Microscopy 1 Biology 101A January 29, 2008

Magnification and Resolution Magnification provides no additional information Resolution often requires magnification

Magnification without resolution

Resolution is a measure of distance Resolution = d = (.61 λ)/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 Red- 700nm Violet- 400 nm λ = wavelength of light

Refractive index Refractive indices: Air- Vacuum 1 (exactly) Vacuum STP AirSTP 0 °C and 1 atm Air [1]Air [1] Helium Helium Water Water Ethyl alcohol (ethanol) Ethyl alcohol (ethanol) Diamond 2.419Diamond Amber 1.55Amber Sodium chloride 1.50 Sodium chloride Other materials Pyrex (a borosilicate glass) [ Pyrexborosilicate glass [ Ruby Ruby Glycerol Glycerol Cubic zirconia Cubic zirconia Diamond Diamond Gallium(III) arsenide Gallium(III) arsenide Silicon 4.01Silicon

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.)