Università Roma Tre

The course aims to provide the student with basic knowledge and technical skills in order to approach the study of biological specimens by ultrastructural morfological methodologies. The student will be able to autonomously choose and adjust experimental protocols, suited to different sample types and to the objective of the study. Knowledge acquired during the course will allow the student to critically analyze morphological results in a functional context.

Upon completion of this course, the student will acquire:
1) Knowledge of basic principles of electron microscopy;
2) Knowledge and competence on methodologies of sample (prokaryotes and eukaryotes) preparation for ultrastructural analysis;
3) Basic competence for utilization of devices for ultrastructural investigation (scnaning, transmission and focusing ion beam microscopy);
3) Competence on the use of appropriate software for microscopic image capturing and processing;
4) Basic competence on qualitative interpretation and quantitative analysis of ultrastructural data.


Course programme
Electron microscopy principles. Trasmission electron microscopy (tem). Scanning electron microscopy (sem). Focussing ion beam microscopy (fib/sem). Use of edax probe for the study of atomic composition.
Biological sample preparation for morphological analysis: general principles. Sample harvesting. Fixing methods: immersion and perfusion. Fixative solutions. Post-fixation methods for electron microscopy. Cryofixation techniques.
Tem: dehydration and embedding in acrylic ed epoxy resins. Ultramicrotomy: glass knives preparing, thin and ultrathin sectioning. Usage of diamond knives. Section harvesting on grids and contrasting methods. Use of tem: lens setting and alignment, sample positioning, focussing, image capturing.
Sem: dehydration methods and use of critical point dryer. Mounting and metallizing of samples, by sputter-coater. Use of sem in the modalities of secondary and back-scattered electrons detection. Image capturing and morphometric analyses.
Fib/sem: use of fib/sem on samples prepared for tem e sem. Milling and deposition techniques. Tem lamellae preparation. Cross sections. Serial images acquisition and processing.
Immunolocalization by pre- and post-embedding methods: immunoenzymatic procedures and usage of antibodies conjugated to colloidal gold nanoparticles.

Booklets of lectures and lab sessions will be provided.

The instructor will be available every working day, from 10 am to 1 pm, even to provide further bibliographic references, on appointment by email:
sandra.moreno@uniroma3.it

PRINCIPLES OF ELECTRONIC MICROSCOPY. TRANSMISSION MICROSCOPY (TEM). SCANNING ELECTRON MICROSCOPY (SEM). ION BEAM MICROSCOPY (FIB / SEM). STUDY OF THE ATOMIC COMPOSITION BY EDAX PROBE.
PREPARATION OF BIOLOGICAL SAMPLES FOR MORPHOLOGICAL ANALYSIS: GENERAL PRINCIPLES. COLLECTION OF SAMPLES. FIXATION METHODS: IMMERSION, PERFUSION MODES. COAGULANT AND NON-COAGULANT FIXATIVES. POST-FIXATION METHODS FOR ELECTRONIC MICROSCOPY. CRIO-FIXATION TECHNIQUES.
TEM: DEHYDRATION AND INCLUSION IN ACRYLIC AND EPOXY RESINS. ULTRAMICROTOMY: PREPARATION OF GLASS BLADES, SECTION OF THIN AND ULTRA THIN SLICES. USE OF DIAMOND BLADES. COLLECTION OF SECTIONS ON GRIDS AND CONTRAST. USE OF TEM: BEAM ALIGNMENT, SAMPLE POSITIONING, FOCUSING, CAPTURE OF IMAGES.
SEM: DEHYDRATION METHODS AND USE OF THE CRITICAL POINT DRYER. SPECIMEN MOUNTING AND METALLIZATION OF SAMPLES BY SPUTTER-COATER. USE OF SEM IN THE DETECTION OF SECONDARY AND BACK-SCATTERED ELECTRONICS. IMAGING AND MORPHOMETRIC ANALYSIS.
FIB / SEM: USE OF THE FIB / SEM MICROSCOPE ON SAMPLES PROVIDED FOR TEM AND SEM. MILLING TECHNIQUES, PLATINUM DEPOSITION. PREPARATION OF TEM SLICES AND CROSS SECTIONS. ACQUISITION OF SERIAL IMAGES AND THEIR PROCESSING.
PRE-AND POST-EMBEDDING IMMUNOLOCALIZATION: IMMUNOENZYMATIC METHODS AND USE OF ANTIBODIES CONJUGATED TO COLLOIDAL NANOPARTICLES.

MATERIAL AND PPT SLIDES SUPPLIED BY THE PROFESSORS.

Office Hours: every day of the week by appointment by email (andrea.digiulio@uniroma3.it)