Program
The course will introduce participant to the light microscopy (Monday, Tuesday, and Wednesday), ultrastructural microscopy (Thursday) and image processing and analysis (Friday). After a thorough explanation of the theoretical essentials, there will be demonstrated microscopic techniques used in modern biomedical laboratories. Due to the limited capacity of the room with microscopes, participants will be divided into 4-5 groups.

PROGRAM (PDF)





MONDAY
   
8:15 - 08:45 Registration
   
8:45 - 09:00 Introductory word
   
9:00 - 09:45

Light microscopy: Instrumentation


Speaker: Pavel Krist

Lecture:
  • Microscope and its parts
  • Lasers, detectors, light sources, types of lenses, optical aberrations, optical elements
   
9:45 - 10:30

Light microscopy: Principles


Speaker: Jaromír Plášek

Lecture:

  • Forming an image in transmitted light
  • Kohler illumination
  • Waves, reflection, diffraction, interference, polarization, image formation, PSF
  • Phase contrast, interference contrast, polarization
   
11:00 - 11:45 Contrast-enhancing techniques in optical microscopy


Speaker: Jaromír Plášek

Lecture:

  • Mathematical methods for improving image quality
  • Deconvolution
   
11:45 – 12:30

Multi-dimensional laser confocal microscopy


Speaker: Pavel Hozák

Lecture:

  • Introduction and principles of confocal microscopy – lasers, objectives, scanning system, non-coherent light sources, electronic light detectors: photomultipliers, interference filters
  • Fluorescence excitation and emission fundamentals, fluorophores for confocal microscopy, spectral bleed-through artifacts (crosstalk)
  • Resolution, signal-to-noise consideration

 

13:30 – 14:15 Resolution and superresolution in fluorescence microscopy overview

 

Speaker: Ivan Novotný

Lecture:

  • Overview of microscopy techniques according the resolution and summarization of the techniques from the practical view.
  • General principles how the superresolution breaks through the Abbe resolution barrier

 

   
14:30 - 17:30 Practical part


Adjusting the microscope: Köhler illumination

Zeiss

Adjusting the microscope: Phase contrast

Pragolab

Adjusting the microscope: Differential interference contrast

Pragolab

Fluorescence microscopy

Anna Malinová

Confocal Microscope (STED)

Ivan Novotný


TUESDAY
   
9:00 - 9:45

Basics of fluorescence microscopy and immunolabeling


Speaker: Pavel Hozák

Lecture:

  • Fluorescence microscopy
  • Sample preparation for light microscopy
  • Selection of antibodies and immunostaining

 

   
9:45 - 10:30

Fluorophores



Speaker: Šárka Pokorná

Lecture:

  • Fluorescence, Jablonski diagram
  • Types of fluorophores and their use in microscopy monitoring processes in living systems
  • A combination of fluorophores

 

   
11:00 - 11:45

Introduction to live cell imaging


Speaker: Ivan Novotný

Lecture:

  • Basic equipment for live cell imaging
  • Visualization of cells in fluorescence microscopy
  • Common methods in live cell imaging
  • How to improve cell vitality / viability


   
11:45 – 12:30 FRAP + FCS


Speaker: Michaela Efenberková

Lecture:

  • Photo-kinetic measurement in live-cell imaging
  • Principles of Fluorescence recovery after photobleaching (FRAP), usage and analysis
  • Fluorescence correlation spectroscopy (FCS) in microscopy kinetic measurements, principles, analysis and its relation to other photo-kinetic techniques

 

 

13:30 – 14:15 FLIM / FRET


Speaker: Aleš Benda

Lecture:

  • Fluorescence Lifetime Imaging (FLIM), principles and use, microscope features
  • Förster resonance energy transfer (FRET) microscopy principles
  • Practical way how to use the techniques

 

   
14:30 - 17:30 Practical part


Fluorescence labeling

Davide Basello

FRET (SP5)

Anna Malinová

FRAP

Michaela Effenberková

Live cell imaging

Ivan Novotný


WEDNESDAY
   
9:00 - 9:45

Super-resolution light microscopy: SIM

 

Speaker: Martin ╚apek

Lecture:

  • Structured Illumination Microscopy (SIM), its principles,  Fourier space, image in Fourier space
  • The image reconstruction
   
9:45 - 10:30

Super-resolution light microscopy: STED


Speaker: Ivan Novotný

Lecture:

  • introduction to the confocal superresolution and principle of STED
  • theoretical background
  • technical solution

 

   
11:00 - 11:45

Super-resolution light microscopy: STORM/PALM


Speaker: Michaela Effenberková

Lecture:

  • Principles
  • Stochastic Optical Reconstruction Microscopy (STORM)
  • Photo-activated Localization Microscopy (PALM)
   
11:45 – 12:30 Spining disc confocal microscope


Speaker: Petr Kolá°

Lecture:

  • introduction to the fast confocal imaging using spinning disc
  • technical solutions – how to
  • practical examples
13:30 – 14:15 Light Sheet microscopy


Speaker: Martin ╚apek

Lecture:

  • how to acquire a large biological object – options
  • why Light Sheet and how it works – idea, principles and solution
  • big data production – how to deal with it
  • lattice light sheet – just few words


   
14:30 - 17:30 Practical part


STED

Ivan Novotný

Total Internal Reflection microscopy - TIRF

Ondrej Horváth

2-photon microscope

Daniel Hadraba

SIM

Martin ╚apek

THURSDAY
   
9:00 - 9:45

Image formation in transmission electron microscope

 

Speaker: Old°ich Benada

Lecture:

  • Electron microscopy basics
  • Properties of electrons, resolution, wavelength of accelerated electrons, the electrons in an electromagnetic field
  • Electron-electron interaction and analytical electron microscopy
  • Transmission electron microscope – design, image creation, interference
  • Image acquisition in the TEM

   
9:45 - 10:30 Scanning electron microscopy


Speaker: Old°ich Benada

Lecture:

  • Scanning electron microscope – the construction
  • Signals and image recording (secondary and back-scattered electron imaging)
  • Scannig Transmission Electron Microscopy
  • SEM image interpretation
   
11:00 - 11:45 Preparing samples for TEM and Immunolabeling


Speaker: Jana Nebesá°ová

Lecture:

  • Physical and chemical principles of sample preparation for electron microscopy
  • Chemical methods - fixation, dehydration, infiltration, embedding, preparation of ultrathin sections, contrasting
  • Physical methods - low-temperature processes, microwaves
  • Ultrastructural immunolabeling (imunogold)
   
11:45 – 12:30 Preparing samples for SEM


Speaker: Jana Nebesá°ová

Lecture:

  • Sample preparation, selection of antibody for correlative microscopy
  • Correlative light-electron microscopy (CLEM)
  • Strategies for biological applications - visualization (changes, diagnostic, clinical proof etc...); analytical morphomics (morphology, identification, content, functionality)

 

13:30 – 14:15 Advanced electron microscopy techniques

 

Speaker: Vlada Filimonenko

Lecture:

  • Cryo Electron Microscopy
  • Analytical techniques
   
14:30 - 17:30 Practical part

Transmission electron microscopy

Jana Nebesá°ová

Scanning electron microscopy

Old°ich Benada

High-pressure freezing, freeze substitution and ultramicrotomy

Dominik Pinkas

EM immunolabeling: detection, clustering and colocalization

Vlada Philimonenko


FRIDAY
   
9:00 - 9:45 Introduction to image deconvolution


Speaker: Ivan Novotný

Lecture:

  • raw data from an optical system – are they ready to use
  • systematical error and PSF
  • the idea of image deconvolution
  • practical aspects, image “requirements”

 

9:45 - 10:30 Methodology for correct microscopy


Speaker: Ivan Novotný

Lecture:

  • garbage in – garbage out
  • critical points in specimen preparation
  • issues – refractive index mismatch, spherical aberration, sample flattening / distortion

 

   
11:00 - 11:45 Atomic Force Microscopy in biology


Speaker: Andre Körnig

  • principles of AFM & imaging modes
  • combination of AFM with optical microscopy
  • characterization of mechanical sample properties with AFM
   
11:45 - 12:30 Introduction to image processing


Speaker: Ji°í JanáŔek

Lecture:

  • Basic concepts (resolution, levels of grey, frame repetition frequency), the advantages and disadvantages of digital processing
  • Types of cameras (analog versus digital) and their important characteristics. The types of capture cards in the PC, so. "Frame grabbers", the basic principle
  • Image parameters (contrast, noise), histogram
  • Data file formats (binary, grayscale, RGB, HSV, Lab) and compression (lossy, lossless)
   
13:30 – 14:15

Image analysis and visualization in 3D


Speaker: Ji°í JanáŔek

Lecture:

  • The data source CLSM and MRI, dimensional calibration
  • Filtering and segmentation data
  • Visualization: volume and surface rendering

14:15 – 15:45

Preparation of digital images for publication and presentation

 

Speaker: Old°ich Benada

Lecture:

  • 300 dpi resolution for printing
  • Allowed image manipulations
  • Some comments on color
  • RGB vs. CMYK
  • ICC profiles

 

15:45 – 16:00

Recap + Evaluation