Question

)Tabulate how transmission electron microscopy (TEM) differs from traditional light microscopy regarding (a) sample preparation (4), (b) processing (4), (c) instrumentation (4), (d) detection (4) and (e) the structures that can be observed (3), (f) staining (3), (g) IHC (4) and (h) ISH (4).

Answer 1

Transmission electron microscopy (TEM)	Light Microscope
Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid.	A light microscope (LM) is an instrument that uses visible light and magnifying lenses to examine small objects not visible to the naked eye, or in finer detail than the naked eye allows.
Sample Preparation: For TEM, samples must be cut into very thin cross-sections. This is to allow electrons to pass right through the sample.	Sample Preparation: Preparation often involves nothing more than mounting a small piece of the specimen in a suitable liquid on a glass slide and covering it with a glass coverslip.
Process: Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen.	Process: After passing through the specimen on the stage, the light enters an objective lens. Most light microscopes have three or four objective lenses on a rotating turret. These lenses magnify the image by 4x to 100x. The light then passes up the body tube to an ocular lens that magnifies the image another 10x to 15x.
Instrumentation: Transmission electron microscope that has three essential systems: (1) an electron gun, (2) the image-producing system, consisting of the objective lens, movable specimen stage, and intermediate and projector lenses, and (3) the image-recording system, The image-recording system usually consists of a fluorescent screen for viewing and focusing the image and a digital camera for permanent records. In addition, a vacuum system, consisting of pumps and their associated gauges and valves, and power supplies are required.	Instrumentation: The main components of light microscopes are: eyepiece, lens tube, objective revolver, stage, table, condenser, fine focus, coarse focus, luminous-field diaphragm, light source, base. An eyepiece is that part of an optical system, which is directed to the viewer. It is a construction of at least one or more lenses.
Detection and structure observed: Electron gun produces the electron beam, and the condenser system focuses the beam onto the object. The image-producing system, focus the electrons passing through the specimen to form a real, highly magnified image, and the image-recording system converts the electron image into some form perceptible to the human eye. The transmission electron microscope is used to view thin specimens (tissue sections, molecules, etc) through which electrons can pass generating a projection image.	Detection and structure observed: A magnified image is formed through the use of a series of glass lenses, which first focus a beam of light onto or through an object, and convex objective lenses to enlarge the image formed You can see most bacteria and some organelles like mitochondria plus the human egg. You can not see the very smallest bacteria, viruses, macromolecules, ribosomes, proteins, and of course atoms.
Staining: The most widely used stains in electron microscopy are the heavy metals, uranium and lead. The double contrast method of ultrathin sections with uranyl acetate (UA) and lead citrate is the standard contrasting technique for electron microscopy.	Staining: During Light Microscopy, Crystal Violet - stains cell walls purple when combined with mordant. Iodine - used as a starch indicator. When in solution, starch and iodine turn a dark blue color. Malachite green - a blue-green counterstain to safranin in Gimenez staining for bacteria.