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Cell and Molecular Biology > Microscopy and Imaging > Flashcards

Microscopy and Imaging Flashcards

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1
Q

How does light travel?

A
  • as a wave, but it is thought of a distinct units (photons)
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2
Q

What is light refraction?

A

change in direction when moving from one medium to another duct to a change in refractive index (measure for the speed of light in a medium)

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3
Q

What is light diffraction?

A

Bending of light around an object

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4
Q

What is light reflection?

A

Bouncing of light off of an object

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5
Q

What is light absorption?

A

Interaction of a photon with an object such that the energy of the photon is taken up by the electrons in that object (usually generates heat)

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6
Q

What is light phase?

A

The occurrence of one whole wave within a specific time period (measured in degrees)

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7
Q

What is light wavelength?

A

The distance between the amplitude peaks of different waves

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8
Q

What is light amplitude?

A

The distance between the middle line and the peak of the wave

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9
Q

What is light polarisation?

A
  • the orientation of oscillation of a wave
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10
Q

What are some imaging technologies?

A
  • Brightfield
  • Phase contrast
  • Differential interference contrast (DIC)
  • Darkfield
  • WIdefield
  • Laser scanning confocal microscopy (LSCM)
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11
Q

What are the different components of a microscope?

A
  • eye piece
  • objectives (different magnifications)
  • stage
  • condenser
  • light source
  • focus dial
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12
Q

What is Kohler illumination?

A
  • Kohler, 1893
  • process that provides optimum contrast and resolution by focusing and centring the light path and spreading it evenly over the field of view.
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13
Q

What is Brightfield microscopy

A
  • the simplest of all the optical microscopy illumination techniques.
  • sample illumination is transmitted white light, and contrast in the sample is caused by attenuation of the transmitted light in dense areas of the sample
  • best suited to viewing stained or naturally pigmented specimens such as stained prepared slides of tissue sections or living photosynthetic organisms.
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14
Q

What is phase contrast?

A
  • Zernike, 1934
  • optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image
  • Phase shifts themselves are invisible, but become visible when shown as brightness variations
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15
Q

What is the light path for phase contrast microscopy?

A

Condenser annulus -> condenser -> specimen -> objective -> 90 degree phase shift ring -> grey filter ring -> image plane

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16
Q

What is differential interference contrast (DIC)?

A
  • Nomarski, 1952
  • used for imaging live and unstained biological samples, such as a smear from a tissue culture or individual water borne single-celled organisms.
  • enhances the contrast in unstained, transparent samples
  • uses the interference between sheared beams and utilises polarised light
17
Q

What is polarised light?

A
  • light waves in which the vibrations occur in a single plane
  • Plane polarized light consists of waves in which the direction of vibration is the same for all waves
  • a plane polarized light vibrates on only one plane
18
Q

What are fluorophores?

A

Molecules that accept a photon and emit a photon as a result
- can be expressed or attached to specific tapes via antibody affinity or via chemical affinity
- examples include green fluorescent protein/mCherry

19
Q

What is fluorescent microscopy?

A
  • instead of light being shone through the sample, light is shone on the sample
  • it does this to look at the emission response
  • the objective is both the sender and the receiver so no condenser is needed
20
Q

How does fluorescent microscopy work?

A
  • introduce fluorophores into the sample through staining or expression
  • illuminate with the specific wavelength of the fluorophore(s) of choice
  • filter for the expected emission wavelength
21
Q

What is the airy disk?

A
  • light coming from a fluorophore starts as a small point (point source) and expands in a sphere
  • passing light through a lens attempts to focus it back to a small point
  • the smallest focussed point is an airy disk which size is limited by diffraction
22
Q

What is the Rayleigh criterion?

A

The minimum resolvable detail dictated by the overlap between the airy pattern of one point source and the airy disk of another

23
Q

What is point spread function (PSF)?

A
  • describes what a single point in the object looks like in the image
  • presents an issue if imaging thicker objects as overlapping PSFs will get in the way of focussed points if they’re in front or behind them
24
Q

What is a confocal microscope?

A
  • uses a confocal pinhole to block out of focus light from reaching the detector
  • only focussed light is let in
  • varying the diameter of the pinhole allows thinner or thicker slices to be taken of the sample
25
Q

What is the difference between confocal and widefield microscopy?

A

Without the optional sectioning of the confocal pinhole, it becomes a wide field microscope which captures out of focus light with focused light - which causes high background

26
Q

What is green fluorescent protein (GFP)?

A
  • discovered in jellyfish, 1962
  • one of the most widely used tools in life sciences as they enable the majority of imaging done
  • first expressed in living e.coli, 1994
  • further work modified the protein to produce different colours
  • development include bio sensors, eg calcium and pH indicators
27
Q

What microscopy is used at St George’s?

A
  1. Confocal microscopy Nikon A1R = sample is illuminated by a scanned laser point, image is generated by high sensitivity photon directors
  2. Widefield microscopy Nikon Ni E = sample is illumined by a metal halide lamp/white light and image is generated by a camera
  3. Brightfield scanner Hamamatsu RS2.0 = sample is illuminated by a white light and image is generated by a camera and stitched together

Cell and Molecular Biology (3 decks)

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