Introduction to Microscopy

General description

This course overviews main modern microscopy techniques used in cell biology. It deals both the theoretical background of the different types of microscopy and with practical aspects of sample preparation, digital data processing and experiment design will be covered. The main objectives of the course are to provide the students with the knowledge necessary to start a practical training on modern fluorescence microscopes and to describe the problems that can be solved by different specific types of microscopy.

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Details of the curriculum

Course: 4 lectures

Lectures: Volodymyr Shvadchak, Yury Kovalchuk, Nina Bondarenko

Lectures

1. Transmission and fluorescence microscopy

Transmission microscopy, phase contrast. Fluorescence microscopy. Principal schemes of microscopes. Lasers. Filters. Dichroic mirrors. Channels. Digital image collection. Image resolution, micrones and pixels. Confocal microscopy. Z-slices. TIRF. ImageJ (Fiji) software for image processing. Colocalization.

Useful links:

Fiji download

2. Advanced fluorescence microscopy

FRET and detection of interactions in microscopy. Fluorescence lifetime and FLIM. Diffraction limit. Superresulution, STORM. PALM. Application to image actin fibrils.

M_L2-001.pdf (download) (open in a new tab) (Size: 0.87M)

3. Fluorophores.

Fluorescent proteins. Small molecule dyes. Channel crosstalk and selection of fluorophores. Membrane trackers, staining of nuclei. Photodegradation during measurements. FRAP. Light intensity and damage to cells. Caged molecules and controllable photorelease. Photoswithchable molecules.

4. Atomic Force Microscopy. Electron microscopy.

Principle and scheme of microscopes. XY and Z resolution. Sample preparation. Scanning speed and sample damage. Application for protein unfolding. Principle. Resolution. Modes. Sample preparation.

Додаткові матеріали

Робота в ImageJ / Fiji

Завантаження безкоштовної програми Fiji для роботи з мікроскопічними зображеннями.

Невеликий підручник по ImageJ/Fiji.

Приклади файлів для самостійного освоєння програми:

jnc15528-sup-0005-Intracellular monomer penetration (Z-scan).avi (download) (open in a new tab) (Size: 17.57M)

Sampe of microscopy files.zip (download) (open in a new tab) (Size: 25.99M)

Lecturers

Nina Bondarenko

Pathologist and Research Scientist with PhD.

Volodymyr Shvadchak

Employee of the Precarpathian University (Ivano-Frankivsk) and the Institute of Organic and Bioorganic Chemistry (Prague). Doctorate degree in Life sciences (PhD) was obtained from Strasbourg University in 2009 for research work on the development of solvatochromic fluorescent labels for studies of protein interactions.