505046 - ADVANCED MICROSCOPY

courses

ID:

505046

Duration (hours):

48

CFU:

SSD:

ANATOMIA COMPARATA E CITOLOGIA

Year:

2025

Date/time interval

Primo Semestre (29/09/2025 - 16/01/2026)

Course Objectives

The course aims to provide an in-depth understanding of the main microscopy techniques, including advanced modalities, as well as associated cytochemical and immunocytochemical techniques. By the end of the course, students will be able to: understand the theoretical principles of the described techniques, identify the most appropriate method for specific biological and biotechnological applications, critically analyze the advantages and limitations of each technique, and to apply correct scientific terminology.

Course Prerequisites

A solid foundation in cellular and molecular biology is required, in particular regarding the structural and functional features of cells. In addition, students are expected to be familiar with key concepts in general chemistry, biochemistry, and physics for the correct understanding of the principles underlying the described techniques.

Teaching Methods

The course includes lectures supported by slide presentations prepared by the teachers. Additional teaching activities will include seminars, as well as a guided visit to a laboratory to observe the practical use of optical, fluorescence, and electron microscopy. These activities are designed to promote autonomy in comprehension through direct observation of laboratory workflow, in line with the formative goals of the course.

Assessment Methods

The final evaluation consists of an individual oral exam (approximately 30 minutes). The assessment will focus on comprehension of the course content, ability to discuss and resolve case studies proposed during the exam, and appropriate use of the discipline-specific scientific vocabulary.

Texts

Lecture slides will be made available to students. There is no mandatory textbook. For further reference, students may consult the following texts:
Chandler and Robertson, Bioimaging, 1st edition 2008, Jones & Bartlett Pub Inc, ISBN-13:‎ 978-1284063165
Spector and Goldman, Basic Methods in Microscopy, 1st edition 2005, Cold Spring Harbor Laboratory Press, ISBN-13: 978-0879697471
Further peer-reviewed articles may be provided by the teachers or specifically suggested upon student request according to individual interests developed during the course.

The course presents microscopy techniques in parallel with cytochemical and immunocytochemical approaches, addressing both theoretical principles and applicative outcomes.
Introduction to microscopy. Concept of resolution.
Optical microscopy: bright-field, dark-field, polarized light microscopy, phase contrast, differential interference contrast microscopy, stereomicroscope, and inverted microscope.
Fluorescence microscopy: principles of fluorescence, fluorochromes, and confocal microscopy.
Electron microscopy: TEM, SEM, Cryo-EM.
Correlative microscopy: Quantum Dots, labeled secondary antibodies, iCLEM, and photoconversion.
Sample preparation for optical and electron microscopy: theoretical principles and technical protocols.
Cytochemical techniques and staining procedures for optical and electron microscopy. Concepts of preferential and specific bindings. Enzyme histochemistry.
Nucleic acids detection: Feulgen reaction, osmium ammine staining, specific fluorochromes.
Immunocytochemistry: monoclonal and polyclonal antibodies, direct and indirect labeling, markers for optical, fluorescence, and electron microscopy.
In situ hybridization: probes, concepts of stringency and specificity.
Flow cytometry: basic principles and applications. Case studies: analyses of diverse cell death pathways.
Dynamic imaging techniques: FRAP, iFRAP, FLIP. Photoactivation and FRET.
Super-resolution microscopy: concepts of deconvolution and point spread function. STED, STORM, PALM, GSDIM, 4Pi, and SIM.
Energy-filtered electron microscopy, EELS, and ESI. Electron tomography and 3D object reconstruction. STM and AFM.
Advances in expansion microscopy, Bonfire, and FlashPaint.