Microscopy.pdf

This and all BIOL200 lecture material is protected by copyright. Textbook and other images whose copyright do not belong to the instructional team remain the property of those that created them, and are used with permission, or under the Fair Dealing Requirement and credited as appropriate. NO BIO...

This and all BIOL200 lecture material is protected by copyright. Textbook and other images whose copyright do not belong to the instructional team remain the property of those that created them, and are used with permission, or under the Fair Dealing Requirement and credited as appropriate. NO BIOL200 material should ever be shared to third party websites without permission of the instructional team.  BIOL200 24WT1 Week 1 Unit 1: Visualizing Cell through Microscopy Image: Super-resolution microscopy of Bovine pulmonary artery epithelium. Credit: Harvard Medical School Dept of Cell Biology - https://sim.hms.harvard.edu  Administrative Notes 1. Questions about the syllabus? 2. Term Project – Your current job is to find a team, name it and email me. The Perusall general discussion board can be used to help with this if you need it. Deadline for team formation is Tuesday Sept 17th!! 3. To submit before next class Syllabus/ Pre-requisite quiz due tomorrow Perusall – First Problem Set annotations are due tomorrow. First attempt for Week 2 Content Quiz due Monday 4. Perusall suggestions: Tag me if you want me to weigh in on your problems solving discussion (use the @ symbol and names will pop up) The general discussion board that can be used for questions that are not related to a specific problem set and also for Term Project team building! Unit 1 – Microscopy  Learning Goals for Unit 1: Visualizing Cells through Microscopy Aligns with Chapter 1 (all topics combined) Distinguish between the four major classes of microscopy: brightfield light microscopy, fluorescence light microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). CLO4 Discuss the major advantages and limitations of each of the four major classes of microscopy. CLO4 Understand the difference between magnification and resolution, and how each one influences what can be visualized in the four major classes of microscopy. CLO1, CLO4 Identify the major cellular organelles in the 4 different types of microscopy. CLO2, CLO4 Identify the type of microscopy that is best suited to detect and study cellular components based on their size and functional aspect being studied. CLO2, CLO4 Interpret the results in experiments using microscopy based on scale, magnification, resolution and plane of section. CLO1, CLO4 How small is a typical eukaryotic cell? A. Larger than 1mm B. Smaller than 1mm, but bigger than 1µm C. Smaller than 1µm, but bigger than 1 nm D. Smaller than 1nm, but bigger than 1pm E. Smaller than 1pm Magnification & resolution determine what can be seen in a microscope, but which is which? Magnification = Resolution = When working with microscopes, which one is going to be more important in terms of what you can see? A. Magnification B. Resolution C. Both are equally important Resolution of different imaging methods This area of overlap covers most of what we care about in cell biology! Textbook Figure 01-02  Use light microscopy to see live cells, colour** and whole tissues. Brightfield Fluorescence Plant root vascular bundle Tetrahymena sp. Ciliated Killer T-cell engaging a Figure 9-11b Molecular Biology of the Cell marine protist cancer cell (A. Ritter et al.) ** Remember that colour can be deceiving, as it is easily photoshopped in & out of the image.  Types of Light Microscopy Transmitted light LM Emitted light LM Light passes through specimen and Coloured light (or lasers) are used to viewed excite photons that are then Bright field emitted by sample and viewed Dark field Fluorescence Phase contrast Epifluorescence Polarized light microscopy Confocal Differential interference-contrast Spinning disk confocal (DIC) Super resolution (many types) Let’s draw the path of photons through the microscope together See also Textbook Figure 01-04  Different types of fluorescence microscopy allow for increased resolution without changing magnification. Confocal Microscopy of microtubules STORM Superresolution Microscopy of microtubules Green Fluorescent Protein (GFP) can be genetically added to gene/ protein of interest, so that it can be located in live cells Originally discovered in Jellyfish, but now known to exist in many bioluminescent organisms Small and versatile. Genetic engineering makes it stable and heritable. Has been extensively modified to suit the needs of the research Addition of targeting signals, Different colours → Split GFP Etc. Difficult to locate more than a 1-2 proteins at once, as they must all be engineered individually.  Immunolabelling uses antibodies to locate proteins of interest in cells. Can be faster/ less difficult that genetic engineering GFP onto a gene Allows more thinks to be labelled in the same cell/ tissue Usually requires cells to be fixed & permeabilized (so they can enter the cell) Thus, live-cell imaging is not possible Can be used in both light and electron microscopy Allows for “correlative microscopy” that looks at the same samples in different ways. There is no real way to tell the difference between different labelling or imaging techniques in fluorescence microscopy, from just a picture. Electron microscopy is used for higher resolution and viewing fine structural details Transmission EM Scanning EM © R.E. Young Muskox tooth Plant microtubules From UBC Biomedia Database Now let’s draw the path of electrons through microscopes  Transmission electron microscopy (TEM) Advantages -Details of cytoplasm can be seen (samples are cut into thin sections) Disadvantages: -Cells must be dead -Complex specimen preparation thin section, coat in heavy metals) Arabidopsis thaliana seed coat cell - difficult to know 3D shape of © R.E. Young structures…. Scanning electron microscopy (SEM) Advantages: -Can view surfaces (images appear 3D) Disadvantages: -Cells must (usually) be dead Muscle tissue from a cow From UBC Biomedia Cell Biology Database Which one of these is a mitochondrion? B C- Both A D- Neither  The only way to get better at understanding microscopy is to practice!! Every time you see a microscopy image, ask yourself 4 things: 1. What type of microscopy do I think this is? 2. What can I see? 3. Does what I think I see match the type of microscopy I think it is? Could it be anything else? 4. What else do I know about this image? For the rest of today’s class (if there’s any time left?) work on the Microscopy quiz on Canvas. This is best done in groups, and by asking lots of questions! Next class... Unit 2: Topic 2.1 - The Features of Membranes (Microscopy Quiz due by the end of tomorrow, as well as your first Perusall Problem Solving)