Foundations of optical microscopy and of image formation, resolution limit of an optical instrument. Contrast methods in microscopy, with special focus on fluorescence. Non-linear (TPF, SHG, etc.) and vibrational (Raman, SRS, etc.) microscopy. Optical and spectroscopic properties of biological samples. Digital images and their analysis. Super-resolution microscopy. Advanced microscopy applications in biology and medicine.
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
Foundations of light-matter interaction gained attending Quantum Mechanics and/or Structure of Matter courses. Basic concepts of Optics.
Teaching Methods
Class lectures, laboratory visits
Further information
None
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
I. Optics of the microscope.
General concepts of geometrical optics and of radiometry (magnification, numerical aperture, conjugate planes, optical invariants). The microscope in physical optics: diffraction of light, vaccum propagator, lens propagator, Fourier optics, PSF and resolution. The axial resolution problem. Optical sectioning. Confocal microscopy, light-sheet microscopy, evanescent wave microscopy (TIRFM). Aberrations in microscopy and their correction.
II. Molecular spectroscopy and contrast methods in microscopy.
Absorption, scattering. Phase contrast microscopy, DIC, digital holography. Optical coherence tomography. Fluorescence spectroscopy (levels diagram, rate equations, saturation, photobleaching). Fluorescence lifetime microscopy (FLIM), resonant energy transfer (FRET). Types of fluorophores, in vivo and ex vivo labeling strategies. Functional sensors (VSD, GCAMP, etc.). Optical properties of biological tissues, clearing techniques. Non-linear microscopy: two- or three-photon excitation, generation of higher harmonics. Vibrational microscopy: spontaneous Raman, stimulated Raman, CARS.
III. Digitalization and analysis of images.
Detectors used in microscopy: point-like (PMT, APD), arrays (CCD, CMOS). General features (linearity, dark noise, electron well, etc.) and specificity of each kind of system. Detection noise. Image analysis principles: formats, filters, deconvolution, segmentation, quantification, main tools (FIJI). Brief outline of machine learning methods.
IV. Advanced developments and applications.
Single molecule microscopy. Super-resolution optical microscopies: structured illumination, PALM/STORM, STED/RESOLFT. In vivo microscopy. High-speed 3D imaging. Optogenetics.
V. Lab.
Alignment and characterization of a wide-field microscope. Characterization of a confocal microscope. Image analysis with FIJI and Python.