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TB4: Microscopy Flashcards

1
Q

What is an image?

A

A matrix of numbers

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2
Q

Brightfield microscopy uses

A

Commonly used in a wide variety of laboratory applications as the standard microscope; produces an image on a bright background.

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3
Q

Darkfield microscopy uses

A

Increases contrast without staining by producing a bright image on a darker background; especially useful for viewing live specimens.

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4
Q

Phase contrast microscopy uses

A

Uses refraction and interference caused by structures in the specimen to create high-contrast, high-resolution images without staining, making it useful for viewing live specimens, and structures such as endospores and organelles.

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5
Q

Fluorescence microscopy uses

A

Uses fluorescent stains to produce an image; can be used to identify pathogens, to find species, to distinguish living from dead cells, or to find locations of molecules within a cell; also used for immunofluorescence.

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6
Q

Confocal microscopy uses

A

Uses a laser to scan multiple z-planes successively, producing numerous 2D, high resolution images at various depths that can be constructed into 3D images by a computer, making this useful for examining thick specimens such as biofilms.

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7
Q

Widefield vs Confocal

A
  • widefield lights up the entire sample whilst confocal has a pinhole
  • confocal has scan mirrors
  • widefield is quicker but can’t do optical sectioning
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8
Q

Experimental Considerations for Microscopy

A
  • living samples need environmental controls
  • always do confocal before super-resolution
  • widefield microscope when studying dynamics
  • thin or thick sample
  • laser lines and filter sets
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9
Q

Rayleigh Criterion (equation)

A

θ = 1.22 x λ(m)/diameter(m)

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10
Q

Abbe Equation

A

r = λ/2NA (numerical aperature)

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11
Q

Focal Point

A

When a beam is transmitted through a lens, all the rays entering parallel will converge to a single spot called the focal point which resides on the focal plane.

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12
Q

Snell’s law

A

sin(incident angle)/sin(refractive angle)=n (refractive index)

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13
Q

Chromatic aberration

A

Caused by different wavelengths passing through a lens as each of these will have a different refractive index, and thus have differing focal points.

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14
Q

Spherical aberration

A

Occur even when there’s only one wavelength passing through, stemming from the fact that spheres of lenses aren’t perfect and so won’t focus the light to a perfect point. This isn’t as bad as chromatic aberrations.

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15
Q

Objective lens

A

Located closest to the object, this relays the real image of the object to the eyepiece. It’s needed to produce the base magnification.

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16
Q

Eyepiece lens

A

Located closest to the eye/sensor, projects and magnifies the real image and yields a virtual image of the object.

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17
Q

Brightfield illumination

A

Backlit illumination where incident light floods the object with light from behind. It uses two lenses (collector and condenser) to provide bright and even illumination on the object plane and image plane. This ensures the user doesn’t image the filament from the light bulb.

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18
Q

Airy Rings

A

Lenses don’t form perfect images of points of light due to waves having 3D dimensions. They instead distort the images in 3D to form airy rings. The out of focus light from this reduces contrast and resolution.

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19
Q

Rayleigh Resolution Limit

A

‘The separation where the central maximum of the Airy pattern of one point is directly overlapping with the first minimum of the Airy pattern of another’. Gives a rough idea of the resolution limit. It’s dependent on the wavelength and angle of the light.

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20
Q

Full-Width-Half-Maximum

A

If you know the wavelength and aperture of a lens, you can use FWHM to determine the resolution limit. The thinner the peak, the better the SNR you can achieve.

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21
Q

Abbe’s Resolution

A

A simplified view on resolution: high frequencies correspond to small structures which diffract light further than larger structures.

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22
Q

Point Spread Function

A

The spread of the Airy rings in a diffraction pattern.

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23
Q

Deconvolution

A

Reassignment of out-of-focus information to increase contrast and resolution.

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24
Q

FISH (fluorescence in situ hybridization)

A

Fluorescent probes bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity.

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25
PALM and STORM
Widefield fluorescence microscopy imaging techniques that allow obtaining of images with a resolution beyond the diffraction limit by distinguishing the emissions of two neighbouring fluorescent molecules so that their PSF don't overlap.
26
Shot noise
Randomness of counting; the more photons you fire, the more evenly distributed the fluctuations of photons (I.e. the brightness), like tossing a coin 5 times vs 1 million times. This cannot be avoided, so must trade off between a strong laser and not destroying the sample.
27
Read noise
Noise generated in the detector
28
Background noise
autofluoresence from the specimen
29
Sources of noise
1. Shot noise 2. Read noise 3. Background noise 4. Stray light
30
Multiplexing
The use of multiple distinct fluorochromes in the same experiment.
31
How do fluorochromes work?
Absorb light of a specific wavelength and then rapidly emits light of a longer wavelength with a lower energy.
32
Visible light spectrum order (shortest to longest wavelength)
Purple --> Blue --> Green --> Red
33
DAPI
Recognizes AT-rich DNA
34
EdU
Recognizes newly synthesized DNA
35
Alexa555
Recognizes microtubules
36
Alexa488-phalloidin
Recognizes actin and blocks depolymerization
37
Stokes Shift
The difference (nm) between the excitation and emission maxima for a given fluorophore. It's always shifted to the right.
38
Organic dyes (pros)
- extremely bright - very photostable - lots of choice for multiple-colour imaging
39
GFP (pros & cons)
- not as bright as organic dyes - can be bleached - genetically encoded so precise
40
Quantum dot (pros & cons)
- not usually used - very bright and small - emission wavelength is determined by the size of the dot
41
HALO tags
Only fluorescent when you add a dye that fluoresces so you get the advantages of the dye alongside the precision of genetically modifying the tag onto the protein of interest.
42
Widefield Microscopy
Entire specimen is exposed to the light source. Gives a characteristic haze that effects the contrast.
43
Dichroic mirrors
used to direct light onto the sample but don't allow any light other than the emission light (has a longer wavelength) onto the detector.
44
Confocal microscopy
use of a pinhole which rejects out-of-focus light, increasing SNR and resolution, and laser-scanning
45
Advantages of confocal microscopy
- can define regions to be studied | - can undergo optical sectioning to create a 3D image
46
Mercury arc lamps (pros & cons)
- broad emission profile - excitation filters required to isolate wavelengths wanted - almost 50% is in the UV range - lifetime of 200 hours; shortened by incorrect use These are now being replaced by LEDs which have a fairly narrow range, thus don't need excitation filters.
47
LASER
- required in confocal microscopes - monochromatic light source - point source of light
48
Longpass filters
Only allow long wavelengths through
49
Shortpass filters
Transmit only short wavelengths
50
Bandpass filters
transmit certain bands of wavelengths
51
Bleedthrough
(Overlap of emission) when the emission of one fluorophore is detected in the channel meant for another
52
Crosstalk
(Overlap of excitation) when a second fluorophore is excited while attempting to excite another.
53
What is single-molecule localization microscopy?
This technique is based on spatial coordinates of fluorophores being determined with high precision if their PSFs don’t overlap. This can be achieved by separating the fluorescent emissions of distinct molecules in time, often via photo switching.
54
Stimulated emission depletion
A diffraction-limited spot is excited at one wavelength while a super-imposed, red-shifted, second laser beam depletes almost all emission laterally leaving only a central focal spot with a dimension below the diffraction limit. The size of the centre of the focal spot and hence the resolution can be tuned by changing the intensity of the depletion laser, reducing the PSF.
55
Structure illumination microscopy
This uses patterned illumination, usually stripes, to excite a sample. The emitted fluorescence is then recorded for a range of stripe positions and orientations, giving rise to moiré fringes, allowing capture of high frequency information. In Fourier space, the information is decoded, however this requires far more data than other techniques.
56
Vital stains
can be applied on living cells without killing them
57
histological stains
used to colour different structures within the cells
58
immunolabelling
antibodies with fluorophores attached recognize specific epitopes
59
What can cause intrinsic fluorescence of a protein?
caused by phenylalanine, tryptophan and tyrosine
60
Why is indirect immunofluorescence used over direct methods?
Allows for amplification of small signals and only one species of secondary antibody is needed to be made to detect many varieties of primary antibodies
61
Nanobodies
Only have one type of variable domain so labelling is closer to the actual site of interest and thus background noise is reduced
62
Describe an epifluorescent microscope.
Light passes through an excitation filter and is projected onto the specimen by a dichroic mirror, allowing only the emitted fluorescence to pass through it and into the detector, passing through an emission filter first.
63
FRET
The principle of FRET relies on the transfer of excitation energy of a donor fluorophore to a nearby acceptor fluorophore via long-range dipole-dipole interactions (~2-10nm). This means that rather than the donor emitting a fluorescent photon, the acceptor does. By measuring the ratio of the donor to acceptor fluorescence, you can understand the interaction between the two fluorophores.
64
When can FRET occur?
FRET can only occur if the emission spectrum of the acceptor overlaps with the excitation spectrum of the donor.
65
Forster Distance
the distance between the two fluorophores where 50% of the excitation energy is being transferred from the donor to the acceptor.
66
What is TIRF?
TIRF uses the phenomenon of total internal reflection to reduce background noise. It only excites fluorophores on the surface of molecules, so other fluorophores aren’t excited and don’t emit light. As a result, background noise is significantly reduced.
67
What can TIRF be used for? How?
TIRF can be used to measure binding kinetics at the single-molecule level. Using a TIRF microscope where one molecular species is immobilized on the cover slip and the other is fluorescently labelled in solution, you can directly resolve when the fluorescently labelled species binds to the cover slip. It appears as a bright spot and when it unbinds, the fluorescence disappears. You can then directly measure on and off rates for binding kinetics.
68
What are optical traps?
These can very precisely measure forces, such as those seen in the folding and unfolding of proteins. Two laser beams each trap a bead, and a protein is tethered in the middle of the two beads. From this, you can perform different experiments.
69
Optical traps: constant force
Set the force between the beads as constant and find the distance between the two beads changes as the protein spontaneously folds and unfolds. From these durations in each state, you can decipher the folding landscape of the protein.
70
Optical traps: constant position
Keep the position constant and measure how the forces change.
71
Optical traps: pull beads apart
Measure how the force changes as you pull the beads apart. a. Force increases until the protein unfolds b. Unfolding results in a decrease in force until you get the unfolded protein back under tension c. You can then relax the force to allow the protein to refold
72
Force jump experiment
the force is suddenly increased resulting in the protein going through several stages of extension where it moves from the folded gradually to an unfolded state with several discrete transitions that involve the unfolding of particular domains of the protein
73
Why can you overcome the resolution limit for single molecule microscopy?
If you have an individual molecule and it makes a diffraction limited focus on your camera, you can tell the centre of the spot with much greater precision than the overall width of the spot. Whereas conventional diffraction limit is due to multiple molecules, as you are only looking at single molecules, this doesn’t apply. Thus, the precision of the localization is limited only by the number of photons collected.
74
Localization precision equation for single molecule microscopy
``` σ = s /√N σ: localization precision s: point spread function width N: number of photons collected NOTE: this is the equation for the standard error of the mean which is much smaller than the individual measurement of error. ```
75
How does super resolution localization microscopy work?
Instead of recording all the signals at once, you isolate a subset of fluorophores and repeat with other subsets, and then reconstruct the image.
76
Why is thiol used with photoswitchable fluorophores?
Using photoswitchable fluorophores, you can image just a subset of fluorophores that are present in a sample, rather than all of them at once. Often thiol is used as this disrupts delocalized electron systems and forces the fluorophore into the dark state.
77
How is contrast generated in EM?
Density differences within the sample and general stains
78
Specimen requirements for TEM
1. stable in the vacuum 2. very thin 3. well preserved 4. electron dense
79
TEM specimen prep
1. Primary fixation with aldehydes 2. Secondary fixation with osmium tetroxide 3. Tertiary fixation with uranyl acetate 4. Dehydration using ethanol/acetone 5. Resin infiltration 6. Heat polymerization 7. Ultramicrotomy (resin sectioning) 8. Post-staining 9. TEM 10. Optimize prep and repeat
80
What is paraformaldehyde? Pros and cons?
small molecule that readily permeates into cells without affecting antigenicity, but doesn't sufficiently preserve cells at the ultrastructure level
81
Glutaraldehyde
strong fixative but penetrates slowly
82
Antigenicity
the ability to specifically combine with the final products of the immune response
83
Osmium tetroxide
acts as both a secondary fixative and an electron stain to significantly improve specimen preparation (especially of membranes) and contrast
84
Uranyl acetate
acts as both a tertiary fixative and an electron stain (used for nucleus and protein complex staining)
85
What is dehydration?
The process of gradually replacing water in the sample with a solvent to cause shrinkage and extraction of cytoplasmic components, which can be minimized by dehydrating at low temperatures.
86
What is EM tomography?
thicker sections of resin are placed on grids with reference points, and then tilted to build up a reconstruction of the structure
87
How does SEM differ from TEM in terms of electron scattering?
In SEM, electrons are scattered by high atomic number elements in the sample
88
SEM specimen prep
1. Primary fixation with aldehyde 2. Secondary fixation with osmium tetroxide x2 3. Tertiary fixation with uranyl acetate 4. Dehydration with ethanol/acetone 5. Resin infiltration 6. Heat polymerization 7. Mount onto SEM pin, coat and polish block face 8. TEM
89
FIB-SEM
Sample is tilted so you can mill trench into it and mill resin away, firing the electron beam to image the fresh block face.
90
SBF-SEM
Each block face is scanned before being thinly sliced away by a diamond cutter. The resolution isn't as good as FIB-SEM.
91
How does protein localization differ in EM?
Like immunofluorescence labelling, but the secondary antibody is conjugated to a small colloidal gold particle so that it can be viewed in the EM.
92
Why aren't permeabilizing agents used in EM?
For light microscopy, cells are usually permeabilized with detergents to allow antibodies to access cells. However, this damages the ultrastructure and results in poor preservation at the EM level. Therefore, for EM, post-embedding labelling is usually the best option as it avoids the use of permeabilization agents.
93
Cryo-processing method
1. Cryo-fixation and high pressure freezing 2. Freeze substitution with solvent 3. Resin infiltration 4. UV polymerization 5. Ultramicrotomy 6. Post-staining 7. TEM
94
Correlative microscopy
the same sample is imaged using two or more microscopy techniques
95
CLEM
(correlative light and electron microscopy) first use fluorescence imaging and then use EM

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