SUBJECT
Fluorescence Microscopic Techniques
lecture
master
2
Semesters 1-4
Autumn/Spring semester
1. Basic concepts in fluorescence – Jablonski energy diagram – The stokes law
2. Quantum yield – Fading, Quenching, Photobleaching – Inhibition of photobleaching and specimen fading – Fluorescence Photobleaching Investigations (FRAP, FLIP) – Fluorescence resonance energy transfer
3. The Fluorescence Light Budget – Fluorescence Light Sources (mercury and xenon burners), Fluorescence Lamp Specifications
4. Basic Aspects of Light Filters – dichroic filters – Filter terminology
5. Detecting Single Molecules – Total Internal Reflection Fluorescence Microscopy. Setup with prism and Setup through the objective lens illumination
6. The evolution of fluorescence microscopy – Transmitted light illumination. Specimen illumination with darkfield condensers – Fluorescence microscopy with episcopic illumination
7. Reflected Light Fluorescence Microscopy Light Pathways – Basic Strategy of Epi-Fluorescence Microscopy – Köhler Illumination in Fluorescence Microscopy
8. Laser Scanning Confocal Microscopy – Comparing Confocal and Widefield Fluorescence Microscopy (Resolution and Contrast in Confocal Microscopy) – Advantages and Disadvantages of Confocal Microscopy – Acousto-Optic Tunable Filters (AOTFs) and deflectors – Fundamentals and Applications in Multiphoton Microscopy
9. Confocal Microscope Scanning Systems (Beam scanning and Stage scanning) – Spinning Nipkow disk – Tandem scanning, Monoscanning – Point and area–scanning confocal system configurations
10. Introduction to lasers – stimulated emission, Population inversions – Laser systems for optical microscopy (Gas lasers, Solid-state lasers, Dye lasers, Semiconductor (diode) lasers) – Disadvantages of confocal microscopy
11. The history of synthetic fluorescent probes
12. Classification and applications of fluorescent probes – Important characteristics of fluorophores, Spectral profiles of popular traditional fluorophores – Traditional and modern fluorescent dyes. Fluorescent Proteins: Aequorea victoria and GFP. Mutagenesis experiments with green fluorescent protein (enhancedGFP, BlueFP, CyanFP), fluorescent protein derivative (YellowFP). Discosoma striata & Heteractis crispa (DsRed, HcRed) – Optical Highlighter Fluorescent Proteins (photoactivation and photoconversion)
13. Organelle Probes: nuclei, mitochondria, lysosomes, Golgi apparatus, and endoplasmic reticulum – Specimen preparation using synthetic fluorophores and immunofluorescence – Quantum Dots
14. Specifications and Identification, Adjustment of Objective Correction Collars, Numerical Aperture & Resolution, Mechanical Tube Length, the effective working distance of the objective and cover glass thickness, Immersion Media – Eyepieces (Oculars)
15. The semester is closed by a practical course (2–2.5 days).
Alberto Diaspro: Optical Fluorescence Microscopy, Springer Science & Business Media, 2010
Basic Concepts in Optical Microscopy