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A level Biology xoyzq > Microscopy > Flashcards

Microscopy Flashcards

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1
Q

Describe what a microscope does

A

A microscope is an instrument which enables you to magnify an object thousands of times. It enables us to see unicellular objects.

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2
Q

Name 4 different types of microscope

A
  • light microscope
  • scanning electron microscope
  • transmission electron microscope
  • laser scanning confocal microscope
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3
Q

Main components of a light microscope (should be able to label one in the exam)

A 
eye piece lens
nose piece
coarse focusing knob
fine focusing knob
stage
light bulb
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4
Q

SEM stands for

A

scanning electron microscope

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5
Q

TEM stands for

A

transmission electron microscope

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6
Q

How does a SEM work?

A

A beam of electrons (wavelength < 1nm) is sent across the surface of a specimen and the reflected electrons are collected.

  • resolution not as good as TEM (3-10nm)
  • produces 3D images
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7
Q

How does a TEM work?

A

A beam of electrons (wavelength < 1nm) is transmitted through a specimen and focused to produce an image

  • very high resolution (0.5nm)
  • produces 2D images
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8
Q

How does a laser scanning confocal microscope work?

A

A higher intensity of light is used to illuminate a specimen that has been treated with a fluorescent dye.

A single spot of focused light is moved across a specimen, causing fluorescence from the components labelled with a dye. The light emitted from the specimen is filtered through a pinhole aperture. Only light radiated close to the focal plane is detected (unwanted radiation does not pass through the pinhole and so is not detected).

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9
Q

What is the definition of fluorescence?

A

The absorption and re-radiation of light

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10
Q

Why is a laser used instead of a light in laser scanning confocal microscopes? (1)

A

To get higher light intensities which improve the illumination

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11
Q

Explain the purpose of the pinhole aperture in confocal microscopy (3)

A
  1. only light radiated very close to the focal plane is detected
  2. unwanted radiation does not pass through the pinhole so is not detected
  3. this reduces blurring and improves the resolution of the image
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12
Q

Why can’t confocal microscopy be used for deep tissue imaging? (1)

A

The light penetration of the sample is limited as it is non invasive

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13
Q

Resolving power of LM, SEM and TEM

A

LM: 200nm (low!!)
SEM: 3-10nm
TEM: 0.1-0.5nm

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14
Q

What is the maximum useful magnification of LM, SEM and TEM?

A

LM: x1500
SEM: x500,000 - x2,000,000
TEM: x100,000 - x500,000

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15
Q

In light microscopy specimens are…

A

living or dead

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16
Q

In electron microscopy specimens are…

A

dead

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17
Q

Is sample preparation complex and does it lead to distortion in LMs and EMs?

A

LM: simple, does not usually cause distortion
EM: complex, often distorts material leading to the creation of artefacts

18
Q

Type of specimen stain used in LMs and EMs

A

LM: simple stains eg. iodine. Natural colouring is fine.
EM: heavy metal salts

19
Q

Explain how to use a light microscope to view a specimen at high and low powers (7 steps)

A
  1. Rotate nosepiece so that lowest power objective lens is in place
  2. Place specimen to be viewed onto stage and position so that specimen is in the centre of the field of view. Fasten with stage clips.
  3. Set up lamp to direct light beam through specimen on stage
  4. Move the coarse focusing knob so that the lower power objective lens is as close as possible to the stage
  5. Use eyepiece lens and focussing knobs to bring specimen into clear view by moving stage away from the lenses
  6. Rotate nosepiece to bring the medium power objective into position and adjust fine focus to view specimen clearly
  7. Repeat for higher power objective lenses
20
Q

Describe how to produce a temporary wet mount of living tissue

A

Place sample on a microscope slide
Add a drop of water
Cover with a coverslip placed on at an angle
Dab away excess water

21
Q

Explain why slide preparations need to be thin

A

So that light can pass through it. This means it can be seen by a light microscope

22
Q

Explain how to use a stage micrometer to work out the distance represented by the small divisions in an eyepiece graticule under 3 different objective lenses

A

.

23
Q

Resolution definition

A

The ability to see two objects (that are close together) as seperate objects

24
Q

Why are samples stained using a light microscope?

A

Dyes enable us to distinguish between organelles by increasing the contrast between them. Chemicals can bind to structures to allow them to be seen.

25
Q

Equation for calculating magnification

A

Magnification = Image size/ Actual size

26
Q

Magnification definition

A

The number of times bigger an object appears compared to its original/ real size

27
Q

How are samples prepared for electron microscopes?

A
  • in vacuum to ensure electrons travel in straight lines
  • fixation using chemicals/freezing
  • staining with heavy metals

TEM -> set in resin
SEM -> fractured inside and coated with heavy metals

28
Q

High resolution allows us to see the … of the cell

A

ultrastructure

29
Q

How many micrometres in a millimetre?

A

1mm = 1000um

30
Q

diameter of a lysosome (um)

A

0.5um

31
Q

length of a flagellum (um)

A

> 10um

32
Q

diameter of a centriole (um)

A

0.2um

33
Q

length of a cilia (um)

A

<10um

34
Q

length of a chloroplast (um)

A

4-10um

35
Q

diameter of a nucleus (um)

A

3-20um

36
Q

width of a plasma membrane (nm)

A

5-7nm

37
Q

width of a plant cell wall (um)

A

0.1um

38
Q

length of a mitochondrion (um)

A

2-5um

39
Q

Rules for biological drawings

A
  1. fill half the space provided
  2. sharp pencil to make sharp lines
  3. thin, single, unbroken lines
  4. no shading or colouring
  5. label lines drawn in pencil with no arrow heads
  6. annotate in pencil
  7. include scale line and magnification
40
Q

ratio of mm to um and nm

A

1mm : 1000um : 1,000,000nm

41
Q

Order of size of organelles

A 
nucleus
chloroplast
mitochondrion
lysosome
centriole
ribosome
42
Q

Explain the advantages and disadvantages of using a TEM to study cells (6)

A
  1. TEM uses beam of electrons
  2. which has a short wavelength
  3. Which allows a greater resolution/ more detail to be seen/ greater useful magnification
  4. Electrons scattered (by molecules in air)
  5. vacuum established

Limitations

  1. Cannot examine living specimens
  2. Many procedures used in preparing specimens eg. fixing/staining/sectioning
  3. May result in artifacts

A level Biology xoyzq (33 decks)

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