fairSIM and ThunderSTORM open source systems
- Code – MB100P04
Guarantor – Msc. Zuzana Burdíková, Ph.D
All lecturers – Msc. Zuzana Burdíková, Ph.D, Msc. Zdeněk Švindrych, Ing. Martin Schätz, Ph.D. , MSc. Ondřej Šebesta, MSc. Peter Hohoth, Ph.D., Msc. Marian Novotny, MD. Robert Haase, Ph.D., Msc. Karel Stepka
Faculty, department – Faculty of Science, Laboratory of Fluorescent and Confocal Microscopy, Charles University
Credits – 02 ECTS
Language of instruction - English
Flagship and/or transversal skills – Flagship 4, Critical thinking
Capacity - 15
Examination – project
Minimal requirements, prerequisites, conditions for selection, and enrolment of students: Basic knowledge of Image J is required. The course aims to explain the workflow in Image Analysis, and processing, and it is assumed that the student is interested in Image Analysis.
Virtual mobility - yes
How the course will be taught (one week) and the starting date –block; on august 23. -25. of the SUMMER semester of 2022,
Syllabus
Day 1
Introduction to superresolution microscopy: methods, principles, theoretical background image formation in Fluorescence Microscopy
Resolution and Noise
Super-resolution Localization Microscopy (STORM, PALM, DNA-PAINT, …)
Structured Illumination Microscopy (SIM)
Introduction to image processing in FIJI, ImageJ
Two-channel colocalization (mitochondrial and membrane labeling)
p-Value of colocalization, data filtering
Quantitative analysis
Quantitative data (filtering, thresholding, background separation)
Filter on photon count
Data visualization, 3D visualization
Histogram, measurement of different parameters
Pseudo-colors, pixel size, rendering mode, multicolor image
Image export
Image reconstruction of superresolution SIM data in ImageJ: fairSIM Super-resolved structured illumination microscopy (SR-SIM)
illumination patterns
SR-SIM image reconstruction algorithms, access to the plugin, and source code
fairSIM, ImageJ plugin that provides SR-SIM reconstructions
FairSIM reconstruction of data sets
Automated reconstruction parameter estimation for data sets of adequate quality
Practical part: ImageJ, fairSIM hands-on
Day 2
ThunderSTORM: a comprehensive ImageJ plug-in for SMLM data analysis and super-resolution imaging https://zitmen.github.io/thunderstorm/
Single Molecule Localisation (briefly)
The idea behind ThunderSTORM
Workflow - localization, filtering, rendering
Simulation engine
3D STORM - astigmatism method
Scientific lecture Case Study
Study Methods for the quantitative analyses of SMLM data, Coordinate-based colocalization / Nearest Neighbor Distance (NND) analysis in ThunderSTORM
Voronoi tesselation in Coloc-Tesseler software
quantitative evaluation of the spatial organization in the cell nucleus
Practical Part ThunderSTORM hands-on sessions: Individual work with ThunderSTORM software
Day 3
Customizing Fiji/ImageJ with ImageJ Macro, Hands-on
Interactive Design of GPU-accelerated Image Data Flow Graphs in Fiji
Introduction to Data Management and FAIR principles
Pixel Classification Using ILASTIK
Object Detection Using StarDist
Practical part : ILASTIK or StarDist Hands-on
