Learning objectives

The course aims to provide students with a knowledge of the fundamental principles of the main imaging technologies applied to translational research, including optical and fluorescence microscopy, nuclear magnetic resonance imaging, and flow cytometry. The course is structured so that, starting with an introduction to fundamental scientific and technological concepts, students will develop an understanding of the main applications of imaging technologies in translational research and diagnostics, through laboratory exercises conducted in working groups. The course is structured into two distinct modules: - Principles of (bio)Imaging Module provides students with the physical concepts underlying the construction of images in modern biomedical imaging techniques; it illustrates concepts related to image quality and contrast, spatial and temporal resolution, and allows students to understand the main biomedical applications of various technologies; it provides the skills necessary to independently read and understand scientific papers in the field, and present their main results; - Biomedical Applications of Imaging Module allows students to delve deeper into the uses of bioimaging techniques in biomedicine, both clinically and in research. The course covers the main techniques for visualizing cellular structures and ultrastructures using optical, fluorescence, and multiphoton microscopy, as well as the applications of flow cytometry for assessing the heterogeneity of cellular samples. It also provides the necessary morpho-functional substrate for evaluating the results obtained. Upon completion of the course, students will have acquired: a) in-depth knowledge of modern diagnostic techniques used in the biomedical field (ultrasound, X-rays, nuclear medicine, magnetic resonance tomography, and optical imaging) and microscopy techniques; b) comprehensive knowledge of analysis methodologies (e.g., segmentation of various organs and volume calculation, parameter extraction, signal-to-noise ratio and contrast measurement, etc.); c) the ability to apply the acquired knowledge to design and execute a simple experiment aimed at collecting data with a real acquisition system (e.g., a magnetic resonance tomography scanner for experimental studies); d) the ability to work in a team, interpret the results of experimental analyses, and communicate them according to the standards of the scientific community; e) ability to analyze a scientific article in the field, understanding the imaging techniques used, the main results obtained, and the limitations of the article itself; f) adequate morpho-functional bases for a correct interpretation of the images and the biological significance of the parameters investigated; g) ability to plan further bioimaging studies in preclinical models of pathology, both oncological and of the CNS.

Prerequisites and basic notions

Knowledge of mathematics, physics and biology at high school level

Program

Introduction, classical imaging techniques, pixel, voxel, segmentation, clusterization, bit-byte, gray levels, pixel dimension, dot per inch, contrast resolution, spatial uniformity, and linearity.
Analog photography, analog to digital conversion. Dynamic range and tone, file size, and compression. Color images: real and pseudo-color images. Receiver operating characteristic (ROC) curve.
Spatial resolution, line spread function, point spread function. Signal-to-noise ratio, co-added scans, contrast-to-noise ratio. Image filtering: high pass, low pass, convolution.
Histograms (Number of pixels vs. gray levels): underexposure, overexposure.
Analog to digital converters, amplifiers. Image artifacts.
DETECTORS
Charge Coupled Device (CCD), the photoelectric effects on metal surface and in silicon bulk, quantum efficiency, well capacity, and dark current, back-thinned back-illuminated CCD, CCD for color acquisitions, the readout process.
CMOS camera, photodiode area
Photodiodes, Photomultipliers, Photomultiplier’s gain, offset
OPTICAL MICROSCOPY
Single lens, lens maker’s equation, focal length, magnification, simple and compound microscope, total magnification, Abbe’s equation, refractive index, numerical aperture, working distance, depth of field, digital microscopes, fluorescent microscopes, fluorescent process, fluorescent dyes (cyanines, proteins, nanoparticles, quantum dots), stokes shift, filter cubes.
Confocal microscopes, pinhole, point spread function, z-stack.
Immunofluorescence, photobleaching, phototoxicity. Diffraction and Airy disk, resolution, Opto-Acoustic Beam Splitter (AOBS). Two-photon microscopy and Light-sheet microscopy.
ELECTRON MICROSCOPY
Overview, de Broglie’s equation, resolution, charged particle in a magnetic field, and in an electrical field, electromagnetic lenses
TEM instrument structure, thermionic effect, electron gun, lenses, apertures, screen detectors, vacuum pumps, mass-thickness contrast, diffraction contrast, sample preparation
SEM instrument structure, detectors, secondary electrons (SE), reflected or back-scattered electrons (BSE), characteristic X-rays, light (cathodoluminescence) (CL), sample preparation, cryo-microscopy, magnification, X-ray microanalysis. SEM application in the analysis of wounds in red meat, chicken breast, fat, and bone produced by laser interaction.
X-RAYS
X-rays: energy (electronvolt), wavelength, frequency. X-ray production: X-ray tube, Joule effect. Bremsstrahlung radiation, characteristics X-rays. Absorption, Lambert Beer law, absorption coefficient, mean free path before absorption, diffusion, photoelectric effect, Compton effect, pair production, annihilation. Primary and secondary radiation. Image contrast in the case of the photoelectric effect. Image formation. Antiscatter grid. Detector plate and Cr reader. Computed tomography, instruments. Contrast agents. Housenfield scale.
NUCLEAR MEDICINE
Radioactivity: alpha, beta (minus and plus), and gamma. Radioactive decay law, half-life. Radiotracers.
Planar scintigraphy; Tc99 m radiotracers, production, applications, other radiotracers, gamma camera, scintillating crystals, collimators, photomultipliers. Single-photon-emission computed tomography (SPECT): instruments.
PET: detectors and PET/CT instruments, acquisition of PET images, Line of Resolution (LOR), 18-FDG, other radiotracers, cyclotrons, Standardized Uptake Value (SUV). Application in the case of various brain stimuli.
ULTRASOUNDS
Elastic waves, Huygens’s principle, Intensity, sound and ultrasound definition, sound level, piezoelectric transducer, sound speed, acoustic impedance, backscattered signal, time gain compensation. Absorption, beam divergence and penetration, lateral and axial resolution. Ultrasound contrast agents. Doppler effect for eco-doppler imaging.
MAGNETIC RESONANCE IMAGING
Introduction, Damadian principal results, nuclear spin, parallel and antiparallel orientation, energy levels, Boltzmann statistics, Energy transition, Larmor frequency, gyromagnetic ratio, spin packets, magnetization vector, T1 and T2 relaxation times, Ts*, magnetic gradients, slice selection, frequency encoding, back projection, the receiver signal, examples of sequences, applications, contrast agents, USPIO contrast agents, relaxivities (r1 and r2).
OPTICAL IMAGING TECHNIQUE
Introduction, light transport in biological tissues. Absorption, the Lambert-beer equation, absorption coefficient, mean free path before absorption, bio-molecules responsible for the absorption and range of wavelength. Scattering process, Rayleigh scattering, Mie solution. Absorption and scattering together, case of blood. Photon propagation in Monte Carlo simulations.
Fluorescence imaging, fluorescent dyes, instrumental set-up and some preclinical applications. Bioluminescence imaging and some experimental investigations used in the oncological field. Description of the instruments for optical imaging acquisitions: technical equipment and image acquisition parameters.

Bibliography

Didactic methods

Frontal lessons, flip classroom

Learning assessment procedures

Written exam with numerical exercises (bring your calculator), open-ended questions and multiple-choice questions on imaging techniques and their applications

Assessment

Knowledge of the physical principles underlying imaging techniques, accuracy in exposure and correct use of language will be assessed.

Criteria for the composition of the final grade

Weighted average of the marks obtained in the two respective modules with the CFU