Laser Microdissection systems allow for the procurement of specific populations of cells from tissue and cytology and live cell culture samples containing heterogeneous populations of cells. The specificity of analyses is therefore much more representative of the disease process being studied. This approach to microdissection ensures that biological molecules, such as RNA and DNA and proteins, remain undamaged during the microdissection process. Downstream molecular analysis of these molecules produces accurate and assured results that have led to over 2,000 peer-reviewed publications by independent researchers.
In this training program, participants learn to prepare tissue specimens for microdissection, then select and acquire homogenous cell populations using the mmi-CellCut, Leica LMD, Arcturus XT, and PALM microdissection systems. Instruction emphasizes operation of these LM systems, appropriate tissue handling and sample preparation for subsequent DNA, RNA or protein analysis, and methods for proper molecular extraction. Lecture and detailed instructions to prepare samples for several downstream molecular analyses are presented.
Overview of Laser Microdissection Technology (History, Theory, Applications); Tissue Sample and Slide preparation; Project set up and QC of microdissected samples, genomic analysis, mRNA analysis including quantitative RT-PCR gene expression analysis and microarrays from microdissected tissue samples, microRNA analysis from microdissected samples, and proteomic analysis from frozen and formalin-fixed, paraffin-embedded microdissected samples using Mass Spec. Special microdissection techniques such as immuno-guided LM and live cells dissections will be also covered.Â The lectures also include examples of actual scientific and clinical applications of LM-based studies. The hands on sessions include tissue slide preparation and histology review for LM, and practice on Arcturus XT, PALM, Leica and mmi-CellCut platforms.
This 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
Imaging of cells and tissue through the microscope in years past had embraced the use of film to capture pertinent events. Improvements in recent years have led to the insurgence of digital imaging techniques which allows for considerably more flexibility and accuracy in obtaining these images. Digital imaging effectively addresses the demands of high resolution, color accuracy speed of acquisition, imaging flexibility and low cost all of which are demanded by the field today. Areas to address in this course will include information related to effective imaging through the microscope as it relates to the camera itself as well as appropriate microscope setup to allow for optimal results in the lab situation.
Characteristics of a wide range of digital cameras (ie resolution vs sensitivity); Appropriate filter selection for the microscope (ie narrow band vs wide band and imaging techniques to optimize signal localization); Advantages of microscope motorization for digital imaging and 3D rendering (ie Z-stack acquisition); Optical sectioning (ie Apotome and Deconvolution). These topics will all be discussed in lecture format by specialists in the individual fields from both the research field as well as from industry where appropriate. Daily laboratory classes will be included for part of each day to allow for hands on experience with a variety of imaging systems.
Super Resolution Microscopy represents a group of recently developed light microscopic techniques that are able to exceed diffraction-limited resolution (less than 200um). This course will focus on three types of Super Resolution Microscopy, Structured Illumination Microscopy (SIM), Stochastic Optical Reconstruction Microscopy (STORM) and Stimulated Emission Sepletion (STED). The course is designed for cell biologists with prior experience in light microscopy who wish to add super resolution microscopy to their research program. Participants will acquire both a theoretical understanding of super resolution microscopy and practical experience using state-of-the-art super resolution microscopes.
Introduction to Super Resolution Microscopy; Advances in Super Resolution Microscopy; Theoretical Background of SIM, PALM/dSTORM, and STED Imaging; Advances in Fluorescent Protein and Organic Dye Technology; Applications of Super Resolution Microscopy using SIM and dSTORM; Challenges Associated with Obtaining Good SIM, STORM and STED Images; Potential Artifacts Common to Super Resolution Imaging.
Working in groups of five, students will rotate through six work stations (Leica gSTED, GE- SIM, Nikon STORM, Zeiss PALM, Shroff Lab – Instant SIM, Shroff Lab – Dual View Plane Illumination Microscope).
During the laboratory sessions, the following topics will be covered: Introduction and Feature Highlights of the Instrument; Imaging Acquisition Procedures; Sample Preparation Requirements and Recommendations (Dye Choices and Imaging Solutions); Imaging Data Analysis and Presentation; Trouble-shoot Common Problems in SIM Imaging; Acquisition of 2D and 3D STORM Images.