Image Analysis and Data Processing in Super-Resolution Microscopy 2020#
Program 24.–26.8 2020
Day 1#
Location: KONFMI Microscopy Center, Viničná 7, Praha 2, study room 311
Lecture schedule
9:00 – 9:10 Introduction and practical information (Ondřej Šebesta, KONFMI, PřFUK)
9:10 – 9:40** Introduction to dSTORM, fPALM, SIM superresolution microscopy: methods, principles, theoretical background** (Zuzana Burdíková, KONFMI, PřFUK)
Super-Resolution Localization Microscopy (The Diffraction Limit of Light, Airy Disks,
The Rayleigh Criterion, Sub-Pixel Localization)
Super-resolution localization microscopy techniques (Photoactivated Localization Microscopy (PALM), Stochastic Optical Resolution Microscopy (STORM), DNA-Based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT), Structured Illumination Microscopy (SIM)
DETERMINING RESOLUTION IN SUPER-RESOLUTION LOCALIZATION MICROSCOPY (Localization Accuracy, Molecular Density)
9:10 – 9:40 ** Introduction to dSTORM, fPALM, SIM superresolution microscopy: methods, principles, theoretical background** (Karel Štěpka, Faculty of informatics, MU)
10:20 – 10:50 Coffee break
10:50 – 11:20 Image processing in FIJI, ImageJ (Karel Štěpka, Faculty of informatics, MU)
Data quantification and image analysis basics
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
11:20– 11:50 Scientific lecture: Super-resolved nuclear landscape defined by dSTORM (Peter Hoboth, UMG)
optimizations of 2-colors labelling and dSTORM acquisition
quantitative analysis of spatial nuclear organization
nanoscale map of nuclear PI(4,5)P2 defined by 2-colors dSTORM
11:50 – 12:30 Lunch
12:30 – 13:30 SHORT TALKS: Presentations of the participants’ projects
13:30 – 14:00 Coffee break
14:00 – 14:30 Image reconstruction of superresolution SIM data in ImageJ: fairSIM
(Martin Schätz, KONFMI, PřFUK)
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
14:30 – 15:00 SIM check tool FIJI plugin and nano-squirrel (Martin Schätz, KONFMI, PřFUK)
Three-dimensional structured illumination microscopy, 3D-SIM
SIMcheck, a suite of ImageJ plugins, advanced calibration tools
Integration of SIMcheck functionalities to the SIM imaging workflow
NanoJ-SQUIRREL, an ImageJ-based analytical approach providing a quantitative assessment of super-resolution image quality
15:00 – 15:30 Coffee break
Practical part
15:30 – 18:00 ImageJ, fairSIM hands-on
Group 1: beginners (Zuzana Burdíková, KONFMI, PřFUK)
Group 2: intermediate (Ondřej Šebesta, KONFMI, PřFUK, Karel Štěpka, Faculty of informatics, MU )
Group 3: advanced (Martin Schätz, KONFMI, PřFUK)
18:00 – 18:30 Optional: Facility tour - KONFMI (Zeiss Elyra PS, Zeiss Axio Scan) Test Samples for Optimizing STORM Super-Resolution Microscopy (Ondřej Šebesta, KONFMI, PřFUK, Zuzana Burdíková, KONFMI, PřFUK)
Refreshment included
Day 2#
Location: KONFMI Microscopy Center, Viničná 7, Praha 2, study room 108
Lecture schedule
09:00 – 9:30 Image Deconvolution and NIS-Elements (Martin Berger, LIM)
09:30 – 10:00 Scientific lecture: Receptor clustering in super-resolution microscopy - how to capture reality on the cell membrane (Barbora Kalousková, KONFMI, PřFUK)
10:00 – 10:15 Coffee break
10:15 – 10:45 ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging (Zdeněk Švindrych, Dartmouth College) https://zitmen.github.io/thunderstorm/
Analyzing single-molecule data, filtering, detection, drift correction, rendering
examples of different filters, thresholds, see detection molecules,
2D, 3D analysis, simulations, documentation, updates
FIJI- help online with software, web site, supplementary data, choice of filters
Non-fitting methods, fitting methods, fitting analysis, statistical tests, rendering
10:45 – 11:30 ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging (Zdeněk Švindrych, Dartmouth College)
Challenges of settings combinations, formats, interactive histogram, drift corrections
Tools for creation and simulation – density mask, background mask, results table
Realistic simulations (comparing real and simulated data)
3D STORM - astigmatism method
11:30 – 12:30 Lunch
Practical part
12:30 – 19:00 ThunderSTORM hands-on sessions: Individual work with ThunderSTORM software (Ondřej Šebesta, Zuzana Burdíková, Martin Schätz, KONFMI, PřFUK)
Coffee break included
Group 1: beginners (Zuzana Burdíková, KONFMI, PřFUK)
Group 2: intermediate (Ondřej Šebesta, KONFMI, PřFUK)
Group 3: advanced (Martin Schätz, KONFMI, PřFUK)
19:00 – 22:00** Informal discussion in the Academic Club**
Day 3#
Location: KONFMI Microscopy Center, Viničná 7, Praha 2, study room 108
Lecture schedule
09:00 – 10:00 Acquisition and localization: From raw pixel data to molecules localizations, and preparing data for external work (Štěpán Svoboda)
10:00 – 10:15 Coffee break
10:15 – 11:30 Analysis and visualization options of NIS-Elements (Štěpán Svoboda)
11:30 – 12:30 Lunch
Practical part
12:30 – 17:00 STORM module in NIS-Elements** :** Individual work
(Ondřej Šebesta, Zuzana Burdíková, Martin Schätz, KONFMI, PřFUK)
Coffee break included
A Group 1: beginners (Zuzana Burdíková, KONFMI, PřFUK)
A Group 2: intermediate (Ondřej Šebesta, KONFMI, PřFUK)
B Group 3: advanced (Martin Schätz, KONFMI, PřFUK)
17:00 – 17:10 Final evaluation and closing remarks
Refreshment included