Imaging in living cells

A.Y. 2021/2022
Overall hours
BIO/04 FIS/07
Learning objectives
The student will acquire the cultural and methodological tools to properly design and carry out in vivo imaging experiments by using the state of the art molecular and technological tools to answer fundamental biological questions. The description of the current methodological approaches will provide the student with the cultural tools to understand the diverse approaches that can be exploited in cellular and molecular biology research projects and to understand which scientific questions can be answered with modern imaging technologies as well as understand their limitations. The student will also gain a deep insight into how to translate images into quantitative data. To reach the goal of this teaching the student will attend traditional classes followed by practical sessions with the different available microscopy tools and imaging software.
Expected learning outcomes
Students will have a knowledge - comprising the understanding of the technical foundations and a hands-on experience - of the most advanced solutions in optical imaging available to the study of cellular phenomena. They will thus become able to (i) appreciate the accuracy and relevance of the results obtained with the various techniques (ii) select the most appropriate imaging techniques for the topics of their future activities and (iii) critically evaluate the best imaging approach to use to answer a given biological question.
Course syllabus and organization

Single session

Lesson period
First semester
The lessons will be held synchronously on the Microsoft Teams keeping the set times.
The lab activities and microscopy training will be done remotely, by sharing the screens.
The program and reference material will not be affected.
Lessons will be also recorded and the files made available to the students.
The exam will take place in oral form using the Microsoft Teams platform, or where the regulations allow, in presence, always in oral form.
Course syllabus
The teaching will cover principal molecular, technical and software solutions to learn how to design, perform and analyse experiments based on imaging.

The teaching will be divided into three modules that partially overlap.

Module 1 (Bellini) Conceptual bases of resolution and super-resolution and introduction to the quantitative analysis of images. After introducing the notions of diffraction limit and its effect on traditional and confocal microscopy imaging, we will consider the main solutions proposed to overcome it (2 photons, STED, PALM, TIRF, SIM).

Module 2 (Nava) The digital formats of the images, and all the concepts essential for their quantitative analysis, will be introduced. With the help of open-source software (ImageJ), analyses will be performed on some types of simple images.

Module 3 (Costa) The teaching will also provide the theoretical and practical bases for the use of molecules and fluorescent proteins in different biological systems as well as the knowledge of using advanced optical microscopes for the study of cellular phenomena (wide field; single and multiphoton confocal, super-resolution with structured light). The theoretical/practical sessions will consider the use of live or fixed biological preparations with which to perform different types of experiments including FRET, FRAP etc.

In the teaching, the student will acquire the following specific expertise.
Knowledge of basic concepts of traditional wide-field and confocal microscopy imaging (point scanner and spinning disk).
Conceptual bases of resolution and super-resolution.
Technical and software solutions to overcome the resolution limits of optical imaging (2 photons, STED, PALM, TIRF, SIM).
Direct experience in the use of advanced optical microscopes for the study of biological phenomena.
Use of fluorescence biosensors for the in vivo study of cellular and subcellular metabolic processes.
The digital formats of the images, and all the concepts essential for their quantitative analysis, will be introduced. With the help of open-source software (ImageJ), analyses will be performed on some types of simple images.
At the end of the course, students will be able to understand which technology to use for different types of imaging analysis to answer a given biological question. They will be able to autonomously use advanced microscopy instrumentation and extrapolate quantitative data from the acquired images.
Prerequisites for admission
Students are required to have some knowledge of: basics of optics (basic properties of light: speed, frequency, wavelength, refraction), basic properties of lenses (focal length, image formation), basic structure of the optical microscopes (objective, eyepiece, magnification, upright or inverted structure, illumination), basic concepts of fluorescence. Those who do not have these elements of knowledge are required of an effort of personal study. During the first hour of the class, the required elements of knowledge will be listed, together with web sites that can be used for the study material, in case no other reference book is available to the students.
Teaching methods
The lectures will have both the classical format with lessons given by the teachers by using PowerPoint slides as well as practical sessions with exercise at the PC or the use of different microscopes (wide-field, confocal, multiphoton, spinning disk and SIM). During the lectures, the students will be encouraged to actively participate with questions and comments related to the considered subjects and will actively participate in the practical sessions. Course attendance is highly recommended.
Teaching Resources
The teaching does not have a dedicated book and all the material will be provided by teachers. All the PowerPoint material used for the lectures will be made available to the students at the dedicated Ariel website. High quality web-based tutorials, among which will be suggested. The reading of selected research papers, reviews or attending to online webinars (e.g. iBiology Microscopy Series: will also be suggested to the students at every lecture and they will be made available through the Ariel website. In case of need by the students of basic information regarding microscopy solutions the following book can be considered:
Basic Confocal Microscopy. W. Gray (Jay) Jerome, Robert L. Price Springer, 30 oct 2018 - 368.
Assessment methods and Criteria
Learning assessment will be through a written exam at the end of the course.
The text of the exam includes open questions (30%), charts and graphs to complete (20%) and multiple choices tests (50%). These proportions broadly reflect their contribution to the composition of the final score. Multiple-choice tests are aimed to broadly verify the understanding of concepts and definitions taught during the course whereas open questions/charts are designed to evaluate problem-solving skills.
The duration of the written test will be 2 hours.
Practicals: 32 hours
Lessons: 24 hours
Educational website(s)
Thursday 14:00-16:00
Department of Biosciences 3rd Floor Tower C