Image Analysis and Data Processing in Super-Resolution Microscopy 2021#

Program 30.8. – 1.9. 2021

Day 1#

Location: KONFMI Microscopy Center, Viničná 7, Praha 2, room P09

9:00 – 9:10 Welcome note (Ondřej Šebesta)#

9:10 – 9:30 Introduction of new applications in KONFMI#

(Ondřej Šebesta, Martin Schätz)

9:40 – 10:20 ** Introduction to superresolution microscopy: methods, principles, theoretical background** (Zuzana Burdíková)#

  • Image formation in Fluorescence Microscopy

  • Resolution and Noise

  • Super-resolution Localization Microscopy (STORM, PALM, DNA-PAINT, …)

  • Structured Illumination Microscopy (SIM)

10:30 – 11:00 Introduction to image processing#

(Karel Štěpka, Faculty of informatics, MUNI)

11:00 – 11:30 Coffee break#

11:30 – 12:10 Image processing in FIJI, ImageJ**#

(Karel Štěpka, Faculty of informatics, MUNI)

  • 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

12:20 – 13:10 Image reconstruction of superresolution SIM data in ImageJ: fairSIM (Martin Schätz)#

  • 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

13:10 – 14:30 Lunch#

Practical part

14:30 – 16:30 ImageJ, fairSIM hands-on#

(Ondřej Šebesta, Zuzana Burdíková, Martin Schätz, Peter Hoboth)

Group 1: beginners (Zuzana Burdíková, KONFMI, PřFUK)

Group 2: intermediate (Ondřej Šebesta, KONFMI, PřFUK, Karel Štěpka, Faculty of informatics, MUNI)

Group 3: advanced (Martin Schätz, KONFMI, PřFUK)

15:00 – 15:30 Coffee break#

16:30 – 17:30 Optional: Facility tour - KONFMI: Zeiss Elyra PS, Zeiss Axio Scan (Ondřej Šebesta, Zuzana Burdíková)#

Refreshment included

Day 2#

Location: KONFMI Microscopy Center, Viničná 7, Praha 2, Study room P09

9:00 – 10:30 ThunderSTORM: a comprehensive ImageJ plug-in for SMLM data analysis and super-resolution imaging#

(Zdeněk Švindrych, Dartmouth College) https://zitmen.github.io/thunderstorm/

  • Single Molecule Localisation (briefly)

  • The idea behind ThunderSTORM

  • Workflow - localization, filtering, rendering

  • Simulation engine

  • 3D STORM - astigmatism method

10:40 – 11:20 Scientific lecture C ase Study part I: Nanoglance at the phospholipids of the gene expression compartments (Peter Hoboth)#

  • phosphoinositides of the nuclear speckles

  • clustering of RNA polymerase II (RNAPII) in the nucleoplasm and nuclear speckles

  • phosphoinositides and RNA polymerase II foci

11:20 – 11:40 Coffee break#

11:40 – 12:20 Scientific lecture Case Study II (Peter Hoboth)#

  • phosphoinositides of the nuclear speckles, methodology development

  • image acquisition troubleshooting

12:20 – 14:00 Lunch#

14:00 – 14:30 CASE Study Methods for the quantitative analyses of SMLM data (Peter Hoboth)#

  • 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

**14:30 – 16:30 ThunderSTORM hands-on sessions: Individual work with ThunderSTORM software#

(Ondřej Šebesta, Zuzana Burdíková, Martin Schätz, Peter Hoboth)

Group 1: beginners (Zuzana Burdíková, KONFMI, PřFUK)

Group 2: intermediate (Ondřej Šebesta, KONFMI, PřFUK)

Group 3: advanced (Martin Schätz, KONFMI, PřFUK, Peter Hoboth, KONFMI, PřFUK)

The coffee break included, Refreshment included

19:00 – 22:00** Informal discussion in the Academic Club**#

Refreshment included

Day 3#

Location: KONFMI Microscopy Center, Viničná 7, Praha 2, study room P09

09:00 – 9:30 Customizing Fiji/ImageJ with ImageJ Macro#

(Robert Haase)

09:30 – 10:00 Hands-on#

(Robert Haase; assisting: Zuzana Burdíková, Ondřej Šebesta, Martin Schätz)

10:00 – 10:15 Coffee break#

10:15 – 10:45 Interactive Design of GPU-accelerated Image Data Flow Graphs in Fiji (Robert Haase)#

10:45 – 11:30 Hands-on#

(Robert Haase; assisting: Zuzana Burdíková, Ondřej Šebesta, Martin Schätz)

11:30 – 12:30 Lunch#

12:30 – 13:30 Introduction to Data Management and FAIR principles#

(Marian Novotny)

13:40 – 14:20 Scientific lecture Data interpretation and quantification of complex dynamical systems#

(Yoonsang Lee, Dartmouth College)

14:30 – 14:50 Pixel Classification Using ILASTIK#

(Martin Schätz)

15:00 – 15:20 Object Detection Using StarDist#

(Martin Schätz)

Practical part

15:30 – 17:00 ILASTIK or StarDist Hands-on#

(Ondřej Šebesta, Zuzana Burdíková, Martin Schätz, Peter Hoboth)#

Group 1: beginners (Zuzana Burdíková, Ondřej Šebesta KONFMI, PřFUK)

Group 2: intermediate (Zuzana Burdíková, Ondřej Šebesta, KONFMI, PřFUK)

Group 3: advanced (Martin Schätz, KONFMI, PřFUK)

The coffee break included, Refreshment included