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z - BIO103 - Microbiology > Lecture 3 - Microscopy and Staining > Flashcards

Lecture 3 - Microscopy and Staining Flashcards

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Biology 101 flashcards
1
Q

basic tool for viewing cells

A

microscope

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2
Q

microscopre is a specialized optical instrument designed to generate englarged, visible __ of specimens

A

images

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3
Q

key features of microscopes

A
  • magnifying
  • resolving power
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4
Q

ability of an optical instrument or type of film to separate or distinguish small or closely adjacent images

A

resolving power

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5
Q

Antonie van Leeuwenhoek’s original occupation

A

draper and haberdasher

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6
Q

eyes can see up to __ mm

A

0.2 (mm)

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7
Q

Why are cells so small?

A

for the cell membrane to accomodate the volume of the cell

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8
Q

suface area to the volume ration gets __ as the cell gets __

A

ratio - smaller
cell - larger

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9
Q

if the cell grows beyond a certain limit, not enough material will be able to cross the __ fast enough to accomodate the increased cellular volume

A

cell membrane

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10
Q

General Principles of Microscopy

A
  1. wavelength of radiation
  2. magnification
  3. resolution
  4. contrast
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11
Q

the bigger the wavelength, the __ it is and the __ it can resolve image

A
  • weaker
  • lower
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12
Q

produces an image of the object upon which it is focused

A
  • simple microscope
  • magnifying lens
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13
Q

bi-convex means that they are __ at the center

A

thicker

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13
Q

simple magnifier lenses are __

A

bi-convex

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14
Q

__ must be accompanied by __ __

A
  • magnification
  • improved resolution
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15
Q

other terms for resolution

A
  • resolving power
  • limit of resolution
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16
Q

capacity of microscope for discerning detail

A

resolution

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17
Q

resolution formula:

A

d= 0.61λ / n sin θ

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18
Q

resolution formula:
λ

A

wavelength of light source

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19
Q

resolution formula:
n

A

refractive index of air or liquid between objective lens and specimen

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20
Q

resolution formula:
θ

A

aperture angle

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21
Q

a measure of the light-gathering ability of the lens

A

aperture angle

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22
Q

the expression of n sin θ is called the __ __

A

numerical aperture

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23
Q

lowerst wavelength of light visible to human eyes

A

400 nm

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24
Q

differences in intensity between two objects, or between an object and background

A

contrast

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25
Q

often utilized to increase contrast

A

staining

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26
Q

in ____ microscopy, contrast results when cells absorb or scatter light differently from their surroundings

A

bright-field microscopy

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27
Q

theoritcal limit for light microscope is __

A

0.2 µm

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28
Q

Types of light microscopes

A
  1. bright-field microscope
  2. dark-field microscope
  3. phase microscope
  4. fluorescent microscopes
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29
Q

Two (2) types of bright-field microscope

A
  1. Simple
  2. Compound
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30
Q

Simple microscope contain a __ magnifying lens

A

single

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31
Q

used simple microscope to observe microorganisms

A

Leeuwenhoek

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32
Q

Compound microscope is a __ __ system

A

2 lens

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33
Q

In compound microscope, light rays pass through __ and into __ __

A
  • specimen
  • objective lens
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34
Q

specimens in compound microscopes are illuminated directly from __ or __

A
  • above
  • below
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35
Q

oil immersion lens increases __

A

resolution

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36
Q

total magnification = magnification of __ __ x magnification of __ __ (x magnification of __ __)

A
  • objective lens
  • ocular lens
  • (condenser lens)
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37
Q

most have __ to direct light through specimen

A

condenser lens

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38
Q

advantages of compound microscopes

A
  • convenient
  • relatively inexpensive
  • available
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39
Q

disadvantages of compound microscopes

A
  • resolving power is 0.2 µm at best
  • can recognize cells but not fine details
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40
Q

compound microscopes needs __ to view cells

A

contrast

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41
Q

easiest way to view cells is to __ and __

A

fix and stain

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42
Q

the __ __ and __ __ combine to produce a magnified image of the specimen

A
  • objective lens
  • eyepiece lens
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43
Q

Light rays from the specimen AB pass through the objective lens to give a __, __ and __ __ __

A
  • magnified
  • inverted
  • real primary image
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44
Q

the eyepiece lens magnifies this further to produce a __ __ of the specimen

A

virtual image

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45
Q

transillumination light source, commonly a __ __ in the microscope stand

A

halogen lamp

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46
Q

focuses light from the light osurce onto the sample

A

condenser lens

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47
Q

collect light from the sample and magnifies the image

A

objective lens

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48
Q

__ and/or __ to view the sample image

A
  • ocular
  • camera
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49
Q
  • real, magnified and inverted
  • optical system with a series of lenses, images formed prior to the final focal plane
A

intermediate image

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50
Q

4X scanning

A

find the object

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51
Q

find the object

A

4X scanning

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52
Q

10X low-dry

A

coarse focusing

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53
Q

coarse focusing

A

10X low-dry

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54
Q

40X high-dry

A

fine focusing

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55
Q

fine focusing

A

40X high-dry

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56
Q

100x oil immersion

A

fine focusing and improved resolution

57
Q

fine focusing and improved resolution

A

100X oil immersion

58
Q

used to improve the resolution

A

immersion oil

59
Q

immersion oil has the same __ __ as glass

A

refractive index

60
Q
  • best for observing pale objects
  • only light rays scattered by specimen enter the objective lens
  • specimen appears light against dark background
  • increases contrast and enables observation of more details
A

dark-field microscopes

61
Q

dark-field microscopes are best for observing __ __

A

pale objects

62
Q

in dark-field microscopes, only light rays scattered by the specimen __ the objective lens

A

enter

63
Q

in dark-field microscopes, specimen appears __ against __ background

A
  • light
  • dark
64
Q

dark-field microscopes increases __ and enables observation of more details

A

contrast

65
Q
  • occludes direct light, passes wide angle light
  • angle too wide to enter objective
A

special condenser diaphragm

66
Q

used to examine living organisms or speciments that would be damaged or altered by attaching them to slides or staining them

A

phase microscopes

67
Q

phase microscopes are used to examine __ __ or __ that would be damaged or altered by attaching them to slides or staining them

A

living organisms or specimens

68
Q

phase microscopes are best for __ __ __that do not absorb __ __

A
  • highly transparent specimens
  • much light
69
Q

light rays __ produce brighter image

A

in phase

70
Q

light rays in phase produce __ __

A

brighter image

71
Q

light rays __ produce darker image

A

out of phase

72
Q

light rays out of phase produce __ __

A

darker image

73
Q

in phase microscopes, contrast is created because light waves are __ __ out of phase

A

1/2 wavelength

74
Q

What are the two specific phase microscope components

A
  1. condenser annulus
  2. objective phase plate
75
Q

condenser annulus and objective phase plate create a _____ that results in an image with greater contrast perceived by the observer

A

phase shift of light

76
Q

Two (2) types of phase microscopes

A
  1. phase contrast microscope
  2. differential interference contrast microscope (DICM)
77
Q

light rays through objects of different wavelength

A

phase contrast microscopy

78
Q

phase contrast microscopy –> change in __, not __

A

change in phase, not intensity

79
Q

human eyes are not able to pick up details with the use of diffracted light that is out of step by about __ of a __ with un-diffracted light

A

1/4 of a wavelength

80
Q

technique used to enhance the contrast of images of transparent and colorless specimens

A

Phase contrast microscopy

81
Q

the phase contrast technique slows the ___ light by __ of a wavelength making the waves further out of phase by a net difference of __ a wavelength

A
  • un-diffracted
  • 1/4
  • 1/2
82
Q

with the 1/2 wavelength difference, the human eye is able to see much more __ on the image produced by the microscope

A

contrast

83
Q
  • change phase differences to intensity differences
  • can view transparent objects as light on dark background
A

special glass disc in objective

84
Q

what is the first component in the phase contrast microscope

A

phase contrast condenser with condenser annulus

85
Q

the condenser in phase contrast contains a phase annulus which has what?

A
  • flat back light absorbing plate
  • transparent annular rings
86
Q

next component in the phase contrast microscope

A

phase contrast objective/s

87
Q

phase contrast objectives are built with a __ __

A

phase plate

88
Q

phase plate works in conjunction with the condenser annulus to achieve __ __ required

A

phase shift

89
Q

phase contrast objectives have a phase plate built into the __ of the objective

A

rear

90
Q

happens when two waves overlap in such a way that they combine to create a larger wave

A

Constructive interference

91
Q

happens when two waves overlap in such a way that they cancel each other out

A

Destructive interference

92
Q

flow diagram of phase contrast microscope

A
  1. light source
  2. annular diaphragm
  3. condenser
  4. specimen
  5. rear focal plane
  6. objective
  7. eyepiece
93
Q

Rays which are not deviated by the object pass through the phase-altering pattern of the phase plate and acquire __ __

A

longer wavelength

94
Q

Rays which are deviated by the objects pass through the phase-plate __ __ by the __ __ __

A
  • not covered
  • phase altering pattern
95
Q

In fluorescent microscopes, direct __ __ is used at the specimen

A

UV light

96
Q

UV light at specimen causes the specimen to radiate energy back as a __, __ __

A

longer, visible wavelength

97
Q

UV light increases __ and __

A
  • resolution
  • contrast
98
Q

UV light wavelength

A

100-400 nm

99
Q

visible light wavelength

A

400–700 nm

100
Q

Fluorescent microscopes are used in __ to identify pathogens and to locate and make visible a variety of proteins

A

immunofluorescence

101
Q

utilizes the specificity of antibodies with fluorescent dyes to recognize their antigen

A

Immunofluorescence

102
Q

Immunofluorescence is used primarily on biological samples and is classically defined as a procedure to detect __ in __ __ using __

A
  • antigens
  • cellular contexts
  • antibodies
103
Q

property of certain dyes absorbing light rays at one particular wavelength and emitting them at a different wavelength

A

fluorescence

104
Q

gives yellow-green fluorescence

A

fluorescein isothiocyanate

105
Q

immunofluorescence tests are also termed as __ __ __

A

fluorescent antibody test (FAT)

106
Q

Fluorescent dyes that emit
- blue-green
- orange-red fluorescence

A
  • fluorescein isothiocyanate
  • lissamine rhodamine
107
Q

passes light directly through specimen; unless cell is naturally pigmented or artificially stained, image has little contrast

A

brightfield microscopy (unstaied specimen)

108
Q

staining with various dyes enhances contrast, but most staining procedures require that cells be fixed (preserved)

A

brightfield microscopy (stained specimen)

109
Q
  • shows the location of specific molecules in cell
  • made by tagging the molecules of interest with fluorescent dyes or antibodies
A

fluorescence microscopy

110
Q
  • enhances contrast in unstained cells by amplifying variation in refractive index within specimen
  • especially useful for examining living, unpigmented cells
A

phase contrast microscopy

111
Q

uses optical modifications to exaggerate differences in refractive index

A

differential interference contrast microscopy

112
Q
  • uses lasers and special optics to focus illuminating beam on a single plane within the specimen
  • only those regions within a narrow depth of focus are imaged
  • regions above and below the selected plane of view appear black rather than blurry
A

confocal microscopy

113
Q

light microscopes cannot resolve structures smaller than __ nm because the shortest wavelength of visible light is __ nm

A
  • 200 nm
  • 400 nm
114
Q

measure of the ability of the objective lens to separate in the image adjacent details that are present in the object

A

limit of resolution (or resolving power)

115
Q

type of very powerful microscope that uses electrons instead of light to produce a magnified image of something

A

electron microscope

116
Q

electrons produce wavelengths of __ to __ nm

A

0.01 nm to 0.001 nm

117
Q

electron microscopes have greater __ and __

A
  • resolving power
  • magnification
118
Q

electron microscopes magnify images __ to __

A

10,000X to 100,000X

119
Q

Two types of electron microscopes

A
  1. Transmission electron microscopes (TEM)
  2. Scanning electron microscopes (SEM)
120
Q
  • form images using electrons that are transmitted (pass) through a specimen
  • see the inside
A

transmission electron microscope (TEM)

121
Q

utilizes electrons that have bounced off the surface of the specimen

A

scanning electron microscope (SEM)

122
Q

flow diagram of electron microscopes

A
  1. electron gun
  2. condenser lens (magnet)
  3. specimen
  4. objective lens (magnet)
    (intermediate image)
  5. projector lens (magnet)
  6. final image on fluorescent screen (TEM) or electron detector (SEM)
123
Q

Similarities between light and electron microscopes

A
  1. form larger (magnified) and more detailed (highly resolved) images of small objects
  2. used in study and research in biology and medical sciences
  3. specimens must be carefully prepared using techniques appropriate for both the equipment and sample
124
Q

Differences between light and electron microscopes:
radiation type

A
  • light microscope use light (~400-700 nm)
  • electron microscope use beams (~ 1nm)
125
Q

Differences between light and electron microscopes:
control of image formation

A
  • light microscope via glass lenses
  • electron microscope by electromagnets due to negative charge of electrons
126
Q

Differences between light and electron microscopes:
color images

A
  • light = range of wavelength
  • electron = greyscale, “false color”
127
Q

damage caused in specimen preparation and can be confused with specimen ultrastructure

A

Artifacts

128
Q

Why should electron microscopes have a vacuum tube

A

limit interference by air molecules

129
Q

use minuscule, pointed, electronic probes to magnify more than 100,000,000 times

A

probe microscopes

130
Q
  • What do probe microscopes use to magnify?
  • How high can it magnify?
A
  • minuscule, pointed electronic probes
  • more than 100,000,000 times
131
Q

Two types of probe microscopes

A
  1. Scanning tunneling microscopes (STM)
  2. Atomic force microscopes (AFM)
132
Q

detects the surface structure of the objects based on the tunnel effect of the quantum mechanics

A

scanning tunneling microscopes (STM)

133
Q
  • gives 3D topography
  • provides various types of surface measurements to the needs of scientist and engineers
A

atomic force microscopes (AFM)

134
Q

long beam with a tip located at its apex

A

cantilever

135
Q

ion or molecule which donates a pair of electrons to the central metal atom or ion to form a coordination complex

A

ligand

136
Q

probe microscopes allow the study of how __ attach to others

A

bacteria

137
Q

optical and electron microscopes can easily generate __ images of a sample surface, with a magnification as large as __ for an optical microscope, and __ for an electron microscope

A
  • 2D
  • 1000X
  • ~100,000X
138
Q

However, optical and electron microscopes cannot measure the __ dimension of the sample, the __ or __ of the surface features

A
  • vertical
  • height
  • depth
139
Q
  • How does AFM, using a sharp tip, probe the surface features?
  • How high can it magnify?
A
  • raster scanning
  • 1,000,000X
140
Q

the measurement of AFM is made in __

A

three dimensions

141
Q

vertical dimension

A

z-direction

z - BIO103 - Microbiology (18 decks)

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