PHYSICAL METHODS FOR BIOMEDICAL INVESTIGATION
The course aims to be self-consistent. A heuristic preparation in atomic and molecular structure and the semi-classical theory of light-matter interaction, as well as notions of basic optics, laser and detectors physics will be helpful, although not propaedeutic. Basic notions in chemistry and molecular biology are also welcomed.
The students will be required to prepare a short (15 minutes) presentation, also with the help of slides if they deem it suitable, on one technique at their own choice. The presentation will be followed by a question time (about 15 minutes) in which the teachers will assess the in-depth comprehension of the physical and technical bases of the chosen technique. Lastly, the student will be tested as to her/his knowledge of the other topics treated at lesson by means of a short oral interview (roughly 15 minutes).
The main aims of the Course are: - developing in the students the knowledge and understanding of the working principles of selected spectroscopic techniques; - developing in the students the knowledge and understanding of the nanomechanical response of biomolecules to sub-nanonewton stimuli - presenting to the students selected methods for cellular and tissue imaging - evidencing the usefulness of the taught techniques in the elucidation of systems and phenomena of biomedical and pharmaceutical interest; - develop in the students the ability of critically examining exemplary papers excerpted from the scientific literature. At the end of the course, the students will inherit a comprehensive panorama of some of the most advanced biophysical techniques used in modern molecular biophysics, including time-correlated single photon counting, fluorescence fluctuation spectroscopy, fluorescence resonance energy transfer, spontaneous and coherent Raman spectroscopy, Fourier-transform infrared spectroscopy, atomic force microscopy, optical and magnetic tweezers. Moreover, they will gain some insight on selected bioimaging methods, both based on fluorescent staining, such as confocal microscopy, multiphoton-excited microscopy and total internal reflection microscopy, and label-free, such as confocal Raman microscopy.
The course will be divided in three sections. The first one (24 h) will be focused on giving an overview on the state of art in fluorescence spectroscopy. Namely: - a semi-quantitative excursus on light-matter interactions, focused on the investigation of molecular electron-state transitions with particular care to the fluorescence phenomenon will be undertaken. Hints on rotovibrational transitions will also be made - steady-state fluorimetry will be overviewed and the information yielded by fluorescence spectra analysis will be outlined - time-resolved fluorescence advantages over steady-state fluorimetry will be stressed and the technical implementation of time-correlated single-photon counting discussed at length - fluorescence fluctuation spectroscopy techniques will be introduced, and the confocal setup will be described at length - the most widespread fluorescence-based bioimaging techniques (confocal, multiphoton-excited, and total internal reflection microscopy) will be outlined During the second section (18 h) Raman and infrared spectroscopy will be treated. Namely: - molecular vibrations and their excitation through electromagnetic radiation: absorption and Raman scattering. Molecular symmetry and excitability of vibrational transition: selection rules for absorption and Raman scattering. Frequency ranges for the detection of vibrational absorption and Raman features. Similarity and complementarity of the information retrieved through vibrational absorption spectroscopy and Raman spectroscopy - practical implementations of vibrational and spontaneous Raman spectrometers: sources, sample preparation, detection and data analysis. Why and how the implementation details determine the applicability of both techniques. Examples of application of vibrational and Raman spectroscopy - variations on the Raman technique: electronic resonance Raman spectroscopy, coherent Raman spectroscopy. Their advantages and disadvantages, exemplary use cases - Raman imaging: spontaneous vs coherent Raman, confocal Raman microscopy, recent advances in spontaneous and coherent Raman microscopy. Examples and outlook of biomedical applications. Finally, a third section (6 h) will be devoted to briefly overview the main nanomechanical features of DNA and proteins and to describe three nanomechanical manipulation techniques allowing to probe single biomolecules’ response to sub-nanonewton stimuli, namely: - Atomic force spectroscopy and microscopy will be introduced - The working principles of molecular (optical and magnetic) tweezers will be outlined, the information which can be achieved on systems of biomedical interest by their implementation will be discussed and their analogies and differences critically evaluated
The lectures will be as interactive as possible and, although slides will be used with the intention to give a reference frame of the fundamental competences to be acquired, discussions and digressions arising from the student personal experiences and curiosity will be encouraged. Moreover, if the number of students will allow this solution, visits to the laboratory and direct observations on the research setups will be scheduled.
The teachers can be contacted by e-mail at their institutional addresses: luca.nardo@uninsubria.it; marco.lamperti@uninsubria.it. Meetings for additional clarifications and explanations, further information on the examination mode, or simply to satisfy the student curiosity on topics connected to the course are welcomed upon appointment both in presence and online.