TRAC 35: Immunofluorescence and Confocal Microscopy
This lecture and "hands on" laboratory course will focus on two important methods that are used extensively in biomedical research. Fluorescence microscopy is a useful tool for observing cellular morphology and function that is readily available and relatively simple to learn. Any specimens prepared for fluorescence microscopy can be analyzed in further detail and with improved resolution using confocal or laser scanning microscopy. Confocal microscopy is a powerful technique for examining 3-dimensional localization and dynamics of cellular components.
Fluorescence Microscopy: Introduction and description of principles; Specimen preparation; Applications; Confocal Microscopy in Biomedical Research: Different types of confocal microscopes and their uses; META multicolor; Two Photon imaging live animals such as mice; Spinning Disk improved resolution for examining movement of organelles and molecules in living cells; Colocalization and Interaction of Proteins: Differences between colocalization and interaction; Importance of preventing emission and excitation crosstalk; Using FRET to show protein-protein interactions; Live Cell Studies: Using FRAP and FLIP to look at cell dynamics; Using time series to look at translocation of proteins
Sample preparation for fluorescence microscopy; Acquisition of Basic Confocal Scans using LSM 510, META, Multiphoton, and Spinning Disk; Colocalization using multifluorescence imaging; Protein-Protein Interaction (Fluorescence Resonance Energy Transfer FRET); Live Cell Imaging: Translocation and localization of proteins Using fluorescent fusion proteins and time lapse imaging; Protein Dynamics Using fluorescence loss in photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP); Photosections of thick specimens; Using (fli1:EGFP) Zebrafish
Related Bio-Trac Course: Bio-Trac 38: Digital Imaging in Microscopy