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Microscopy 1 Biology 101A January 29, 2008 Magnification and Resolution • Magnification provides no additional information • Resolution often requires magnification Magnification without resolution Magnification without resolution Magnification without resolution Magnification without resolution Magnification without resolution Magnification without resolution Resolution is a measure of distance • Resolution = d = (.61λ)/N.A • d = distance between 2 pts. • λ = wavelength of light • N.A. = Numerical Aperture • N.A. = n sin α • n = refractive index • α = half-angle of cone of light Light travels in waves • White light is a mixture of several wavelengths • ROYGBIV Red---Violet • Red700nm • Violet400 nm • λ = wavelength of light Refractive index • • • • • • • • • • • • • • • • • • • • Refractive indices: AirVacuum Air @ STP Gases @ 0 °C and 1 atm Air Helium Water Ethyl alcohol (ethanol) Diamond Amber Sodium chloride Other materials Pyrex (a borosilicate glass) Ruby Glycerol Cubic zirconia Diamond Gallium(III) arsenide Silicon 1 (exactly) 1.0002926 1.000293 [1] 1.000036 1.333 1.361 2.419 1.55 1.50 1.470 [ 1.760 1.4729 2.15 - 2.18 2.419 3.927 4.01 Field of View • Actual diameter of microscope image at a certain mag. • As magnification increases, field of view _______. Depth of field • A measure of the thickness of the focal plane of an image • As magnification increases, depth of field _______________. Depth of field in Photography • Shallow depth of field prevents an entire object from being in focus Depth of field • Can be exploited for identifying layers in a substance Phase-contrast Electron Microscopes • Use electrons instead of light • electron wavelengths are much shorter than those of light • TEM- sends electrons through a specimen • SEM- specimen spraypainted with gold TEM SEM • Only looks at surfaces • Generates 3-D image • Often color-retouched Visualizing Fluorescence Green Fluorescent Protein • discovered in 1960s by Dr. Frank Johnson and colleagues • closely related to jellyfish aequorin • absorption max = 470nm • emission max = 508nm • 238 amino acids, 27kDa • “beta can” conformation: 11 antiparallel beta sheets, 4 alpha helices, and a centered chromophore • amino acid substitutions result in several variants, including YFP, BFP, and CFP 40 Å 30 Å More fluorescence Lab Report • Titles: • Which do you think is best? – Superpurple – Permeability permutations of purple anion membrane penetration – Properties of Nonliving Membranes Introduction • Some questions to consider for your lab report introduction: – What was learned in the previous lab that was pertinent to this one? – What structures exist in a normal cell membrane to regulate passage of things into and out of the cell? (this can be revisited in the discussion/conclusion sections) – What do the processes of diffusion, osmosis and active transport have to do with the lab at hand? • You do not need to answer all these questions Quiz Thursday! • Microscope care and maintenance (how to keep from breaking them) • Microscpe anatomy (labelling of parts) • Microscope principles (wavelength, magnification, etc.)