Introduction to Light Microscopy

Abstract

The course provides basic knowledge in light and electron microscopy, as well as important applications of different methodical approaches in microscopy down to the molecular level. The students will be able to incorporate these techniques into their experimental planning e.g. for Master theses, also to understand and judge new developments in this field from the literature.

Objective

The course will provide basic knowledge in light and electron microscopy, as well as important applications of different methodical approaches in microscopy down to the molecular level. The students will be able to incorporate these techniques into their experimental planning e.g. for Master theses and understand and judge about new developments in the field from the literature in order to update knowledge independently.

Content

Content and responsible teachers consecutively: • Roland Gebert will teach the basics: elements of a lens, the eye, simple and compound microscope (stereomicroscopes, macroscopes, upright and inverted microscopes),Abbe’s theory of image formation, including Köhler illumination. Limitations of bright field light microscopy (lens aberrations etc, resolving power of the microscope, dry and immersion lenses, useful range of magnification). Experiments with the diffraction apparatus will illustrate the theory. Contrast of the microscopic image (dark field, phase contrast, polarized-light microscopy, differential interference contrast DIC). Control of wavelength, vibration direction and amplitude of the light. Fluorescence contrast: Autofluorescence and secondary fluorescence, equipment of the fluorescence microscope (light sources, filters, objectives), illuminated and self-luminous objects, Airy’s theory of image formation, resolving power of the fluorescence microscope (Rayleigh-criterion), lateral and axial resolution, linear measurements; link with the confocal laser scanning microscope (CLSM). • Christof Sautter: Epifluorescence microscopy, Confocal Laser Scanning Microscopy (CLSM). Overview about different approaches to detect specific molecules on sections (chemical stains, immunocytochemistry, and in situ hybridization). Basics of fixation (chemical fixation, plastic embedding, sectioning for light microscopy). Basics of image processing. • Heinz Gross and Roger Wepf: Basic electron microscopy (EM): Cellular Methods, molecular methods, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Molecular electron microscopy: cryo-techniques and image processing in EM. • Gabor Csucs: Advanced CLSM will deal with different aspects of confocal microscopy. We will use both scanning confocal (single photon and multi-photon) and spinning disk confocal systems, and image both fixed and (probably) living samples. Special applications linked to CSLM like Fluorescence Recovery after Photobleaching (and its variants) photo-activation and Fluorescence Correlation Spectroscopy. • Vahid Sandoghdar will review modern optical methods to detect and study single molecules and small particles. This includes single fluorescent molecules to study dynamics of molecules in real time by examples of intensity measurements, fluorescence lifetime, polarization, and of emission pattern, study of molecular interaction by fluorescence resonant energy transfer (FRET) and its application in biology. Finally non-fluorescent nano-objects such as gold particles, and unlabelled viruses or microtubule will be detected and tracked. • Renato Zenobi: Analytical approaches combining near-field optical methods with spectroscopy (e.g. vibrational spectroscopy, mass spectrometry) have a great potential to fill the need of chemical / molecular information of nanostructures. Raman spectroscopy (TERS) and near-field laser ablation mass spectrometry will present the state-of-the art. • Christof Sautter: Advanced methods of invasive technique on the microscopic level: Micro-projectiles as a tool to deliver any biologically active material to plant cells and laser dissection as a tool to collect very small tissue parts for isolation and study of genomics and proteomics in homogenous cell populations. The last day will offer the chance to review a recent important paper from the microscopy field and refer it to the colleagues.

Resources