USE AND CARE OF THE MICROSCOPE LECTURE 1

MICROSCOPY u Light Microscopy: any microscope that uses visible light to observe specimens u Compound Light Microscopy: u utilizes a series of lenses u uses visible light as the source of illumination u allows you to observe a specimen as a magnified image u Resolution=0.2 um u Brightfield Illumination: visualization of dark objects in a bright field

Fine adjustment Coarse adjustment

Key Terms u Illuminator: light source u Condenser: series of lenses that direct light through the specimen of interest u Objective lens: first magnification, lens located closest to the specimen, usually 4 different objective lenses (scanning, 10X, 40X, 100X) u Ocular lens: located in the eyepiece, last magnification lens u Coarse adjustment: used with low-power objectives (scanning and 10X) u Fine adjustment: used with high-power objectives (40X and 100X)

Key Concepts u Total Magnification: Multiply the objective lens magnification by the ocular lens (usually 10X) magnification u Resolution/Resolving Power: Ability of the lens to distinguish fine detail and structure or the ability to distinguish two points a specified distance apart. Compound Microscope = 0.2 um. Shorter the wavelength, the greater the resolution. u Oil Immersion: Same effect of increasing the objective lens, therefore it improves the resolving power of the lens u Refractive Index: The measure of the light bending ability of a medium (Ex:Water). Therefore, specimens must be made to contrast their medium. u Use of Stains, Dyes

Other Types of Microscopy u Darkfield: Specimen is light against a dark background, used to examine live organisms u Phase-Contrast: uses special condenser to enhance intracellular details of specimen can be visualized in living organisms/ takes advantage of differences in refractive indexes of cell structures u Differential Interference Contrast (DIC): Similar to Phase-Contrast, higher resolution, image appears 3-D u Fluorescence: Ability of specimens to absorb short wavelengths of light (ultraviolet) and give off long wavelengths of light (visible). Used in diagnostic procedures called fluorescent antibody technique u Confocal: specimen is stained w/ fluorescent dye, computer process the image-uses laser light to illuminate one plane of a specimen at a time. Higher resolution, clear images

Electron Microscopy u Electron Microscopy: u Used for specimens smaller than 0.2 um u Electrons used instead of a beam of light u Higher Resolution due to shorter wavelengths of electrons (~100,000X shorter than visible light) u Types of Electron Microscopes: u Transmission u Scanning

TEM u Transmission Electron Microscopy (TEM): u A beam of electrons is passed through an ultra-thin section of specimen u electromagnetic lenses are used to direct the beam u a copper mesh grid is used to view the sample u a projector lens is used to focus the image on a fluorescent screen u Resolving Power =2.5 nm u Total Magnification =10, ,000X u best for virus particles, flagella and proteins u resulting photo is black and white, no 3-D

SEM u Scanning Electron Microscopy (SEM): u Uses an electromagnetic lens to direct electrons over the surface of a specimen u Results in a 3-D image u Resolving Power= 20 nm u Total Magnification = 1,000-10,000X u Best for observing the surfaces of microorganisms

IMPORTANT TIPS TO REMEMBER: u 1-OCULAR AND OBJECTIVE LENSES SHOULD ONLY BE CLEANED WITH LENS PAPER (NOT BIBULOUS PAPER) ALWAYS CLEAN THEM BEFORE AND AFTER USE u 2-OIL SHOULD ONLY BE USED WITH 100X OBJECTIVE u 3-RETURN MICROSCOPES ON SCANNING OBJECTIVE WITH STAGE UP AND SLIDE CENTERED u 4-ALWAYS HANDLE AND CARRY YOUR MICROSCOPE USING THE BASE AND ARM