The main objectives of this course are: 1. Knowledge and understanding: Students are provided with a basic knowledge of microscope techniques with emphasis on the recently developed methodologies. The course is designed to familiarize the students with the scientific possibilities given by microscopy techniques. 2. Applying knowledge and understanding: The practical and theoretical lectures aim at explaining the optimal usage of different microscopes. The students will be able to choose the microscopic technique that best allows the study of a given biological problem in terms of spatial resolution, temporal dynamics, interaction and sample damage and spectral and compositional chemical information, mechanical and morphological structure. 3. Making judgements: The students will have to acquire independence in the evaluation of the best protocols to perform scientific relevant image acquisition and quantitative microscopy. 4. Communication skills: The students are highly encouraged to engage in the revision part of the lessons (first 20 min of a lesson are dedicated to discussion of the content of the previous lesson) to allow familiarity and active usage of technical language. 5. Learning skills: The student, given a biological question, will be invited to design and present an experiment and the necessary microscopy setup to achieve the most relevant results.

The student should have basic knowledge of physics and cell biology.

The purpose of the course is to give an overview of microscopy techniques used in different scientific areas from cell biology to material sciences with an introduction to digital image processing and analysis. Specific topics of this course: 1. Optical microscopy: physical principles, technics and applications 2. Fluorescence microscopy: principles and applications in cellular biology and biotechnology; with laboratory (4h) 3. Super-resolution microscopy: principles and applications in biotechnology 4. Image analysis and quantification 5. Scanning probe microscopy 6. Contact less manipulation techniques at molecular and cellular level; with laboratory (4h) 7. Electron microscopy and X-Ray microscopy

All the material presented and discussed in class will be made available to students. Some arguments are partly taken from the following textbooks: - Guy Cox, Optical Imaging Techniques in Cell Biology, Taylor and Francis Jacobs, Huang and Kwon, Introduction to Cell Mechanics and - Mechanobiology, Garland ScienceMurphy, D. B. and Davidson, M. W. (2012) - References in Fundamentals of Light Microscopy and Electronic Imaging, Second Edition, John Wiley & Sons, Inc., Hoboken, NJ, USA. doi: 10.1002/9781118382905.refs - O'Farrell, M. Basic Light Microscopy, in Cell Biology Protocols (eds J. R. Harris, J. Graham and D. Rickwood), John Wiley & Sons, Ltd,Chichester, UK. doi: 10.1002/0470033487.ch1(2006) - Fluorescence Microscopy, in Fluorescence Microscopy: From Principles to Biological Applications (ed U. Kubitscheck), Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. doi:10.1002/9783527671595.ch3 (2013)

1. Optical microscopy: 1.1. Physical principles - Image formation; magnification and diffracted limited resolution - Optical aberrations and image quality - Digital camera image acquisition (formats, properties, signal/noise ratio - SNR) 1.2. Techniques to image non-stained samples and applications in biotechnology - Brightfield and dark field microscopy - Phase contrast - Quantitative phase imaging: digital holography microscopy - Polarization microscopy - Non-linear optical microscopy - Photoacoustic microscopy 2. Fluorescence microscopy: principles and applications in cellular biology and biotechnology 2.1. Fluorescence principles, fluorescent dyes vs fluorescent proteins, direct vs indirect labelling (antibodies, probes) 2.2. Autofluorescence, quenching and photobleaching 2.3. Sample preparation: fixation vs live-cell imaging and immunofluorescence protocols 2.4. Confocal fluorescence microscopy: spinning disk and laser scanning; with laboratory (4h) 2.5. Advances techniques: FRET, TIRF and FRAP 3. Super-resolution microscopy: principles and applications in biotechnology 3.1. STED, PALM, MINFLUX, . 3.2. DNA-PAINT and DNA nanotechnology-based nanoscopy 4. Image analysis and quantification 4.1. Acquisition and control software 4.2. Preprocessing: background correction, temporal alignment 4.3. Quantitative measurements and statistical data analysis 5. Scanning probe microscopy 5.1. Atomic Force Microscopy 5.2. Scanning Tunneling Microscopy 5.3. Near-Field Scanning Optical Microscopy 6. Contact less manipulation techniques at molecular and cellular level 6.1. Optical Tweezers (OT) and scissors; with laboratory (4h) 6.2. Magnetic and acoustic tweezers 7. Electron microscopy and X-Ray Microscopy 7.1. SEM and TEM 7.2. Synchrotron radiation and phase contrast and fluorescence X ray microscopy 7.3. Free electron laser and coherent imaging

1. Frontal lessons with aid of powerpoint slide presentations including short videos and demonstration of image processing and analysis with ImageJ. 2. Hands-on experience in the optical microscopy laboratory. 3. Demonstration of confocal microscopes, optical tweezers and digital holographic microscopy.

Dan Cojoc, PhD CNR - Istituto Officina dei Materiali, Trieste Tel: 040 376 6413 E-mail: cojoc@iom.cnr.it Elena Ambrosetti, PhD Department of Life Sciences University of Study of Trieste E-mail: eambrosetti@units.it The detailed program and the ppt presentations used to support teaching will be available on Moodle2 website of the course.

The details of the exam will be explained to the students by the teacher during the presentation of the course in the first lesson. Assessment of the students’ learning will take place in an oral exam that includes a 15 min Powerpoint presentation by the student on a paper they are provided with a few days prior to the exam, followed by questions on the main microscopy techniques employed within the paper. The student will need to show knowledge of the topic and the ability to discuss the various microscopy techniques pointing out pros and cons of each and to select the correct technique for a given microscopy problem. The exam will produce a note up to 30 cum laude, a result under 18/30 will be considered as not passed. The following criteria will be used for assessment and marking: - Excellent (30 -30 cum laude): excellent knowledge of the topics, excellent usage of the technical language, brilliant application of theoretical knowledge to concrete cases. - Very good (27 -29): good knowledge of the topics, remarkable usage of the technical language, ability to correctly apply theoretical knowledge to concrete cases. - Good (24-26): good knowledge of the main topics, good command of the technical language; adequate ability to apply theoretical knowledge to concrete cases. - Satisfactory (21-23): the student does not show full command of the main topics of teaching, while possessing the fundamental knowledge; satisfactory knowledge of the technical language and sufficient ability to apply theoretical knowledge to concrete cases. - Sufficient (18-20): minimum knowledge of the main subjects of teaching and of the technical language, limited ability to adequately apply theoretical knowledge to concrete cases. - Insufficient (<18): the student does not have an acceptable knowledge of the contents of the various topics of the program.

This course explores topics closely related to one or more goals of the United Nations 2030 Agenda for Sustainable Development (SDGs) 3- salute e benessere 4- istruzione di qualità 5- parità di genere