Foundations of optical microscopy, treated both in a geometrical optics and in a physical optics framework. Digital images and their analysis. Contrast methods in microscopy, with a focus on fluorescence. Non-linear (TPF, SHG, etc.) and vibrational (Raman, SRS, etc.) microscopy. Optical and spectroscopic properties of biological samples. Super-resolution microscopy. Imaging and manipulation of single molecules.
J. Mertz, “Introduction to Optical Microscopy”, Roberts and Company Publishers (2009)
J. Pawley (ed.), “Handbook of Biological Confocal Microscopy”, Springer (2006)
Articles and notes provided during the course.
Learning Objectives
This course aims at introducing the theoretical foundations and the main biomedical applications of optical microscopy, with reference to the most recent developments in the field. At the end, the student should know advantages and limitations of the main techniques, and should be able to understand and critically evaluate a scientific publication in the field.
Prerequisites
Quantum Mechanics, Structure of Matter, Optics
Teaching Methods
Class lectures, laboratory visits
Type of Assessment
Oral exam. During the exam, the student will present and discuss a scientific paper. The evaluation will focus on the critical analysis skills, the mastery of the subject and the communication efficacy.
Course program
1. Introduction
2. The microscope: general concepts of geometrical optics and radiometry (magnification, numerical aperture, conjugate planes, optical invariant)
3. The microscope in physical optics: light diffraction, vacuum propagator, Fourier optics, PSF and resolution
4. Aberrations in microscopy and their correction
5. Detectors used in optical microscopy: single-point (PMT, APD) and array (CCD, CMOS). Main features (linearity, dark noise, electron well, etc.) and specs for the different detector types. Detection noise.
6. Principles of image analysis: formats, filters, deconvolution, segmentation, quantification, main tools (FIJI)
7. The problem of axial resolution. Optical sectioning. Confocal microscopy, light-sheet microscopy, total internal reflection microscopy
8. Contrast methods in microscopy: bright field, dark field, fluorescence. Fluorescence spectroscopy (levels scheme, rate equations, saturation, photobleaching).
9. Different types of fluorophores and labeling techniques for in vivo and ex vivo. Functional sensors (VSD, GCAMP, etc.) and Optogenetics.
10. Optical properties of biological tissues. Introduction to clearing methods.
11. Phase contrast microscopy and DIC.
12. Non-linear microscopy: two-photon absorption and second-harmonic generation
13. Advanced contrast methods: fluorescence lifetime (FLIM), resonant energy transfer (FRET), spontaneous Raman, stimulated Raman, coherent anti-Stokes Raman scattering (CARS)
14. Optical coherence tomography and digital holography
15. Single-molecule microscopy
16. Super-resolution optical microscopy: structured illumination, PALM/STORM, STED/RESOLFT
17. Single molecule manipulation with optical tweezers
18. Lab: alignment and characterization of a wide-field microscope. Image analysis with FIJI. Optical tweezers.