Unit 1-3 Flashcards
What is the “cell doctrine”?
The idea that all plant and animal tissues are aggregates of cells.
1p1-05
Bacteria and mitochondria are the smallest entities visible by light microscopy. Any attempt to increase magnification will result in fuzziness, due to what kind of effects?
Interference effects.
1p1-06
True or False:
2 waves out of phase will result in a bright light.
False.
Will result in a dim light.
1p1-06
What is the objective lens?
It collects a cone of light rays to create an image.
1p1-07
What is the condenser lens?
It focuses a cone of light rays onto each point of the specimen.
1p1-07
What is the equation for resolution?
0.61λ / nsinθ
1p1-07
What is nsinθ?
Numerical aperture (NA). 1p1-07
What does the limit of resolution depend on?
Wavelength of light and the numerical aperture.
1p1-08
What does a larger NA mean?
- Better resolution
- Brighter image
- Shorter working distance
1p1-08
True or False:
Structures smaller than 0.2um cannot be observed.
False. They can be observed, but they’re always blurred and appear to be at least 0.2um thick.
1p1-08
In order to better visualize cells, what do we need to enhance?
Contrast.
1p1-09
True or False:
Unstained cells show very little contrast.
True.
1p1-09
What is “differential-interference-contrast (DIC, or Nomarski) microscopy”?
A more complex optical system that also exploits interference effects to impart contrast.
1p1-09
What is dark-field microscopy?
Light is coming in from the side - only scattered light will enter the objective.
1p1-10
True or False:
Reduction of amplitude = Reduced brightness
True.
1p1-11
What are the benefits of using “electronic imaging systems”?
- Can overcome difficulties in detecting very dim light.
- Detecting small differences in light intensity in a very bright background.
1p1-12
What does light microscopy require?
Samples thin enough for light to pass through them - chromosome spreads are simply “squashed”.
1p1-13
Thicker tissues must be…
1) Fixed
2) Embedded
3) Sectioned
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What are FIXED tissues?
Killed, immobilized, preserved.
1p1-13
What are the most common fixatives?
Formaldehyde and glutaraldehyde. They form covalent bonds with free amino groups of proteins.
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What are EMBEDDED tissues?
Prior to sectioning, tissues are placed in supporting medium (wax or plastic resin).
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What is a benefit of fluorescence microscopy?
It allows the visualization of specific molecules within cells.
1p1-15
What will happen if a fluorescent compound is illuminated at its absorbing wavelength and viewed through a filter that only allows light of the emitted wavelength to pass?
The object will appear to glow brightly against a dark
background.
1p1-15
True or False:
Fluorescent molecules absorb light at one wavelength and emit it at a different, shorter, wavelength.
False.
Longer wavelength.
1p1-15
In the fluorescence microscope, what does the first barrier filter do?
It lets through only blue light with a wavelength between 450 and 490 nm.
1p1-16
In the fluorescence microscope, what does the second barrier filter do?
It cuts out unwanted fluorescent signals, passing the specific green fluorescein emission between 520 and 560 nm.
1p1-16
In the fluorescence microscope, what does the beam-splitting mirror do?
It reflects light below 510 nm but transmits light above 510 nm.
1p1-16
DAPI is a fluorescent stain that is specific to which compartment of the cell?
DNA.
1p1-17
Fluorescent molecules (such as fluorescein or rhodamine) are more often coupled to molecules that do what?
That selectively bind certain proteins within the cell.
1p1-17
What are antibodies and what do they bind to?
They’re proteins produced by the vertebrate immune system.
They bind to specific target molecules (antigen).
1p1-19
How are antibodies generated?
By injecting an animal with preparation of an antigen (usually purified protein) and collecting the blood serum, which is antibody rich and known as the antiserum (polyclonal).
1p1-20
True or False: Antibodies are always polyclonal.
False. Can be polyclonal or monoclonal.
1p1-20
How are monoclonal antibodies recovered?
They’re recovered from hybridoma cell lines and can be finicky since they recognize a single antigenic determinant.
1p1-20
True or False: Monoclonal antibodies can be produced in unlimited amounts.
True.
1p1-20
Antibodies can be conjugated to:
1) Enzymes
2) Colloidal gold
3) Fluorescent molecules
1p1-20
What types of antibodies are used in indirect immunocytochemistry?
Labelled (=conjugated) “secondary” antibodies.
1p1-21
In conventional light microscopy, what is light from above and below the plane of focus observed as?
Out-of-focus blur.
1p1-23
What are the 2 different approaches for reducing the blur?
1) Confocal (an optical solution)
2) Image deconvolution (a computational solution)
1p1-23
What is a Z stack?
A series of such optical sections taken at different depths through a specimen.
1p1-23
What is a Z stack projection?
A reconstructed image of the specimen that is essentially an overlay of many individual Z stack images.
1p1-23
How does deconvolution microscopy work?
Z stack sections are processed computationally to “deblur” the images.
1p1-24
True or False: In confocal microscopy, decreasing the detector pinhole size results in a thinner optical slice.
True.
1p1-25
The pinhole aperture in front of the detector is confocal with the illuminating pinhole. What does this mean?
This means that it’s located at exactly the point that the fluorescence coming from the specimen is focused.
1p1-26
Which can penetrate deeper into a specimen: Confocal or deconvolution?
Confocal.
1p1-27
What is multi-photon imaging?
Multi-photon imaging use 2 or more photons of lower energy, BUT they must be w/i ~ a femtosecond of each other.
Can penetrate deeper by using longer wavelength excitation.
1p1-28
How is the GFP chromophore formed?
Proper protein folding permits formation by autocatalysis.
1p2-4
True or False: GFP is an enzyme.
False.
1p2-4
Why are specific amino acid sequences added to GFP?
To target specific subcellular locations.
1p2-4
What can GFP be used for?
- Report gene expression.
- To act as a tag for specific cell types or tissues.
1p2-5
What does a fusion protein result from?
Chimeric genes that fuse a gene of interest to the GFP coding sequence.
1p2-5
What can GFP fusion proteins be used for?
To observe subcellular localization of specific proteins in living cells.
1p2-5
Why live-cell imaging?
- High resolution imaging of live tissues (or cells) reveals phenomena & structures that are difficult to observe in fixed material.
- Can interrogate a protein/organelle in real time.
- Highlight movement/changes in morphology while cells are doing everything that need to do.
1p2-6
How can GFP and GFP spectral variants be used to evaluate molecular interactions?
Via Fluorescence Resonance Energy Transfer (FRET).
1p2-8
Two molecules of interest are labelled with different fluorophores, with the emission spectrum of one overlapping with the excitation spectrum of the other. If the 2 fluorophores are in close proximity, what does that mean?
It means there can be a transfer of energy from the first fluorophore to the second.
1p2-8
If there are no protein interactions in FRET, what colour light would you detect?
No excitation of GFP, therefore, blue light detected.
1p2-9
If there ARE protein interactions in FRET, what colour light would you detect?
Fluorescence resonance energy transfer, therefore, green light detected.
1p2-9
What is photoactivation used for?
To visualize protein trafficking. Can control exactly where/when the molecule becomes fluorescent.
1p2-10
What can happen if a portion of a cell is exposed to the light source/laser?
Can lead to photobleaching of the fluorescent marker.
1p2-11
What can we determine from the recovery of fluorescence (FRAP) within a bleached area?
The rate of movement of fluorescently-labelled molecules.
1p2-11
What are 2 types of fluorescence “indicators”?
1) Ca2+ indicators
2) Indicators of intracellular pH
1p2-13
What are some examples of supperresolution microscopy?
- SIM
- STED
- PALM
- STORM
1p2-14
What is the purpose of the electron microscope?
To resolve ultrastructure of cell.
1p2-15
Resolution is a function of ______________?
Wavelength.
1p2-15
Describe TEM (Transmission Electron Microscopy)?
- The cathode emits electrons from top of column.
- Electrons pass through a tiny hole, creating an electron beam, focused by magnetic coils.
- Electrons may pass directly through specimen or be scattered by electron dense structures.
1p2-16
In TEM, why do you need a vacuum?
Because electrons will collide with air molecules and scatter.
1p2-16
What are the steps towards preparing samples for TEM?
- Samples must be thin.
- Fixation
- Embedding
- Cutting sections
- Staining
1p2-17
Why are specimen sections stained with electron-dense material to achieve contrast?
Tissue composed of atoms of low atomic number (C, O, N, H) = low contrast by TEM.
Make them visible by staining with salts of heavy metals (=high contrast by TEM).
1p2-17
What does flash freezing tissue result in?
Non-crystalline vitreous ice (a glass) such that ice crystals do not disrupt structure.
1p2-18
What is the method of choice for immuno EM?
Cryo-fixation.
1p2-18
What is immunogold EM?
Secondary antibodies can be attached to very small particles prepared from colloidal gold.
1p2-19
True or False: In EM immunolabelling, only the exposed surface of the thin section is accessible to antibody labelling.
True.
What is serial reconstruction?
If you’re really good at sectioning, it’s possible to recover several consecutive ribbons of ultra-thin sections.
From this, a 3-D image can be projected.
1p2-20
What is 3D EM reconstruction (tomography)?
Information of the third dimension can be recovered by viewing the specimen from several different angles.
1p2-21
How does SEM work?
SEM detects electrons that are scattered or emitted from the surface of a specimen.
1p2-22
What is the resolution of SEM?
~10 nm.
20,000X magnification.
1p2-22
How can large macromolecules be visualized?
If they’re shadowed with heavy metal.
1p2-24
What must be done to molecules in order for them to be visualized at high resolution?
Molecules to be visualized are biochemically purified, mixed with an aqueous solution of heavy metals, spread over a thin carbon film, and then dried.
1p2-24
When the electron dense stain is absorbed by the background but not the molecules of interest, what do the molecules appear as (light or dark)?
The molecules of interest appear as light areas surrounded by dark.
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What is single particle reconstruction?
An averaging method of taking tens of thousands of images of the same molecule combined to produce an “average” image.
Will reveal structures hidden by “noise”.
1p2-25
What is “noise”?
Inherent random variability that obscures the underlying image.
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