ch 2 microscopy

Question 1

light microscopes

Answer 1

first microscopes invented and still widely used today
resolution limit is 0.2um

Question 2

refractive index

Answer 2

a measure of how much a substance slows the velocity of light

Question 3

lenses

Answer 3

focus light rays at a specific place called the focal point
- distance between center of lens and focal point is the focal length
- short focal length = more magnification

Question 4

objective lens

Answer 4

the lens closest to the specimen
forms a magnified image that is further enlarged by one or more additional lenses

Question 5

bright-field microscope

Answer 5

produces a dark image against a brighter background

Question 6

resolution

Answer 6

ability of a lens to distinguish two objects from each other, rather than a single larger object
shorter wavelength = greater resolution

Question 7

numerical aperture

Answer 7

n sin theta
n = refractive index of the medium in which the lens works
theta = 1/2 the angle of the cone of light entering an objective lens

Question 8

working distance

Answer 8

distance btw the front surface of lens and surface of cover glass or specimen when it is in sharp focus

Question 9

dark-field microscope

Answer 9

produces detailed images of living, unstained cells and organisms by producing a bright image against a dark background
- used to observe living, unstained preparations
- used to observe internal structures in eukaryotic microorganisms

Question 10

phase-contrast microscope

Answer 10

produces an image of a darker microbe against a lighter background
- observe microbial movement and detecting bacterial structures
- usually have a heated stage so the specimen doesn’t die

Question 11

differential interference contrast microscope

Answer 11

creates image by detecting differences in refractive indices and thickness of different parts of the specimen
- used to observe cell walls, endospores, granules, vacuoles, nuclei
- produces a more 3D image

Question 12

fluorescence microscope

Answer 12

produces image by exciting a specimen with a wavelength of light that triggers the object to emit fluorescence light
- stained with fluorochromes
- used for localization of specific proteins in cells

Question 13

fluorochromes

Answer 13

absorbs light energy from the excitation light and emits visible fluorescent light that travels up through the objective lens into the microscope

Question 14

confocal microscopy

Answer 14

uses a laser beam to illuminate fluorescently labeled specimen
- creates sharp, composite 3D image of specimens by using an aperture that eliminates stray light outside of the plane of focus
- used to study biofilms

Question 15

fixation

Answer 15

the process by which the internal and external structures of specimens are preserved and fixed in position

Question 16

heat fixation

Answer 16

preserves overall morphology but destroys subcellular structures

Question 17

chemical fixation

Answer 17

protects fine cellular substructures and morphology

Question 18

chromophore groups

Answer 18

chemical moieties with conjugated double bonds that give the dye its color

Question 19

simple staining

Answer 19

a single stain is used
- used to determine size, shape, and arrangement of bacteria

Question 20

differential staining

Answer 20

distinguishes organisms based on their staining properties
- used to detect presence or absence of structures

Question 21

gram stain

Answer 21

most widely used differential staining procedure
positive - stay purple
negative - pink

Question 22

acid-fast staining

Answer 22

used for staining members of the genus mycobacterium
- have cell walls with lipids that prevent dyes from binding to cells
loses stain = not acid fast
retains stain = acid fast

Question 23

capsule staining

Answer 23

reveals the presence of capsules
- negative staining - capsule itself doesn’t get stained

Question 24

flagella staining

Answer 24

revels presence and distribution pattern of flagella

Question 25

electron microscope

Answer 25

electrons replace light as the illuminating beam that is focused
resolution is 0.2nm

Question 26

transmission electron microscope (TEM)

Answer 26

uses a heated tungsten filament in the electron gun to generate a focused beam of electrons on the specimen
electrons that pass through the specimen form the image
- can only see very thin slices

Question 27

shadowing

Answer 27

preparation method
specimen is coated with a heavy metal on one side

Question 28

freeze-etching

Answer 28

rapidly freeze cells in liquid nitrogen
- allows for observations of shapes of intracellular structures
very brittle

Question 29

scanning electron microscope (SEM)

Answer 29

produces a realistic 3D image of specimens surface features
uses electrons excited from the surface of a specimen to create detailed image

Question 30

cryo-electron microscopy

Answer 30

used to visualize biomolecules and generate high resolution structures
samples are rapidly frozen, then multiple TEM images are captured at different angles
each protein gets a specific color

Question 31

scanning probe microscopy

Answer 31

measures surface features of an object by moving a sharp probe over an objects surface

Question 32

scanning tunneling microscope

Answer 32

view atoms on the surface of a solid
magnification of 100 million times
- type of SPM

Question 33

atomic force microscope

Answer 33

up and down movement of sharp probe as it maintains constant distance from specimen
deflection of a laser beam that strikes the level holding the probe is measured
- used to image protein interactions
- used on surfaces that don’t conduct electricity well
- type of SPM