Fluorescence Microscopic Techniques

Type of instruction




Part of degree program


Recommended in

Semesters 1-4

Typically offered in

Autumn/Spring semester

Course description

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