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Cell and Developmental Biology > MICROSCOPY > Flashcards

MICROSCOPY Flashcards

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1
Q

What do you use upright microscopes for?

A

Large organisms and cells

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2
Q

What do you use inverted microscopes for?

A

Mostly single cells

- looking at a dish with thin layers

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3
Q

How do you find the magnification of modern microscopes?

A

Multiply the magnification of the ocular ( lens) by the magnification of the eyepiece

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4
Q

What is Snell’s Law?

A

The angle of reflection is equal to the angle of incidence (regardless of the surface material)

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5
Q

What is the difference between the refracted beam and reflected beam?

A

Refracted beam is the beam that is transmitted and bent when passing from one medium to another. Reflected is the beam that bounces back

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6
Q

How does the refracting beam enable the sample to emit light?

A

The refracted beam causes a dispersion of colours. The colours will heat the sample which will emit the light and highlight the sample/ specimen.

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7
Q

What is the refractive index?

A

How fast the light propagates through

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8
Q

What does the refractive index have to be to produce a perfect image compared to a blurry image?

A

The refractive index has to be the same throughout the apparatus and the medium/sample for the image being produced to not turn out blurry.

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9
Q

What is the numerical aperture (NA)?

A

it is a dimensionless number that characterises the range of angles over which the system can accept or emit light
- cone of light is wide or narrow dependent on the numerical aperture

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10
Q

How is resolving power related to numerical aperture (NA)?

A

Directly related

- the higher the NA the greater the resolution

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11
Q

How do you work out NA?

A 
NA = n sin y
n= lowest refractive index between the object and first objective element
y= 1/2 the angular aperture of the objective
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12
Q

Define resolution.

A

Limit for the smallest resolvable distance between 2 points

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13
Q

How do you work out the smallest resolvable unit (d)?

A

d= wavelength of light / 2NA

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14
Q

What NA will you see 2 dots as 1 dot?

A

2NA

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15
Q

Define a Point Spread Function (PSF).

A

Describes 3D light distribution in an image of a point source

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16
Q

How does PSF relate to resolution?

A

If you have a v good clear PSF - you are at the highest resolution. If it is large, then it is a low resolution. The lower wavelength, better resolution.

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17
Q

What does PSF dependent on?

A

Numerical Aperture and the wavelength of fluorescent emission

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18
Q

What does bright field illumination show?

A

Different brightness intensities

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19
Q

What does phase contrast show?

A

Structural details emerge by staining of components

  • black/white/greyscale contrast
  • can see shape
20
Q

What does DIC show?

A

Edge effects producing contrast and details (doesn’t need staining)

21
Q

What does Fluorescent microscopy show?

A

Can identify cells and sub-microscopic cellular components

  • add dyes/ fluorescent proteins or tracers that will emit light
  • need to label the sample
22
Q

How do you choose fluorophores?

A
  • Use bright, stable fluorophores
  • Shorter wavelength gives you better resolution
  • Longer wavelength gives you less photo-toxicity/photobleaching
  • Minimise the spectral overlap
23
Q

What is the difference between Widefield vs Confocal vs TIRF Microscopy?

A

Widefield is the normal usual one
Confocal has a slicing effect used for more detail
TIRF is more used for cell movement and trafficking

24
Q

How does fluoerescence microscopy work?

A
  1. sample gets excited by lights
  2. lights get de-fracted
  3. light is bent
  4. captured
25
Q

What are some Pro’s of fluorescence microscopes?

A
  • uses light sources of much lower power
  • cheaper than confocal systems
  • give high quality photographic images
26
Q

What is Laser Scanning Confocal Microscopy (LCS) used for?

A
  • localisation of proteins
  • imaging multiple fluorescent stains
  • 3D visualisation of stainings
  • Quantification of protein expression`
27
Q

What is the main difference between LCS and normal microscopy?

A

For LCS you select specific plane of sample and hide everything else
- only one layer

28
Q

What is Total Internal Reflection Fluorescent (TIRF) microscopy?

A

Microscopy that you look at interface of the sample and the glass slide

29
Q

How does TIRF microscopy work?

A

Laser will have a specific path that will bend

  • creating a wave
  • evanescent wave
30
Q

What is an evanescent wave?

A

an optical phenomenon that can occur when light strikes the interface between two media of different refractive index

31
Q

What are some TIRF microscopy applications?

A
  • endocytosis / exocytosis
  • dynamics of membrane-associated proteins
  • protein arrangement
  • focal adhesions
  • growth cone migration
  • receptor - ligand interactions
  • single molecule behaviour
32
Q

What is Single Plane Illumination Microscopy (SPIM)?

A

Microscope with 2 objective lenses on the sides - one is illuminating and one is capturing.

  • embed sample into a glass capillary
  • Rotating capillary will be present - containing our sample (360 view)
33
Q

What is superresolution microscopy?

A

Group of microscopy techniques that allow these objects to be distinguished as separate identities

34
Q

What techniques are part of Super Resolution microscopy?

A

STED, SIM and dSTORM

35
Q

Which Super Resolution microscopy technique is used for live samples and which is used for fixed samples?

A

STED and SIM are used for live and fixed whereas dSTORM is used for just fixed

36
Q

How does the SIM technique in Super Resolution Microscopy work?

A

Mixing the image with pattern grids that rotate to produce a cleaner image

37
Q

What is the dStorm technique in Super resolution microscopy associated with?

A

Blinking events

38
Q

How does the STED technique in Super Resolution Microscopy work?

A

De-activation of the fluorophore will allow highlighting of the particles inside the sample

39
Q

How does electron microscopy work?

A

Beams of electrons are used to produce images.

- Wavelength of electron beam is much shorter than light therefore is a much higher resolution

40
Q

How does Transmission Electron Microscopy (TEM) work?

A

Electrons scatter when they pass through thin sections of a specimen.
Transmitted electrons are used to produce image
Denser regions in specimen, scatter more electrons and appear darker

41
Q

How does Scanning Electron Microscopy (SEM) work?

A

Uses electron reflected from the surface of a specimen to create image
Produces a 3D image of specimens surface features
- can avoid staining

42
Q

What are some microscopy and Imaging Applications?

A 
Live Cell Imaging
Ionic Flux Determinations
FRET
FRAP
DNA Paint
Expansion Microscopy
43
Q

What is FRET?

A

Interaction between two molecules

  • One molecule can change its conformation to allow an open channel or to allow an interaction to another protein
  • Energy of fluorophore will transfer energy from one protein to another to emit light
44
Q

What is FRAP?

A

Bleach protein

  • Waiting for recovery of the fluorophore over time
  • Recovery due to behaviour or chemicals
45
Q

What is DNA Paint?

A

Label (with dyes) DNA
- Image the DNA throughout time
Useful:
Can do SIM or STED microscopy

46
Q

What is Expansion Microscopy?

A

Possibility to insert the cell into a polymer gel

  • Gel will expand
  • Can look at it in more detail

Cell and Developmental Biology (16 decks)

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