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

Basics of fluorescence microscopy and immunolabeling


Speaker: Pavel Hozák

Lecture:

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

 

13:30 – 14:15 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

 

   
14:30 - 17:30 Practical part

 

Macroscope: AxioZoom.V16
Zeiss

Adjusting the microscope: Köhler illumination, Phase contrast, Differential interference contrast
Pragolab

Fluorescence microscopy
Anna Malinová

Confocal Microscope (SP8-STED)
Ivan Novotný




TUESDAY
   
9:00 - 9:45

Fluorophores


Speaker: Jan Sýkora

Lecture:

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

 

   
9:45 - 10:30

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

 

   
11:00 - 11:45

Spinning disc confocal microscopy


Speaker: Michaela Efenberková

Lecture:

  • introduction to the fast confocal imaging using spinning disc
  • technical solutions – how to
  • practical exercises


   
11:45 – 12:30 Light Sheet Microscopy


Speaker: Anna Malinová


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

 

 

13:30 – 14:15 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

 

   
14:30 - 17:30 Practical part


Confocal Spinning Disc (Dragonfly)

Ivan Novotný

Light Sheet (ZEISS Z.1)
Anna Malinová

FRAP (DV OMX)
Michaela Effenberková

Live cell imaging (DV Core)
Anastasiya Klebanovych



WEDNESDAY
   
9:00 - 9:45

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

 

   
9:45 - 10:30

Super-resolution light microscopy: SIM, STORM, STED


Speaker: Ivan Novotný

Lecture:

  • Structured Illumination Microscopy (SIM), its principles,  Fourier space, image in Fourier space
  • The image reconstruction
  • introduction to the confocal superresolution and principle of STED
  • theoretical background
  • technical solution
  • Stochastic Optical Reconstruction Microscopy (STORM) – principles, solution

 

   
11:00 - 11:45

Quantitative phase microscopy


Speaker: Martin ╚apek

Lecture:

  • Principles – hologram construction
  • Quantitative Phase Imaging
  • Automatic segmentation of cells based on phase image
   
11:45 – 12:30 Computative high resolution methods


Speaker: Michaela Efenberková

Lecture:

  • introduction to the computative methods
  • principles
  • deconvolution enhanced methods
  • SRRF
13:30 – 14:15 Methodolgy for correct microscopy


Speaker: Ivan Novotný

Lecture:

  • What kind of data I need to publish? What you need to show?
  • Which method would be most suitable for it?
  • How the sample should be prepared in respect to used method?
  • The image acquisition crucial points – bit depth & using of dynamic range, linear response to intensity


   
14:30 - 17:30 Practical part


STED (SP8-STED)

Ivan Novotný

SIM (DV OMX)
Anna Malinová

SRRF (Dragonfly)
Michaela Efenberková

Quantitative Phase microscopy
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


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
   
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:

  • Ultrastructural immunolabeling (imunogold)
  • Volume electron microscopy
  • 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:

  • What is the image deconvolution
  • The image is convolved - Point Spread Function (PSF)  ….  And the noise
  • Resolution and sampling (widefield, confocal, superresolution…)

 

9:45 - 10:30 Image aquisition by two-photon microscopy


Speaker: David Vondrášek

 

   
11:00 - 11:45 Optical projection tomography


Speaker: Martin ╚apek

 

   
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

14:30 – 16:00

Preparation of digital photographic documentation for publication

 

Speaker: Old°ich Benada

Lecture:

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

 

16:00 – 16:15

Recap + Evaluation