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BIOL 112 > 2 - Imaging Cells > Flashcards

2 - Imaging Cells Flashcards

1
Q

What was important in the release of Hooke’s essays in 1665?

A

He gave a detailed description of a section of cork as being a honeycomb of chambers or cells.

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2
Q

What was significant about Leeuwenhoek’s work in 1675?

A

His microscopes could resolve to about 1.5 microns. He reported ‘a host of little animals’ in a drop of rain water, which was protozoa.

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3
Q

What important events happened in the 19th century for microscopes?

A

The maximum theoretical resolution of the light microscope was attained (0.25 microns)

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4
Q

What important event happened in the 1930s for microscopes?

A

The electron microscope was developed = cell organelles could be seen.

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5
Q

Ultrastructure

A

The level of detail obtainable with the electron microscope.

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6
Q

What are the 3 important parameters of microscopy?

A

magnification, resolution, contrast

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7
Q

Magnification

A

The ratio of an object’s image size to its real size.

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8
Q

Resolution

A

The measure of the clarity of the image or the minimum distance of two distinguishable points.

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9
Q

Contrast

A

Visible differences in brightness or colour between parts of the sample.

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10
Q

Light microscope

A

The light passes through the specimen, which must be at least partially transparent.
Stained to enhance contrast and must be fixed.

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11
Q

Advantages of light microscopes

A

The ability to image living cells.

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12
Q

Disadvantages of light microscopes

A

Limited resolution (0.2 microns)

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13
Q

What is the only way to improve resolution of a microscope?

A

Use a shorter wavelength of radiation.

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14
Q

Fluorescence microscopy

A

Shows the location of specific molecules in the cell by absorbing short wavelength, ultraviolet radiation and emit longer wavelength, visible light. Usually by tagging molecules.

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15
Q

phase contrast light microscopy

A

Enhances contrast in unstained cells by amplifying variations in density within specimen.
Useful for living unpigmented cells.

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16
Q

Differential-interference contrast light microscopy (Nomarski)

A

Uses optical modifications to exaggerate differences in density.

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17
Q

Confocal Light Microscopy

A

Uses lasers and special optics for ‘optical sectioning’.
Narrow depth are imaged.
Above and below plane of view = black rather than blurry.
Usually fluorescently stained.

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18
Q

Sample preparation for light microscopes

A
  • Whole mounts
  • Tissue sections
  • Fixation
  • Dehydration and clearing
  • Embedding
  • Sectioning
  • Staining
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19
Q

Whole mounts in LM

A

Small relatively transparent specimens can be mounted directly onto slides.

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20
Q

Tissue sections in LM

A

Most tissues need to be sectioned before they can be examined.

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21
Q

Fixation in LM

A

using chemical fixatives to prevent cell autolysis and to preserve the structure of the tissue.

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22
Q

Dehydration and clearing in LM

A

Removes water from tissue in preparation for wax impregnation.

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23
Q

Embedding in LM

A

Specimen is infiltrated with molten wax after which it’s transferred to a mould.

24
Q

Sectioning in LM

A

Thin sections approx 5 microns thick are cut on a microtome and collected onto a glass slide.

25
Q

Staining in LM

A

Wax removed and tissue stained with coloured dye

26
Q

Eosin

A

Coloured dye used for the cytoplasm

27
Q

Haematoxylin

A

A coloured dye used for nuclei.

28
Q

Confocal scanning microscopy 3D

A

Generated 3D images of living cells.

Removes out of focus images by optical sectioning and can look inside thick specimens e.g. embryos.

29
Q

Deconvolution microscopy

A

Algorithms remove out of focus light and sharpens the image to improve resolution

30
Q

How is the resolution limit broken?

A

Super resolution gathers light from individual fluorescent molecules and records their position, combining these breaks the limit.

31
Q

Who developed electron microscopes?

A

Ruska and Knoll

32
Q

What is the theoretical limit of electron microscopes?

A

~0.002nm

33
Q

Why is the electron microscope kept under vacuum?

A

Electrons have poor penetrating power.

34
Q

What can electrons be focused by in the electron microscope?

A

Magnetic fields

35
Q

Electron gun

A

Heated tungsten filament which produces electrons by thermionic emission

36
Q

How do transmission electron microscopes work?

A

Electrons from electron gun > passes through specimen > focused and magnified.

37
Q

How is the image from a TEM focused and magnified?

A

By magnetic objective and projector lenses.

38
Q

What happens to the electron image after it is focused and magnified?

A

Converted into a visible image by a fluorescent screen, which is viewed through a glass window.
Photos taken with digital camera.

39
Q

Sample preparation for TEM

A
  • Whole mounts
  • Fixation
  • Dehydration
  • Embedding
  • Sectioning
  • Staining
40
Q

How can whole mounts be used for a TEM?

A

Bacteria and viruses can be examined directly as well as tissue sections.

41
Q

Fixation in TEM

A

Glutaraldehyde for protein crosslinking and then osmium tetroxide for lipid crosslinking.

42
Q

Dehydration in TEM

A

In an ethanol series

43
Q

Embedding in TEM

A

Embedded in plastic resins such as epoxy resins.

44
Q

Sectioning in TEM

A

50nm thick sections cut using ultramicrotome.

45
Q

Staining in TEM

A

Biological tissue has little contrast under the electron beam, heavy metal stains such as lead are used to improve contrast.

46
Q

X-ray cystallography

A

X-rays scatter as they pass through a crystallized protein, resulting in waves that interfere with each other, creating a diffraction pattern which shows the position of atoms.

47
Q

Cryo-electron microscopy

A

A beat of electron is fired at a frozen protein solution, the emerging scattered electrons pass through a lens to create a magnified image on the detector, allows structure to be found.

48
Q

Scanning electron microscope

A

Electron beam scanned across specimen, used for looking at the surface of specimens.

49
Q

How does the SEM work?

A

Electrons are reflected from the surface of the specimen, collected by a electron detector and converted into an electronic signal, which is displayed on a screen.

50
Q

Sample preparation for SEM

A
  • Fixation
  • Dehydration
  • Critical point drying
  • Coating
51
Q

Fixation in SEM

A

The same as TEM

52
Q

Dehydration in SEM

A

Water is replaced with ethanol.

53
Q

Critical Point Drying

A

Allows all of the ethanol to be removed from the sample in a way that minimises shrinkage.

54
Q

Coating

A

Specimens coated with a thin layer of gold to protect them from electron beam damage.

55
Q

Cell fractionation

A

Allows major organelles to be individually separated out so they can be studied in isolation.

56
Q

What must happen to the cells before the organelles can be released?

A

Must be homogenised.

57
Q

Differential centrifugation

A

Isolates cell components on the basis of size and density by using increasing durations and g forces.

BIOL 112 (12 decks)

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