Microscopy 1 Biology 101A

Announcements Quiz- Wed, not today- 8:10am-8:25am

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

Magnification without resolution

Contrast

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

Quiz Wednesday Microscope care and maintenance (how to keep from breaking them) Microscpe anatomy (labelling of parts) Microscope principles (wavelength, magnification, etc.)

Microscope anatomy Parts 1.Headpiece 2.Eyepieces 3. Nosepiece/turret 4. Objective leses 5. Stage clips 6.Stage 7.Condenser 8. Substage filter holder/iris diapragm lever 9.Mirror 10.Base 11. Condenser adjustment knob 12. Fine focus knob 13. Coarse focus knob 14. Arm

Phase-contrast

Electron Microscopes Use electrons instead of light Use magnets instead of glass lenses 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

Fluorescence microscopes look at a single wavelength of light at a time

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

If you reach your fingers into the area under the microscope stage without looking, which of the following is most likely to be damaged? A. The base of the microscope B. The coarse adjustment knob. C. The fine focus adjustment knob. D. The blue frosted glass filter. E. The mirror

When carrying a microscope, your principal grip should be on its: A. tube. B. nosepiece. C. stage. D. arm. E. coarse adjustment knob.

Iris diaphragms are most easily damaged by: A. being touched by someone’s fingers. B. too high a light intensity. C. having too little oil on their leaves. D. too low a light intensity. E. Not being wiped clean before every use.