microscopy Flashcards
smallest to largest measuremtns
angstrom –> nanometer –> micrometer –> millimeter –> centimeter
refractive index
how much a substance (i.e. water, air) slows the velocity of light
when light transitions through substances it changes speed and bends
focal points and focal lengths
focal point: where the light beams are all focused onto this single point
focal lengths: distance between lens and focal point
brightfield microscope
light source –> condenser lens (w object on it) –> objective lens to see through
resolution
minimum distance between 2 objects so they can still be viewed as seperate from each other
-lower # is better resolution
numerical aperture (N sin theta)
theta is the angle the cone of light is entering an object (max is 90 degrees)
N= refractive index
refractive index of air is 1
oil immersion objective
use oil between lens and object to limit refraction (no air)
change refractive index
Abbe equation
d= 0.5 lambda/ n sin theta
d=resolution (smaller is better)
lambda= wavelength
n= refractive index
theta= angle
minimize resolution: decrease wavelength, or increase n sin theta
3 ways to visualize unpigmented microbes through light microscopy
- dark-field microscopes
- phase-contrast microscopes
- differential interference contrast (DIC) microscopes
dark field microscope
but a “poker chip” right after light source, only peripheral beams reach objective lens
phase-contrast microcopy
have 2 different wavelengths through the wavelengths passing through bacteria (high refractive index) and the ones that dont pass through
-to further perturb the wavelength there is a phase plate
-condenser annulus “poker chip”
and phase plate; exaggerate difference in wavelengths
able to see motility, detailed structures like organelles
fluorescent microscopes
opposite of light microscopes
light source is perpendicular to a tube; emits short wavelengths
there is a dichromatic mirror which reflects short wavelengths and transmits longer wavelengths
so short wavelengths hit mirror and go to specimen which emits longer wavelengths which bypass mirror again and go up to visualize it
fluorochromes: stain DNA
i.e. GFP
confocal scanning laser microscopy
looking at 3D not flat!!!
create z stacks to get many focal planes
light vs electron microscopy
electron microscopy uses electron beams instead of light
and use magnets as lens to bend the beams instead of glass
electron microscope is way higher energy wavelength then light microscope which means its more likely to hit things (i.e. jump rope and throw ball in)
but need to kill specimens and use chemicals