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Cell Bio > Microscopy > Flashcards

Microscopy Flashcards

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0
Q

3 things microscope must do:

A
  1. Produce a magnifies image of the specimen
  2. Separate or resolve the details in the image
  3. Render the details visible to the eye
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1
Q

What does a microscope do?

A

Enhances ability to see objects by making them bigger

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2
Q

Magnification

A
  • process of enlarging the physical appearance of something
  • achieved by placing a biconvex lens into the light path
  • light from object passes through lens and is bent (refracted) towards the eye
  • objects seem bigger than they really are
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3
Q

Magnification that has no increase in detail is called?

A

Empty magnification

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4
Q

What is magnification a combination of?

A

All the lenses in the light path

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5
Q

Resolution

A

Shortens the distance between 2 separate pints in the microscopes field of view that can still be distinguished as distinct entities

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6
Q

What determines microscope resolution?

A

Representation of point spread function of single sources of light

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7
Q

Microscope resolution of airy disks

A

Resolvable: 2 pings separated by more than the radii of airy disks

Not resolvable: 2 points separate by less than the radii of airy disks

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8
Q

Wave length

A

Shorter lamda = better resolution

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9
Q

Numerical aperture

A

Higher NA = better resolution

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10
Q

Resolution equation

A

R = 0.61 lamda/ NA

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11
Q

What is the resolving power of a light microscope under optimal conditions?

A

200 nm

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12
Q

Increasing magnification means decrease of…?

A

FOV (field of view)

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13
Q

2 types of contrast

A
  1. Phase contrast

2. Differential interference contrast (DIC)

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14
Q

Phase contrast

A
  • changes in refractive index causing light rays to shift

- causes a halo around cell

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15
Q

Differential interference contrast (DIC)

A
  • light splits into 2 waves so light passes through different parts of sample
  • gives a 3D Pseudo effect
  • light and dark regions
16
Q

Benefits of using DIC and Phase contrast

A
  • method used living cells and they can be examined at natural state
  • no harm, staining, or killing is necessary
17
Q

DIC goods and bads

A

Goods

  • gives 3D image, more detail
  • important technique for imaging thick plants and animal tissues

Bads

  • more expensive than phase contrast
  • some plastics not suitable for DIC imaging
18
Q

Staining

A

Strain different cellular components different colors to allow for identification of parts

19
Q

Fluorescence Microscopy

A

Emission of light from a compound following adsorption of light of shorter wavelengths

  • fluorescent proteins can be used to tag specific proteins in cells
20
Q

Fluorescent dyes

A

Can be attached to wide range of antibiotics or phalliodin to allow specific structures in the cell to be visualized by microscopy

21
Q

Epifluorescence microscopy (Epi)

A
  • camera based technique
  • fluorophores throughout sample are excited
  • images contain interference from out of focus molecules
  • good for fixed or live cell imaging and multicolor
  • see structures throughout cell
22
Q

2 types of confocal microscopy

A
  1. Spinning disc microscopy

2. Laser scanning confocal microscopy (LSCM)

23
Q

Spinning disc microscopy

A
  • camera based
  • fast and suitable for photo sensitive samples
  • imaging highly dynamic processes
24
Q

Laser scanning confocal microscopy

LSCM

A
  • point scanner

- slower

25
Q

Advantages of confocal microscopy

A
  • ability to control depth
  • eliminates out of focus light using a pinhole
  • optically section though 3D reconstruction
  • image can be zoomed without loss of resolution
26
Q

Disadvantages of confocal microscopy

A
  • sample still illuminated causing phototoxicity
  • slower than a camera
  • resolution limited to 200 nm
27
Q

Total internal reflection fluorescence (TIRF) microscopy

A
  • uses camera to capture image(fast)
  • reduces phototoxicity
  • suitable for fixed and live cells
  • showed skeleton of cell
  • only see structure at adherent membrane
28
Q

Example applications of TIRF

A
  • time lapse in endocytosis
  • focal adhesion dynamics
  • exocytosis
29
Q

Super resolution microscopy

A
  • 2 objects seen as one in diffraction limited microscopy

- Nobel prize winning techniques

30
Q

Direct stochastic optical reconstruction microscopy (dSTORM)

A
  • uses fluorescent antibodies to label proteins
  • used on dead samples
  • increased resolution by x10
  • randomly switching on and off causing “blinks” and nm precision
31
Q

2 color dSTORM

A
  • increased resolution allows finer detail

- proteins can be seen as distinct structures with no overlap

32
Q

Advantage of dSTORM over EM

A

Preparation is easy and multi color 3D images can be taken

33
Q

Electron Microscopy (EM)

A
  • original super resolution microscopy
  • objects as small as 1 atom can be seen
  • uses a beam of electrons to illuminate sample as electrons have smaller wavelengths
  • electromagnetic cools used to focus electron beam instead of glass lens
  • can only be done on dead samples and preparation is complicated
34
Q

What microscope has the highest resolution?

A
  • EM

- behave it uses a beam of electrons instead of light

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