Imaging in Living Cells
A.Y. 2026/2027
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.
Lesson period: First semester
Assessment methods: Esame
Assessment result: voto verbalizzato in trentesimi
Single course
This course can be attended as a single course.
Course syllabus and organization
Single session
Responsible
Course syllabus
The teaching will provide the conceptual, technical and computational foundations required to design, perform and quantitatively analyse imaging experiments in living cells. Particular emphasis will be placed on selecting the most appropriate imaging approach to address specific biological questions and on extracting quantitative information from microscopy data.
The teaching will be organised into three partially overlapping modules.
Module 1. Conceptual bases of optical microscopy, resolution and super-resolution. After introducing the notion of the diffraction limit and its effects on traditional and confocal microscopy imaging, we will consider the main solutions proposed to overcome it (STED and PALM). Two-photon and TIRF microscopy will be presented, highlighting their applications.
Module 2. Introduction to the quantitative analysis of images. With the help of open-source software (ImageJ), analyses will be performed on some types of sample images. Topics will include image enhancement, segmentation, intensity measurements, and object detection. Students will learn how to extract, visualise, and interpret quantitative data from microscopy images.
Module 3. Theoretical and practical aspects of fluorescent molecules, genetically encoded fluorescent proteins and biosensors for the study of cellular and subcellular processes. Practical sessions will cover experimental design, image acquisition and analysis using live and fixed biological samples. Representative applications will include FRAP, FRET and other approaches for investigating molecular dynamics and cellular functions.
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.
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.
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.
The teaching will be organised into three partially overlapping modules.
Module 1. Conceptual bases of optical microscopy, resolution and super-resolution. After introducing the notion of the diffraction limit and its effects on traditional and confocal microscopy imaging, we will consider the main solutions proposed to overcome it (STED and PALM). Two-photon and TIRF microscopy will be presented, highlighting their applications.
Module 2. Introduction to the quantitative analysis of images. With the help of open-source software (ImageJ), analyses will be performed on some types of sample images. Topics will include image enhancement, segmentation, intensity measurements, and object detection. Students will learn how to extract, visualise, and interpret quantitative data from microscopy images.
Module 3. Theoretical and practical aspects of fluorescent molecules, genetically encoded fluorescent proteins and biosensors for the study of cellular and subcellular processes. Practical sessions will cover experimental design, image acquisition and analysis using live and fixed biological samples. Representative applications will include FRAP, FRET and other approaches for investigating molecular dynamics and cellular functions.
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.
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.
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 http://olympus.magnet.fsu.edu/ will be suggested. The reading of selected research papers, reviews or attending to online webinars (e.g. iBiology Microscopy Series: https://www.ibiology.org/online-biology-courses/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.
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.
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.
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PHYS-06/A - Physics for Life Sciences, Environment, and Cultural Heritage - University credits: 1
PHYS-06/A - Physics for Life Sciences, Environment, and Cultural Heritage - University credits: 1
Exercises: 32 hours
Lessons: 24 hours
Lessons: 24 hours
Professor(s)
Reception:
Thursday 14:00-16:00
Department of Biosciences 3rd Floor Tower C