MCB 100 1st Edition Lecture 10 Outline of Last Lecture I. Sugars and polysaccharides II. Fatty acids and lipidsIII. Microscopy: simple vs. compound lens system Outline of Current Lecture I. Magnification II. Resolution III. The limits of resolution with light microscopy IV. Electron microscopesV. Microscopy techniques A. Bright-field microscopyB. Dark-field microscopyC. Phase contract microscopy D. Fluorescent microscopy E. Electron microscopy Current LectureI. MagnificationA. Magnification: the ability of a microscope or a lens to increase the apparent size of the image of an objectB. Total magnification: for a compound microscope the total magnification is the product of the magnifying abilities of all of the lenses in the system C. Total magnification = (magnification of the objective lens) x (magnification of the ocular lens)II. ResolutionA. The ability to discern fine details in an image of an object B. Resolving power: (resolution distance) R.P. is a measure of the smallest distance between two separate objects that can be seen to be 2 separate objects C. The resolving power formula: R.P. = (0.61 x wavelength)/numerical aperture D. Smaller R.P. = ability to see finer details E. To get better resolution (smaller R.P.) you need to increase the numerical aperture ordecrease wavelength of the light used to illuminate the object F. Summary: a shorter wavelength of light gives a smaller and thus better resolving powerThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.II. The limits of resolution with light microscopy A. Typical bacterial cells are 700-3000 nm in length. B. Typical viruses are smaller than 100 nm in diameterC. So for light with wavelength of 480 nm (blue) using a good microscope with a numerical aperature of 1.25, resolving power = (0.61 x 480 nm)/1.25 = 235 nm D. Viruses are too small to see with a light microscope and bacteria are so small you can't see much detailE. (remember that 1000 nanometers = 1 micron, and know other conversions for the metric system) IV. Electron microscopes a. Allow you to see viruses and subcellular structures such as ribosomes, b/c electrons have a shorter wavelength than light and thus smaller resolving power (better) b. With better resolving power, you can use higher magnifications without encountering the phenomena of "empty magnification" c. Can be used at up to 100,000x magnificationV. Microscopy techniquesa. Bright-field microscopy: light shines through specimen and directly into the objective lens and to the observer's eye creating a lighted or bright field of vision. Bacteria and other microorganisms are seen as dark objects on white background. i. Works best when bacteria are stained a bright color and immobilizedii. Very easy to use iii. Works best with fixed and stained microorganisms b. Dark-field microscopy : light shines on specimen from an oblique angle and doesn’t go directly into the objective lens. The background or field of vision is dark. The only light that gets to the observer's eye is the light that is scattered by specimen. i. Specimen doesn’t have to be stained ii. Works for living microorganisms but they move around pretty fast--> difficult tofocus on them b. Phase contract microscopy: invented by Frits Zernike (1934); relies on a complicated optical trick to enhance the visual effect seen when light goes from a medium of lesser optical density (water) to a medium of greater optical density (cytoplasm) i. Beams of light are bent when they cross the boundary between 2 different transparent substances with different optical densitiesii. A lens can diffract a beam b/c light moves slower in glass than in air; optics enhance the contrast seen when light is diffracted at a phase boundary iii. Most useful for examining living protozoa and other eukaryotic cells. iv. Doesn’t require specimen to be stained a. Fluorescent microscopyi. Specimen is illuminated with UV light which cause some chemicals to emit visible lightii. UV light is called black light b/c you cant see itiii. The only visible light seen is that which is produced by fluorescence of naturallyoccurring compounds in the microorganism or special dyes that are used to stain the specimen 1. Natural fluorescencea. Some bacteria make a fluorescent pigmentb. Photosynthetic pigments like chlorophyll are fluorescent c. Methanogenic archaea make a fluorescent pigment 2. Fluorescent dyes a. For DNA (acridine orange) b. For bacillus anthracis (fluorescein isothiocyanate) c. For Mycobacterium tuberculosis (auramine O) 2. Immunofluorescence microscopy a. A fluorescent dye such as fluorescein (yellow-green) or rhodamine (red) is chemically joined to an antibody to make a very specific probe. b. Electron microscopy i. Sample is bombarded with electrons ii. Image is produced using a cathode ray tube iii. Sample must be dehydratediv. Sample must be stained with a heavy metal v. Sample must be In an evacuated chamber 1. TEM: Transmission Electron Microscopy a. Thin sliced exampleb. Used to see cellular organelles such as E.R., mitochondria, chloroplasts, ribosomes, etc. 2. SEM- Scanning Electron Microscopy a. Sample is sputter coated with gold b. Used to look at the surface of objects c. (variant = freeze/fracture