Overview

ABOUT THE COURSE:Different types of electron microscopies and their applications in biological system. Basic introduction of electron microscopy physics and optics. Principles of image formation, electron scattering, phase contrast, electron–biological specimen interactions, electron diffraction. Sample preparation, imaging, data collection techniques of biomolecules (DNA, Protein, RNA, lipids, DNA-protein, RNA-protein, lipid-protein, RNA-lipid complexes) for negative staining TEM and cryo-electron microscopy. Basic principles and introduction to structure determination using single particle cryo-EM and cryo-electron tomography.INTENDED AUDIENCE: UG and PG studentPREREQUISITES: No other courses should be done before this can be done. Good knowledge in Mathematics, Physics, Chemistry and Biology.INDUSTRY SUPPORT:Any pharmaceutical company will be interested about this course.Sun Pharmaceutical Industries Ltd.,Alembic Pharmaceuticals Ltd.,NATCO Pharma Limited,Serum Institute of India Pvt. Ltd., Pune,Mylan Laboratories Limited,

Syllabus

Week 1: Objectives and basic working principles of different types of microscopes, including all the light microscope. Different types of electron microscopies, like SEM, TEM, STEM, HT-TEM and their applications.
Week 2:Basic introduction of electron microscopy physics like electron gun (sources), electromagnetic lenses system.
Week 3:Various types of aberrations of lenses (Spherical aberration, Chromatic aberration, Astigmatism, Coma), resolution limit of the microscope, magnification, and diffraction by electron microscope.
Week 4:Microscope basic vacuum system, and camera (CCD & Direct electron detectors). Principles of image formation, Ray diagram to understand the image formation, Fourier analysis.
Week 5:Contrast Transfer Function and point spread function, electron scattering, phase contrast, electron–specimen interactions, electron diffraction.
Week 6:Study the various advanced sample preparation techniques for TEM. Negative staining TEM analysis of soluble protein, membrane protein, DNA-protein complex, RNA-protein complex, lipid-protein complex, liposome sample preparation and imaging and image analysis. Identify good and bad negative staining images.
Week 7:Cryo-EM sample preparation (grid preparation) using Vitrobot, VitroJet, Chameleon and other techniques; We will learn sample preparation and imaging of soluble protein, membrane protein, DNA-protein, lipidprotein. Sample loading and automatic data collection and data analysis.
Week 8:Sample preparation using ultramicrotomes & cryo-ultramicrotomes-- tissue sample, whole cells, polymer, and nanoparticles.Learn the methodology to transfer the thin sections to transfer to grids and visualize and analyze samples using TEM and cryo-EM.
Week 9:Data collection using cryo-EM. Study ray diagram to understand focus overfocus under focus image collection, importance of defocused data collection/image collection (for 3D reconstruction and only for imaging), electron dose rate, identify good and bad image, understand Signal/Noise ratio.
Week 10:Theoretical, computational and practical aspects of various advanced 3D image processing techniques for all kinds of EM data. Basic understanding of Cross-Correlation and Convolution. Describe whole pipeline of 3D reconstruction and importance of 2D classification, 3D classifications.
Week 11:Basic principle behind 3D reconstruction and reference-free 2D classification, 3D classifications. Resolution and FSC calculation.
Week 12:Cryo-EM map interpretation and data analysis, FSC calculation, validation, molecular rigid-body docking and basic of model building.