Microscopy Overview PDF

8/27/24, 8:22 PM Platform | Study Fetch Microscopy Overview (00:00:24 - 00:00:35) Types of Microscopy There are two main types of microscopy: optical and electron microscopy. Immunohistochemistry (00:00:35 - 00:01:18)...

8/27/24, 8:22 PM Platform | Study Fetch Microscopy Overview (00:00:24 - 00:00:35) Types of Microscopy There are two main types of microscopy: optical and electron microscopy. Immunohistochemistry (00:00:35 - 00:01:18) Tissue Preparation 1. Collect the tissue sample (human, animal, etc.) 2. Fix the tissue by cross-linking the proteins using a chemical like formaldehyde 3. Embed the fixed tissue in a stabilizing medium (e.g., wax, gelatin) to allow sectioning 4. Section the embedded tissue using a microtome or "deli slicer" to create thin slices 5. Mount the tissue sections onto slides Antigen Retrieval After mounting, the proteins in the tissue need to be "opened up" so antibodies can penetrate This is done using heat, acid, or other methods to expose the antigens Blocking The slide surface is coated with a homogeneous layer of protein to prevent non-specific antibody binding Antibody Staining (00:01:31 - 00:01:43) Antibodies specific to the target antigens are added and will bind to the exposed antigens in the tissue Optical Microscopy (00:00:24 - 00:00:35) Overview Optical microscopes use visible light and lenses to magnify small objects Key Features Allows visualization of objects too small to be seen by the naked eye Provides higher magnification than the naked eye https://www.studyfetch.com/platform/studyset/66cd116dd279f5220d947c66/material/66ce6bd3e81bb65113fe36af/document?go=note 1/5 8/27/24, 8:22 PM Platform | Study Fetch Electron Microscopy (00:00:24 - 00:00:35) Overview Electron microscopes use a beam of electrons instead of light to image very small samples Key Features Provides much higher magnification and resolution than optical microscopes Can image structures at the nanometer scale Immunofluorescence Microscopy (00:01:43 - 00:01:56) Your primary antibody is specific to whatever you're trying to identify You then add a secondary antibody that will stick to the primary antibody and carry a fluorescent dye or label (00:01:56 - 00:02:10) You may also add a counterstain or nuclear stain to better visualize the cells on the slide This helps you better see the cells and where the antibody is binding Optical vs. Electron Microscopy (00:02:10 - 00:02:27) Light microscopy uses light as the source to observe the sample Electron microscopy uses magnets and an electron stream to visualize the diffraction and reflection of electrons on the sample (00:02:27 - 00:02:44) Electron microscopy offers higher resolution than optical microscopy Electron microscopy can image smaller structures like lipids, proteins, and even smaller viruses Types of Optical Microscopes (00:02:44 - 00:03:09) Stereo/Dissection Microscopes: Provide low magnification to observe the surface of live specimens Useful for procedures requiring fine motor control, like surgeries (00:03:09 - 00:03:24) Stereo microscopes have two eyepieces, allowing you to work with tools like tweezers under the microscope https://www.studyfetch.com/platform/studyset/66cd116dd279f5220d947c66/material/66ce6bd3e81bb65113fe36af/document?go=note 2/5 8/27/24, 8:22 PM Platform | Study Fetch "I've used these for um doing surgeries or any time that you need to do a procedure that requires very fine motor control. And typically you can even work with, with tweezers um under the dissecting scope because it has two eyes, you're" Microscopy Techniques Compound Microscopes vs. Dissecting Microscopes (00:03:24 - 00:03:38) Compound microscopes are similar to dissecting microscopes, but have a stage that holds multiple magnification lenses This allows them to visualize samples that are one cell thick Brightfield vs. Darkfield Microscopy (00:03:38 - 00:04:08) Brightfield Microscopy: Uses a bright light to illuminate the sample The sample is viewed against a white background Darkfield Microscopy: Only the scattered light from the sample is observed The background appears dark Phase Contrast Microscopy (00:04:08 - 00:04:34) Allows visualization of thin samples containing live cells Observes the phase shift of light between refracted and unrefracted light Can provide more detail on living cells, but may cause a halo effect Confocal Laser Scanning Microscopy (00:04:47 - 00:05:12) Particularly good for observing fluorescence Can visualize endogenous or cell-created fluorescent proteins Fluorescence Microscopy and Electron Microscopy https://www.studyfetch.com/platform/studyset/66cd116dd279f5220d947c66/material/66ce6bd3e81bb65113fe36af/document?go=note 3/5 8/27/24, 8:22 PM Platform | Study Fetch (00:05:12 - 00:05:34) Cells expressing green fluorescent protein can be useful for observing and detecting the presence of the fluorescent protein Fluorescence microscopy allows for visualization of fluorescence without the need for fluorescent labeling (00:05:34 - 00:05:51) Antibodies can be labeled with fluorescent dyes to visualize the location of specific proteins The sample is excited with a specific wavelength, and the emitted fluorescence is detected and visualized (00:05:51 - 00:06:04) There are several types of electron microscopes, including: Scanning electron microscope (SEM) Cryo-scanning electron microscope Transmission electron microscope Electron tomography (00:06:04 - 00:06:21) The scanning electron microscope (SEM) uses an electron gun to excite electrons on the surface of a sample The scattered electrons are then detected, allowing for visualization of the sample's surface (00:06:21 - 00:06:32) Samples for SEM need to be dehydrated before imaging, as the high vacuum environment would otherwise damage the sample SEM provides detailed visualization of the surface of objects (00:06:32 - 00:06:48) SEM can capture the depth and detail of samples, such as pollen grains, with incredible resolution However, the need for sample dehydration is a limitation of this technique Cryo-Electron Microscopy and Transmission Electron Microscopy Cryo-Electron Microscopy (00:06:48 - 00:07:04) Cryo-electron microscopy is a technique that allows you to visualize samples under the scanning electron microscope The sample is frozen with liquid nitrogen, which prevents dehydration and damage that can occur with other sample preparation methods Advantages of Cryo-Electron Microscopy (00:07:04 - 00:07:18) Allows you to get surface images of the sample, but the sample is frozen with liquid nitrogen instead of being dried out https://www.studyfetch.com/platform/studyset/66cd116dd279f5220d947c66/material/66ce6bd3e81bb65113fe36af/document?go=note 4/5 8/27/24, 8:22 PM Platform | Study Fetch Provides images that can visualize things that were not easily visualized before Examples of Cryo-Electron Microscopy (00:07:18 - 00:07:33) Cryo-electron microscopy images of cross-sections of Staphylococcus aureus biofilms, prepared by plunging into liquid ethane and sublimating for 10 minutes Transmission Electron Microscopy (00:07:33 - 00:07:46) Transmission electron microscopy uses an electron gun to fire electrons through the sample This provides better detail of the interior of the objects, rather than just the surface Differences between TEM and SEM (00:07:46 - 00:08:01) Transmission electron microscopy is used for 2D images, while scanning electron microscopy provides more 3D images Transmission electron microscopy gives a cross-sectional view, while scanning electron microscopy requires scanning the whole object to get a 3D image Electron Tomography (00:08:01 - 00:08:24) Electron tomography is not really a type of microscopy, but a way to look at and reconstruct the 3D image of a substance It involves scanning the entire structure to build up a 3D image, similar to how a 3D printer works Key Takeaways (00:08:24 - 00:08:31) You have now mastered the key concepts of different electron microscopy techniques Good luck on your test! https://www.studyfetch.com/platform/studyset/66cd116dd279f5220d947c66/material/66ce6bd3e81bb65113fe36af/document?go=note 5/5