Lecture 3 - Microscopy and Staining Flashcards
1
Q
basic tool for viewing cells
A
microscope
2
Q
microscopre is a specialized optical instrument designed to generate englarged, visible __ of specimens
A
images
3
Q
key features of microscopes
A
- magnifying
- resolving power
4
Q
ability of an optical instrument or type of film to separate or distinguish small or closely adjacent images
A
resolving power
5
Q
Antonie van Leeuwenhoek’s original occupation
A
draper and haberdasher
6
Q
eyes can see up to __ mm
A
0.2 (mm)
7
Q
Why are cells so small?
A
for the cell membrane to accomodate the volume of the cell
8
Q
suface area to the volume ration gets __ as the cell gets __
A
ratio - smaller
cell - larger
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
10
Q
General Principles of Microscopy
A
- wavelength of radiation
- magnification
- resolution
- contrast
11
Q
the bigger the wavelength, the __ it is and the __ it can resolve image
A
- weaker
- lower
12
Q
produces an image of the object upon which it is focused
A
- simple microscope
- magnifying lens
13
Q
bi-convex means that they are __ at the center
A
thicker
13
Q
simple magnifier lenses are __
A
bi-convex
14
Q
__ must be accompanied by __ __
A
- magnification
- improved resolution
15
Q
other terms for resolution
A
- resolving power
- limit of resolution
16
Q
capacity of microscope for discerning detail
A
resolution
17
Q
resolution formula:
A
d= 0.61λ / n sin θ
18
Q
resolution formula:
λ
A
wavelength of light source
19
Q
resolution formula:
n
A
refractive index of air or liquid between objective lens and specimen
20
Q
resolution formula:
θ
A
aperture angle
21
Q
a measure of the light-gathering ability of the lens
A
aperture angle
22
Q
the expression of n sin θ is called the __ __
A
numerical aperture
23
Q
lowerst wavelength of light visible to human eyes
A
400 nm
24
differences in intensity between two objects, or between an object and background
contrast
25
often utilized to increase contrast
staining
26
in ____ microscopy, contrast results when cells absorb or scatter light differently from their surroundings
bright-field microscopy
27
theoritcal limit for light microscope is __
0.2 µm
28
Types of light microscopes
1. bright-field microscope
2. dark-field microscope
3. phase microscope
4. fluorescent microscopes
29
Two (2) types of bright-field microscope
1. Simple
2. Compound
30
Simple microscope contain a __ magnifying lens
single
31
used simple microscope to observe microorganisms
Leeuwenhoek
32
Compound microscope is a __ __ system
2 lens
33
In compound microscope, light rays pass through __ and into __ __
- specimen
- objective lens
34
specimens in compound microscopes are illuminated directly from __ or __
- above
- below
35
oil immersion lens increases __
resolution
36
total magnification = magnification of __ __ x magnification of __ __ (x magnification of __ __)
- objective lens
- ocular lens
- (condenser lens)
37
most have __ to direct light through specimen
condenser lens
38
advantages of compound microscopes
- convenient
- relatively inexpensive
- available
39
disadvantages of compound microscopes
- resolving power is 0.2 µm at best
- can recognize cells but not fine details
40
compound microscopes needs __ to view cells
contrast
41
easiest way to view cells is to __ and __
fix and stain
42
the __ __ and __ __ combine to produce a magnified image of the specimen
- objective lens
- eyepiece lens
43
Light rays from the specimen AB pass through the objective lens to give a __, __ and __ __ __
- magnified
- inverted
- real primary image
44
the eyepiece lens magnifies this further to produce a __ __ of the specimen
virtual image
45
transillumination light source, commonly a __ __ in the microscope stand
halogen lamp
46
focuses light from the light osurce onto the sample
condenser lens
47
collect light from the sample and magnifies the image
objective lens
48
__ and/or __ to view the sample image
- ocular
- camera
49
- real, magnified and inverted
- optical system with a series of lenses, images formed prior to the final focal plane
intermediate image
50
4X scanning
find the object
51
find the object
4X scanning
52
10X low-dry
coarse focusing
53
coarse focusing
10X low-dry
54
40X high-dry
fine focusing
55
fine focusing
40X high-dry
56
100x oil immersion
fine focusing and improved resolution
57
fine focusing and improved resolution
100X oil immersion
58
used to improve the resolution
immersion oil
59
immersion oil has the same __ __ as glass
refractive index
60
- 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
dark-field microscopes
61
dark-field microscopes are best for observing __ __
pale objects
62
in dark-field microscopes, only light rays scattered by the specimen __ the objective lens
enter
63
in dark-field microscopes, specimen appears __ against __ background
- light
- dark
64
dark-field microscopes increases __ and enables observation of more details
contrast
65
- occludes direct light, passes wide angle light
- angle too wide to enter objective
special condenser diaphragm
66
used to examine living organisms or speciments that would be damaged or altered by attaching them to slides or staining them
phase microscopes
67
phase microscopes are used to examine __ __ or __ that would be damaged or altered by attaching them to slides or staining them
living organisms or specimens
68
phase microscopes are best for __ __ __that do not absorb __ __
- highly transparent specimens
- much light
69
light rays __ produce brighter image
in phase
70
light rays in phase produce __ __
brighter image
71
light rays __ produce darker image
out of phase
72
light rays out of phase produce __ __
darker image
73
in phase microscopes, contrast is created because light waves are __ __ out of phase
1/2 wavelength
74
What are the two specific phase microscope components
1. condenser annulus
2. objective phase plate
75
condenser annulus and objective phase plate create a _____ that results in an image with greater contrast perceived by the observer
phase shift of light
76
Two (2) types of phase microscopes
1. phase contrast microscope
2. differential interference contrast microscope (DICM)
77
light rays through objects of different wavelength
phase contrast microscopy
78
phase contrast microscopy --> change in __, not __
change in phase, not intensity
79
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
1/4 of a wavelength
80
technique used to enhance the contrast of images of transparent and colorless specimens
Phase contrast microscopy
81
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
- un-diffracted
- 1/4
- 1/2
82
with the 1/2 wavelength difference, the human eye is able to see much more __ on the image produced by the microscope
contrast
83
- change phase differences to intensity differences
- can view transparent objects as light on dark background
special glass disc in objective
84
what is the first component in the phase contrast microscope
phase contrast condenser with condenser annulus
85
the condenser in phase contrast contains a phase annulus which has what?
- flat back light absorbing plate
- transparent annular rings
86
next component in the phase contrast microscope
phase contrast objective/s
87
phase contrast objectives are built with a __ __
phase plate
88
phase plate works in conjunction with the condenser annulus to achieve __ __ required
phase shift
89
phase contrast objectives have a phase plate built into the __ of the objective
rear
90
happens when two waves overlap in such a way that they combine to create a larger wave
Constructive interference
91
happens when two waves overlap in such a way that they cancel each other out
Destructive interference
92
flow diagram of phase contrast microscope
1. light source
2. annular diaphragm
3. condenser
4. specimen
5. rear focal plane
6. objective
7. eyepiece
93
Rays which are not deviated by the object pass through the phase-altering pattern of the phase plate and acquire __ __
longer wavelength
94
Rays which are deviated by the objects pass through the phase-plate __ __ by the __ __ __
- not covered
- phase altering pattern
95
In fluorescent microscopes, direct __ __ is used at the specimen
UV light
96
UV light at specimen causes the specimen to radiate energy back as a __, __ __
longer, visible wavelength
97
UV light increases __ and __
- resolution
- contrast
98
UV light wavelength
100-400 nm
99
visible light wavelength
400–700 nm
100
Fluorescent microscopes are used in __ to identify pathogens and to locate and make visible a variety of proteins
immunofluorescence
101
utilizes the specificity of antibodies with fluorescent dyes to recognize their antigen
Immunofluorescence
102
Immunofluorescence is used primarily on biological samples and is classically defined as a procedure to detect __ in __ __ using __
- antigens
- cellular contexts
- antibodies
103
property of certain dyes absorbing light rays at one particular wavelength and emitting them at a different wavelength
fluorescence
104
gives yellow-green fluorescence
fluorescein isothiocyanate
105
immunofluorescence tests are also termed as __ __ __
fluorescent antibody test (FAT)
106
Fluorescent dyes that emit
- blue-green
- orange-red fluorescence
- fluorescein isothiocyanate
- lissamine rhodamine
107
passes light directly through specimen; unless cell is naturally pigmented or artificially stained, image has little contrast
brightfield microscopy (unstaied specimen)
108
staining with various dyes enhances contrast, but most staining procedures require that cells be fixed (preserved)
brightfield microscopy (stained specimen)
109
- shows the location of specific molecules in cell
- made by tagging the molecules of interest with fluorescent dyes or antibodies
fluorescence microscopy
110
- enhances contrast in unstained cells by amplifying variation in refractive index within specimen
- especially useful for examining living, unpigmented cells
phase contrast microscopy
111
uses optical modifications to exaggerate differences in refractive index
differential interference contrast microscopy
112
- 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
confocal microscopy
113
light microscopes cannot resolve structures smaller than __ nm because the shortest wavelength of visible light is __ nm
- 200 nm
- 400 nm
114
measure of the ability of the objective lens to separate in the image adjacent details that are present in the object
limit of resolution (or resolving power)
115
type of very powerful microscope that uses electrons instead of light to produce a magnified image of something
electron microscope
116
electrons produce wavelengths of __ to __ nm
0.01 nm to 0.001 nm
117
electron microscopes have greater __ and __
- resolving power
- magnification
118
electron microscopes magnify images __ to __
10,000X to 100,000X
119
Two types of electron microscopes
1. Transmission electron microscopes (TEM)
2. Scanning electron microscopes (SEM)
120
- form images using electrons that are transmitted (pass) through a specimen
- see the inside
transmission electron microscope (TEM)
121
utilizes electrons that have bounced off the surface of the specimen
scanning electron microscope (SEM)
122
flow diagram of electron microscopes
1. electron gun
2. condenser lens (magnet)
2. specimen
4. objective lens (magnet)
(intermediate image)
5. projector lens (magnet)
6. final image on fluorescent screen (TEM) or electron detector (SEM)
123
Similarities between light and electron microscopes
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
Differences between light and electron microscopes:
radiation type
- light microscope use light (~400-700 nm)
- electron microscope use beams (~ 1nm)
125
Differences between light and electron microscopes:
control of image formation
- light microscope via glass lenses
- electron microscope by electromagnets due to negative charge of electrons
126
Differences between light and electron microscopes:
color images
- light = range of wavelength
- electron = greyscale, "false color"
127
damage caused in specimen preparation and can be confused with specimen ultrastructure
Artifacts
128
Why should electron microscopes have a vacuum tube
limit interference by air molecules
129
use minuscule, pointed, electronic probes to magnify more than 100,000,000 times
probe microscopes
130
- What do probe microscopes use to magnify?
- How high can it magnify?
- minuscule, pointed electronic probes
- more than 100,000,000 times
131
Two types of probe microscopes
1. Scanning tunneling microscopes (STM)
2. Atomic force microscopes (AFM)
132
detects the surface structure of the objects based on the tunnel effect of the quantum mechanics
scanning tunneling microscopes (STM)
133
- gives 3D topography
- provides various types of surface measurements to the needs of scientist and engineers
atomic force microscopes (AFM)
134
long beam with a tip located at its apex
cantilever
135
ion or molecule which donates a pair of electrons to the central metal atom or ion to form a coordination complex
ligand
136
probe microscopes allow the study of how __ attach to others
bacteria
137
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
- 2D
- 1000X
- ~100,000X
138
However, optical and electron microscopes cannot measure the __ dimension of the sample, the __ or __ of the surface features
- vertical
- height
- depth
139
- How does AFM, using a sharp tip, probe the surface features?
- How high can it magnify?
- raster scanning
- 1,000,000X
140
the measurement of AFM is made in __
three dimensions
141
vertical dimension
z-direction
