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GSBS Cell Bio > Microscope > Flashcards

Microscope Flashcards

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1
Q

brightfield illumination

A

the light source is positioned below the sample. Light then propagates through the sample, and is observed by the objective lens and sensor, which are positioned above the sample. The darker the sample, the denser the specimen, as denser samples absorb more light.

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2
Q

upright microscope

A

observe samples such as slides placed on a stage through an objective located above the stage.

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3
Q

inverted microscope

A

a microscope with the objective lens placed below the stage on which the sample is located, allowing the sample to be observed from below. This type of microscope is used for observation at higher magnifications than stereoscopic microscopes, with magnification ranging from about 20 to 1500x.

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4
Q

numerical aperture

A

a unitless measurement of a microscope’s objective lens’s ability to gather light and resolve fine details in a specimen.

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5
Q

the higher the NA

A

the better the resolution

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6
Q

rule of thumb for NA

A

do not exceed 1000x the NA of the objective

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7
Q

which obective has a better NA
short and wide
tall and skinny

A

short and wide

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8
Q

why is confocal not the best for colocalization?

A

it has a wider range

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8
Q

if you want to see if two proteins colocalize, what should you use

A

TIRF or STORM

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9
Q

simple vs compound microscope

A

A simple microscope uses a single magnifying lens to produce an enlarged image. In contrast, a compound microscope employs multiple lenses.

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10
Q

achromatic aberration

A

a distortion in color that occurs when light passes through a lens and different wavelengths bend at different angles. This can cause images to appear blurry or have rainbow-like halos around objects.

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11
Q

spherical aberration

A

a phenomenon that occurs when light rays passing through a spherical lens or mirror are not focused to a single point. This results in images that are fuzzy or indistinct around the edges.

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12
Q

darkfield microscopy

A

a technique used in light microscopy to enhance the visibility of unstained specimens, particularly those that are transparent or difficult to see under brightfield illumination.
Use high contrast to make light observation images visible on a dark background.

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13
Q

phase contrast microscopy

A

a technique that uses light to enhance the contrast of transparent, unstained samples by converting phase shifts in light passing through the specimen into visible brightness changes in the image, allowing for detailed observation of structures like living cells that would otherwise be difficult to see under normal light conditions

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14
Q

differential interface contrast microscopy

A

a microscopy technique that uses polarized light to create high-contrast images of transparent, unstained samples.
DIC microscopy uses polarized light to illuminate a specimen.
The light is split into two rays with different polarizations.
The rays pass through the specimen, experiencing different refraction and scattering.
The rays reunite and interfere, becoming elliptically polarized.
An analyzer changes the polarization into an amplitude shift.
The resulting image has a pseudo 3D effect, with the object appearing black to white on a gray background.

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15
Q

fluorescence microscopy

A

a type of light microscopy that uses fluorescence to study the properties of substances. It’s a powerful tool in biology that allows researchers to see the distribution, amount, and localization of molecules and structures inside cells

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16
Q

confocal microscopy

A

is a technique that uses a laser to create high-resolution, three-dimensional images of biological samples of which are stained with fluorescent probes

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17
Q

two-photon microscopy

A

a fluorescence imaging technique that allows researchers to see living cells
can do thick samples

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18
Q

polarized light microscopy

A

a technique that uses polarized light to identify and characterize materials.

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19
Q

birefringence

A

the optical property of a material that causes light to split into two rays when it passes through. It’s also known as double refraction.

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20
Q

Wollaston/Nomarski prisms

A

an optical device that splits a beam of light into two separate, linearly polarized beams with orthogonal polarization, meaning the polarization directions of the two beams are perpendicular to each other

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21
Q

immunofluorescent staining

A

a laboratory technique that uses fluorescent antibodies to identify the location of proteins and other molecules in cells and tissues

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22
Q

FRET

A

a physical phenomenon where energy is transferred non-radiatively from an excited fluorophore molecule (called the donor) to another nearby fluorophore molecule (called the acceptor) when they are positioned close enough together

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23
Q

spectral confocal laser scanning microscopy (CLSM)

A

is a technique that uses multiple laser sources to excite a sample and analyze the emission spectrum

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24
spectral imaging
a digital imaging technique that uses multiple bands of light to capture images of objects. It combines spectroscopy and photography to create images that provide information about the spectrum of light reflected by an object.
25
TIRF
a microscopy technique that uses light to excite and image fluorescent molecules near a cell membrane
26
widefield microscopy
a basic microscopy technique that uses a light source to illuminate an entire sample and create a 2D image. It's a common method for viewing biological structures like cells and tissues.
27
Structured illumination microscopy (SIM)
a super-resolution microscopy technique that enhances the resolution of a standard light microscope by illuminating a sample with a patterned light pattern (like stripes), which creates a "Moiré effect" that allows for the extraction of high-frequency details beyond the normal diffraction limit, effectively providing a significantly improved image resolution compared to conventional wide-field microscopy
28
3D localization microscopy (STORM)
a super-resolution imaging technique that allows for the precise determination of the 3D positions of individual fluorescent molecules within a sample, achieving significantly higher resolution than traditional light microscopy by precisely localizing single emitters in both the lateral and axial dimensions, enabling detailed visualization of complex cellular structures at the nanoscale level
29
What is microscopy?
The technique of using microscopes to view objects that cannot be seen with the naked eye.
30
what are the two main types of microscopes
simple (single lens) compound (multiple lenses)
31
what is magnification
the process of enlarging the apperance of an object
32
what is the normal viewing distance for 1x magnification
250mm from the eye
33
why can younger individuals achieve higher magnification
better eyes, can focus as close as 125mm
34
what is the optical spectrum
The range of wavelengths from infrared (10⁻³ m) to ultraviolet (10⁻⁸ m).
35
what is absorption
the reduction in light intensity as it passes through an object
36
what is refraction
the bending of light when it passes through a different medium
37
how does refraction affect light direction
light bends toward the perpendicular plane when entering a denser medium
38
what is dispersion
the separation of light into different wavelengths as seem in a prism
39
how does light behave when passing through a curved lens
it bends and focuses at differnt points
40
which color is refracted the most
purple
41
which color is refracted the least
red
42
what happens when blue light is absorbed in a sample
the sample appears yellow or red
43
what happens when green light is absorbed
the sample appears pink
44
what is the function of the objective lens
it collects and magnifies the image of the specimen
45
what is kohler illumination
a technique that provides even illumination and high contract
46
what is a parfocal lens
a lens that stays in focus when switching between magnifications
47
what is the mechanical tube length in a standard microscope
160mm
48
what is the object to image distance
195mm
49
what is the focal length of objective
45mm
50
What is the difference between an upright and an inverted microscope?
In an upright microscope, the light source is below; in an inverted microscope, it is above.
51
why are inverted scopes useful?
They allow imaging of cells in liquid environments, such as culture dishes.
52
What is an epi-illumination source?
A light source that illuminates the sample with light.
53
What is infinity optics in modern microscopes?
A system where the tube lens focuses light to an intermediate image plane for flexibility.
54
Why are modern microscopes designed with infinity optics?
They reduce sensitivity to additional optics and allow better focusing.
55
What is resolution in microscopy?
The ability to distinguish two closely spaced points as separate.
56
How does numerical aperture (NA) affect resolution?
Higher NA leads to better resolution.
57
How does immersion oil improve resolution?
It increases the refractive index, reducing light scattering and improving focus.
58
What is chromatic aberration?
A type of distortion where different wavelengths focus at different points.
59
How is chromatic aberration corrected?
By using achromatic or apochromatic lenses.
60
What is spherical aberration?
A distortion where light rays focus at different points along the optical axis.
61
What is field curvature in microscopy?
When an image appears sharp in the center but blurry at the edges.
62
What is the benefit of using a phase contrast microscope?
It enhances contrast in unstained samples by exploiting differences in refractive index.
63
What is differential interference contrast (DIC) microscopy?
A technique that increases contrast by separating and recombining polarized light.
64
What is confocal microscopy?
A technique that uses a pinhole to remove out-of-focus light, improving resolution.
65
Why do electron microscopes have higher resolution than light microscopes?
They use electron beams with much shorter wavelengths than visible light.
66
Why are biological specimens difficult to see under brightfield microscopy?
Many are thin and transparent, providing little natural contrast.
67
What are two solutions for increasing contrast in brightfield microscopy?
Fluorescent staining and brightfield contrast techniques (DIC, Phase, etc.).
68
What is phase contrast microscopy used for?
Enhancing contrast in transparent specimens by converting phase shifts into intensity differences.
69
How does brightfield microscopy generate contrast?
By absorption differences in the sample, often enhanced with stains.
70
What is polarized light microscopy used for?
Observing optically anisotropic (birefringent) specimens.
71
What two components are required for a polarized light microscope?
a polarizer and an analyzer
72
What does birefringence mean?
A material property where the refractive index depends on the light’s polarization and propagation direction.
73
How does a polarizer work?
It filters light so that waves vibrate in a single plane.
74
What happens when crossed polarizers are used?
No light is transmitted unless a birefringent material is placed between them.
75
What is an isotropic material?
A material with uniform optical properties in all directions.
76
What is an anisotropic material?
A material whose optical properties vary depending on the direction of light propagation.
77
How do isotropic crystals interact with light?
Light refracts at a constant angle and passes through at a single velocity.
78
How do anisotropic crystals interact with light?
They split light into two polarized rays traveling at different velocities.
79
What is double refraction?
The splitting of light into two rays in an anisotropic material.
80
What is polarization of light?
The orientation of the electric field (E-field) of light waves.
81
Why do most light sources produce unpolarized light?
Because they emit waves vibrating in multiple directions.
82
What is linearly polarized light?
Light waves oscillating in a single plane.
83
How does a circular polarizer work?
It converts linear polarization into circular polarization by introducing a phase shift.
84
What are three key features of a DIC image?
Directional contrast, edge highlighting, and pseudo-3D appearance.
85
What is an optical path length (OPL)?
The product of the refractive index and the physical path length of light through a material.
86
What optical component in DIC microscopy produces two orthogonally polarized beams?
Wollaston or Nomarski prisms.
87
How does DIC achieve contrast?
By using two beams of light and interference to measure differences in optical path length.
88
What happens in DIC when both beams see the same optical path length?
They emerge in phase, regenerating the initial polarization, and no contrast is visible.
89
What happens in DIC when beams see different optical path lengths?
A phase shift occurs, generating elliptical polarization and contrast.
90
Why does DIC create a pseudo-3D effect?
Because one side of a structure appears brighter while the other appears darker.
91
How does phase retardation affect contrast in DIC?
It alters polarization, allowing light to pass through the analyzer and create contrast.
92
How is contrast in DIC controlled?
By adjusting the prism’s bias retardation.
93
What are the two main light paths in a transmitted light microscope?
The illumination path and the imaging path.
94
What is the function of the aperture iris in a microscope?
It controls the range of illumination angles.
95
What does the field iris control?
The illuminated field of view.
96
Where is the analyzer placed in a polarized light microscope?
Between the objective rear aperture and the camera/eyepiece.
97
What happens when imaging a normal (non-birefringent) sample in polarized light?
The sample appears dark because no birefringence alters polarization.
98
What happens when imaging a birefringent sample in polarized light?
The sample appears bright on a dark background.
99
Why does a birefringent sample appear bright?
Because it splits light into two rays with different refractive indices, creating phase retardation.
100
How does a compensator (wave plate) improve contrast?
It introduces additional phase shifts to enhance birefringence detection.
101
What structures in cells can be visualized using polarized light microscopy?
Spindles, cytoskeletal structures, membranes, and collagen.
102
What is immunofluorescent staining used for?
It identifies patterns of protein expression in cells using antibodies.
103
What does a primary antibody do in immunofluorescent staining?
Binds directly to the antigen of interest.
104
What is the role of a secondary antibody in immunofluorescent staining?
It binds to the primary antibody and is conjugated to a fluorochrome for detection.
105
What happens when a fluorochrome absorbs high-energy light?
It emits light at a longer wavelength, producing fluorescence.
106
What types of samples are suitable for immunofluorescent staining?
Frozen, ethanol-fixed, paraformaldehyde-fixed, and methanol/acetone-fixed cells.
107
What is the first step in immunofluorescent staining?
Culture cells on a glass coverslip and fix them with paraformaldehyde or methanol/acetone.
108
Why is cell permeabilization necessary before staining?
To allow antibodies to penetrate the cell membrane.
109
What is the function of a blocking solution in immunofluorescence?
It prevents non-specific antibody binding.
110
What is a common fluorochrome used to stain nuclei?
DAPI
111
What fluorochrome is used to stain F-actin stress fibers?
Phalloidin-conjugates.
112
What is the role of the excitation filter in fluorescence microscopy?
It selects the specific wavelength of light needed to excite the fluorophore.
113
What does a dichroic mirror do in fluorescence microscopy?
Reflects excitation light while allowing emitted fluorescence to pass through.
114
What is an emission filter used for?
To isolate the specific fluorescence signal from the background light.
115
What is a common problem in fluorescence microscopy?
Overlapping excitation/emission spectra leading to signal "bleed-through."
116
How is fluorescence different from phosphorescence?
Fluorescence occurs almost instantly, while phosphorescence continues after excitation stops.
117
What is a fluorochrome?
A molecule that absorbs light and emits fluorescence.
118
What fluorophore emits at 615 nm?
texas red
119
Why do fluorochromes have overlapping emission spectra?
Because their energy transitions are broad.
120
What is spectral unmixing?
A computational method to separate overlapping fluorescence signals.
121
What is confocal microscopy used for?
Generating high-resolution, 3D images of fluorescent specimens.
122
What is the main advantage of confocal microscopy?
It removes out-of-focus light for clearer images.
123
What optical component is unique to confocal microscopes?
The pinhole aperture.
124
How does confocal microscopy improve signal clarity?
By scanning point-by-point and rejecting out-of-focus light.
125
What type of laser is used in confocal microscopy?
A focused laser beam.
126
How does confocal microscopy allow for more color possibilities?
The computer detects different emission wavelengths with high precision.
127
What is cross-talk in fluorescence microscopy?
When emission from one fluorophore overlaps into another detection channel.
128
How does confocal microscopy reduce cross-talk?
By exciting fluorophores sequentially using different wavelengths.
129
How does confocal microscopy assist in 3D imaging?
It scans optical sections and reconstructs a 3D model.
130
What biological structures benefit from 3D confocal imaging?
Tight junctions, cytoskeletal structures, and neurons.
131
What is FRAP used for in live cell imaging?
To measure protein mobility and recovery after photobleaching.
132
What is GFP fusion imaging?
A method of tagging proteins with green fluorescent protein for live tracking.
133
What is colocalization analysis?
Determining whether two proteins are present in the same cellular location.
134
What biological interactions can be studied using FRET?
Protein-protein interactions and conformational changes.
135
How is spectral imaging used in microscopy?
t captures a full spectrum for each pixel, allowing precise fluorophore separation.
136
What is the advantage of spectral unmixing?
It removes overlapping signals for clearer imaging.
137
What is Total Internal Reflection Fluorescence (TIRF) microscopy?
A technique that excites fluorophores only near the sample surface.
138
What is structured illumination microscopy (SIM) used for?
Enhancing resolution beyond the diffraction limit.
139
How does STORM microscopy break the diffraction limit?
By using stochastic activation of fluorophores.
140
What is the diffraction limit in conventional microscopy?
250nm`
141
How does super-resolution microscopy overcome the diffraction limit?
By localizing individual fluorophores with high precision.
142
What is a spectral CLSM?
A confocal laser scanning microscope that uses spectral detection.
143
What type of microscopy is best for detecting membrane dynamics?
TIRF
144
What does FRAP stand for?
Fluorescence Recovery After Photobleaching.
145
What is the main purpose of FRAP?
To study the mobility and diffusion of fluorescently labeled molecules in living cells.
146
How does FRAP work?
A laser irreversibly bleaches fluorophores in a specific region, and the recovery of fluorescence is monitored over time.
147
What does a fast fluorescence recovery in FRAP indicate?
High molecular mobility or diffusion of unbleached molecules into the bleached area.
148
What does FLIP stand for?
Fluorescence Loss in Photobleaching.
149
What is the purpose of FLIP?
To study molecular transport and connectivity between cellular compartments.
150
How is FLIP different from FRAP?
In FLIP, a region is repeatedly bleached over time to observe continuous fluorescence loss in adjacent areas.
151
What does a slow fluorescence loss in FLIP indicate?
A restricted molecular exchange between bleached and unbleached areas.
152
What does FRET stand for?
Fluorescence Resonance Energy Transfer.
153
What is the main purpose of FRET?
To study molecular interactions by measuring energy transfer between two fluorophores.
154
What is required for FRET to occur?
The donor and acceptor fluorophores must be within 1-10 nm of each other.
155
What happens when FRET occurs?
The donor fluorophore transfers energy to the acceptor, leading to emission at a longer wavelength.
156
How is FRET useful in studying protein interactions?
It allows real-time detection of molecular proximity and conformational changes in living cells.
157
What does TIRF stand for?
Total Internal Reflection Fluorescence.
158
How does TIRF microscopy generate an image?
It uses an evanescent wave to excite fluorophores only near the glass-water interface.
159
What is the main advantage of TIRF microscopy?
It provides high-contrast imaging with minimal background fluorescence.
160
How does TIRF microscopy reduce phototoxicity?
By limiting excitation to a very thin region near the sample surface.
161
What does SIM stand for?
Structured Illumination Microscopy.
162
How does SIM improve resolution?
By using patterned illumination and computational reconstruction to surpass the diffraction limit.
163
What is the typical resolution improvement of SIM?
It achieves about 2x better resolution than conventional light microscopy (~100 nm).
164
What are the advantages of SIM over other super-resolution techniques?
It works with standard fluorophores, enables live-cell imaging, and does not require specialized dyes.
165
What is a limitation of SIM?
It has lower resolution than STORM and requires complex image processing.
166
What does STORM stand for?
Stochastic Optical Reconstruction Microscopy.
167
How does STORM achieve super-resolution?
By using stochastic activation of fluorophores and precise localization of individual molecules.
168
What resolution can STORM achieve?
It can reach ~20-50 nm resolution, significantly beyond the diffraction limit.
169
What is a major limitation of STORM?
It requires special fluorophores and long acquisition times.
170
what techniques can be used on live cells
FRET, SIM, TIRF, two photon
171
what techniques can be used on fixed cells
PLA, TIRF, STORM

GSBS Cell Bio (6 decks)

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