Lab 2 Flashcards
what is the resolution of the following:
human eye?
light microscope?
transmission microscope?
scanning electron microscope?
human eye= .2mm
light microscope= 200 nm
transmission electron microscope=2nm
scanning electron microscope=3-20 nm
general discription of resolution?
it is the clarity of the image. the ability to distinguish objects separated by small distances
resolution is directly proportional to wavelength 1/2 the wavelength.
the limit of resolution of light microscope is approximately .2 micro m
What are some advantages and disadvantages of light microscopes
ADVANTAGES:
relative cheap
can observe living materials
DISADVANTAGES
low reolution
what are some advantages and disadvantages of electron microscopes
ADVANTAGES:
-high resolution
DISADVANTAGES:
- expensive
- cannot observe living materials
light microscopy
- also known as optical microscope
- uses visible light to magnify samples on a slide
- magnify objects objects up to about 1000x
- most cells are between 1-100 micro m, so they can be observed by light microscopy, as can some organelles
Type of light microscopy
- bight field and dark field
- phase-contrast
- confocal
- fluorescene
bright field microcscope
- light passes directly through cell
- cells are often preserved with fixtatives and stained with dyes to enhance the contrast
- this technique cant be used to study living cells
dark field microscopy
- instead of light coming up through the specimen, the light is reflected by particles on the slide
- to view a specimen in dark field, an opaque disc is placed underneath the condenser lens, so that only light that is scattered by objects on the slide can reach the eye
phase-contrast microscopy
- converts variation in density or thickness to differences in contrast that can be seen in the final image
- utilizes differences in refractive index of specimen based on density
fluorescence microscopy
- a fluorescent dye is used to label the MOLECULE OF INTEREST in fixed of living cells
- the fluorescent dye molecules ABSORB LIGHT at one wavelength and EMIT LIGHT at a different wavelength
- the fluorescence is detected by illuminating the specimen with a wavelength of light that excites the fluorescent dye, then using filters to detect the specific wavelength of light that the dye emits
confocal microscopy
- increase contrast and detail by analzying fluorescence from a single point
- a small point of light from a laser is focused on the specimen at a PARTICULAR DEPTH
- the emitted light must pass through a pin-hoe aperture (CONFOCAL APERATURE). thus only light emitted from the plane of focus is able to reach the detector
- scanning across the speciment generates a two dimensional image of the plane of focus.
- a series of images can be used to reconstruct a three-dimensional image
electron microscopy
- can achieve much greater resolution (.2nm) than light microscopy: short wavelength of electrons
- resolution for BIOLOGICAL SAMPLES is about 1 to 2 nm: inherent lack of contrast
transmission electron microscopy
- specimens are fixed and stained with salts of heavy metals, which provide contrast by scattering electrons
- a beam of electrons is passed through the specimen and forms an image on a fluorescent screen
scanning electron microscopy
- the electron beam does not pass through the specimen
- instead, the surface of the cell is coated with a heavy metal, and a beam of electrons is used to scan across the specimen
haemocytometer
- a device originally designed for counting blood cells
- engraved with laser-etched grid of perpendicular lines
