99129 - ELECTRONIC AND OPTICAL MICROSCOPY

Learning outcomes

At the end of the course the student has acquired knowledge of the most common electronic and optical microscopies used for the characterisation of materials and biomolecules and the analysis of the acquired images.

Course contents

The course will start with an introduction on the general concepts of digital imaging, including image formation, resolution, size, digitalization.

We will then proceed introducing the different microscopy techniques using EM waves in the range of the UV, VIS and IR:

• Optical microscopy (transmission - bright field and dark field)

• Fluorescence microscopy: the use of filters in excitation, emission and of dichroic filters. Detection cameras

• Confocal microscopy: the pinhole, the scanner unit, signal multiplexing and decoding. Point and line measurements, dynamic measurements, FCS and correlative microscopies. Multiplexing in wavelength (channels vs spectral module) and in time (FLIM and PLIM, fluorescence and phosphorescence lifetime imaging). Dynamic techniques (FRAP, FLIP).

• Multiphoton microscopy

• Special configurations: light sheet microscopy

• Beyond diffraction limit: super-resolution microscopies

• Brief in-depth information on fluorescent probes that meet specific microscopy needs (brightness, super-resolution, photostability…)

The second course section introduces the students to the principles and applications of imaging technologies based on electron microscopy (EM). Different microscopy techniques will be described: scanning (SEM), transmission (TEM), and scanning transmission (STEM) electron microscopy.

The class will also discuss techniques coupled to EM to get additional information on the sample, i.e., composition or crystallinity. These include energy-dispersive X-ray spectroscopy (EDS), cathodoluminescence (CL), electron backscatter diffraction (EBSD), focused ion beam (FIB), etc.

The course will discuss the technique and theoretical bases behind each of these classes of EMs along with their limitations and range of application. Aside of the basic theory, a strong relevance will be given to practical knowledge as sample preparation and high image quality acquisition.

Finally, the student will learn the basics of image analysis to perform quantitative experiments and measurements.

Readings/Bibliography

Freely available on Virtuale

1. Fluorescence Microscopy - From Principles to Biological Applications
Edited by Ulrich Kubitscheck

2. Optical Microscopy Course - Neil Switz, Daniel Fletcher

Additional readings:

Introduction to confocal fluorescence microscopy / Michiel Muller. Bellingham : Spie press, c2006

Single-molecule optical detection, imaging and spectroscopy / edited by T. Basche; Weinheim: VCH, c1997

Fluorescence spectroscopy, imaging and probes : new tools in chemical, physical and life sciences / Ruud Kraayenhof, Antonie J. W. G. Visser, Hans C. Gerritsen (eds.)
Springer, c2002

Teaching methods

Classroom lectures with workshops

Assessment methods

Oral Examination - Mimicking a practical microscopy session

Teaching tools

Powerpoint slides will be available on Virtuale

Office hours

See the website of Damiano Genovese

See the website of Devis Montroni