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Biology > microscopy > Flashcards

microscopy Flashcards

1
Q

what type of microscopes were first developed

A

light microscopes
16th-17th century

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2
Q

how does a light microscope work

A
  • compound light microscope has 2 lenses
  • objective lens produces a magnified image which is magnified again by the eyepiece lens
  • objective/eyepiece lens configuration allows for much higher magnification and reduced chromatic aberration
  • illumination is provided by a light underneath the sample
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3
Q

sample preparation in light microscopy

A

dry mount
- solid specimens are viewed whole or cut with a sharp blade (sectioning). Cover slip is placed over specimen on the centre of the slide

wet mount
- specimens are suspended in liquid
- cover slip placed at an angle

squash slides
- wet mount is prepared
- lens tissue used to gently press down the cover slip
- damage to cover slip can be avoided by squashing the sample between 2 microscope slides

smear slides
- edge of a slide is used to smear the sample
- creates a thin even coating
- cover slip is placed

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4
Q

purpose of stains

A
  • increase contrast as cell cytosol and structures are often transparent
  • which allows components to become visible so they can be identified
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5
Q

what is magnification

A

how many times larger the image is than the actual size of the object being viewed

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6
Q

resolution

A
  • the ability to see individual objects as separate entities
  • determines the amount of detail that can be seen
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7
Q

what is resolution limited by

A

diffraction of light as it passes through the sample and lens

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8
Q

how can resolution be increased

A
  • using beams of electrons which have a wavelength 1000s of times shorter than light
  • electron beams are still diffracted but the shorter wavelength means that individual beams can be much closer before they overlap
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9
Q

equation for magnification

A

image= magnification x actual size

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10
Q

why do microscopes need to be calibrated

A

the true magnification of the different lenses on a microscope can vary slightly from the magnification stated

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11
Q

what is an eyepiece graticule

A
  • glass disc marked with a fine scale of 1-100
  • scale has no units and remains unchanged whichever objective lens is in place
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12
Q

what is a stage micrometer

A
  • microscope slide with a very accurate scale in micrometers engraved on it
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13
Q

how to calibrate a microscope

A
  • put the stage micrometer in place and the eyepiece graticule in place
  • get the scale on the micrometer slide in clear focus
  • align the micrometer scale with the scale in the eyepiece and take a reading from the two scales in order to calculate what 1 eyepiece graticule unit is equal to.
  • Replace the micrometer slide with the sample slides and use the calibrated scale in the eyepiece to measure the sample
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14
Q

when was the electron microscope invented

A

20th century

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15
Q

how does electron microscopy work

A
  • beam of electrons with a wavelength of less than 1nm illuminates the specimen
  • more detail of cell ultrastructure can be seen because electrons have a much shorter wavelength than light waves
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16
Q

disdvantages of electron microscopes

A
  • expensive to buy and operate
  • can only be used in a controlled environment
  • large and needs to be installed
  • complex sample preparation
  • sample preparation often distorts material
  • artefacts are produced
  • vacuum is required
  • black and white images produced (can be coloured digitally)
  • specimens have to be dead
17
Q

advantages of electron microscopes

A
  • over 500,000x magnification
  • resolving power of transmission electron microscope is 0.5nm and scanning electron microscope is 3-10nm
18
Q

transmission electron microscope

A
  • beam of electrons is transmitted through a specimen and focused to produce an image
  • best resolution and resolving power of 0.5nm
19
Q

scanning electron microscope

A
  • beam of electrons is sent across the surface of a specimen
  • reflected electrons are collected
  • resolving power is 3-10nm
  • 3d image of surfaces are produced
20
Q

advantages of light microscopes

A
  • inexpensive to buy and operate
  • easily available
  • small and portable
  • can be used out in the field
  • simple sample preparation
  • sample preparation does not usually lead to distortion
  • vacuum is not required
  • specimens can be living or dead
21
Q

disadvantages of light microscopes

A
  • only up to 2000x magnification
  • resolving power is 200nm
22
Q

what is an artefact

A
  • visible structural detail caused by processing the specimen and not a feature of the specimen
  • appear in both light and electron microscopy
  • bubbles trapped under cover slips are artefacts

Biology (12 decks)

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