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Biology > Module 2: Microscopy. > Flashcards

Module 2: Microscopy. Flashcards

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1
Q

What is the formula for magnification?

A

Magnification = image size / real size.

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2
Q

millimeter to micrometer?

A

x1000

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3
Q

micrometer to nanometer?

A

x1000

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4
Q

Micrometer to millimeter?

A

divide by 1000.

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5
Q

Nanometer to micrometer?

A

Divide by 1000.

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6
Q

Define Magnification.

A

How much bigger the image is than the actual specimen.

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7
Q

Define Resolution.

A

How detailed the image is.

How well a microscope distinguishes between 2 points that are close together.

(the smaller the number the higher the resolving power)

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8
Q

What is the Maximum Magnification for Compound light microscope?

A

x1500

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9
Q

What is the Maximum Resolution for compound light microscope?

A

0.2 micrometers (200nm)

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10
Q

Describe the compound Light Microscope.

A

A light source shines through the specimen. (This is often below the specimen).

The objective lens produces a magnified image that is then magnified again by the eyepiece.

Resolution is limited by by the wavelength of light and diffraction of light as it passes through the sample.

The images tend to have low contrast as most cells do not absorb a lot of light. This is why cells are often stained to increase contrast.

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11
Q

What are the advantages of Compound Light Microscope?

A

It is inexpensive.

It is portable and it does not require a lot of time to prepare.

Specimens can be living or dead.

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12
Q

What are disadvantages of Compound Light Microscope?

A

It has a low magnification and resolution compared to the Electron microscope.

Internal structures cannot be viewed.

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13
Q

What is the Maximum Magnification for Laser Scanning Confocal Microscope?

A

x2000

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14
Q

What is the maximum Resolution for Laser Scanning confocal Microscope?

A

0.2 micrometers (200nm)

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15
Q

Describe Laser Scanning Confocal Microscope.

A

A laser beam is focused through a lens which is aimed at a beam splitter. This splits the beam and some of the light is directed to the specimen.

When the laser hits the dyes it causes them to give off fluorescent light. This light is then focused through a pinhole onto a detector.

The detector is hooked up to a computer, which generates an image. The Pinhole means that any out of focus light is blocked.

Very thin sections can be examined and a high resolution can be produced.

A 3D image can be produced by creating images at different focal planes.

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16
Q

What are the Advantages of Laser Scanning Confocal Microscope?

A

Simple sample Preparation.

Specimens can be living or Dead.

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17
Q

What are the disadvantages of laser Scanning Confocal Microscope?

A

Expensive to buy.

Has a low magnification and resolution compared to the electron Microscope.

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18
Q

What is the Maximum Magnification for TEM?

A

magnification over x100 000

19
Q

What is the Maximum Resolution for TEM?

A

0.0005 micrometers (0.5nm)

20
Q

Describe Transmission Electron Microscope.

A

TEM’s uses electromagnets to focus a beam of electrons, which is then transmitted through a specimen to produce 3D images.

Denser parts of the specimen absorb more electrons, which makes them look darker on the image you end up with.

The specimen is kept in a vacuum to ensure the electron beams travel in a straight line.

21
Q

What are the Advantages of TEM?

A

It has a high resolution and magnification.

Can view internal structures.

Need to be thinly sliced. The angle at which specimens are cute can affect how they appear.

22
Q

What are the disadvantages of TEM?

A

Expensive to buy and operate.

Large and needs to be installed.

Time consuming.

Complex sample preparation.

Risk of artefacts.

Specimens are dead.

Black and white images are produced.

23
Q

What is the Maximum Magnification for SEM?

A

Magnification over x500 000

24
Q

What is the Maximum resolution for SEM?

A

0.002 micrometers (2nm)

25
Q

Describe Scanning Electron Microscope.

A

SEM’s scan a beam of electrons across a specimen.

This knocks off electrons from the specimen, which are gathered in a cathode ray tube to form an image.

A beam of electrons are sent across the surface of a specimen and the reflected electrons are collected.

3D images of the surface are produced.

26
Q

What are the Advantages of SEM?

A

It has a high resolution and magnification.

27
Q

What are the disadvantages of SEM?

A

Expensive to buy and operate.

Large and needs to be installed.

Time consuming.

Complex sample preparation.

Risk of artefacts.

Specimens are dead.

Black and white images are produced.

28
Q

What is an eyepiece graticule?

A

A small ruler that can be fitted to a microscope allowing structures to be measured under the microscope.

29
Q

What is a stage graticule? (stage micrometer)

A

It is a microscope slide with an accurate measuring scale.

This can be used to calibrate the value of the eyepiece divisions at different magnifications.

30
Q

Method for Calibrating the graticule?

A
  1. Set up the Microscope to the required magnification to view the sample.
  2. Place the stage graticule onto the stage.
  3. Line up the two scales (the eyepiece and stage graticule)
  4. Count the number of divisions on the eyepiece graticule equivalent to each division on the stage micrometer.
  5. 1 division on stage micrometer divide by how much divisions on eyepiece graticule.
31
Q

Why do samples need to stained for Light Microscope?

A

Coloured dye binds to structures.

Helps visualise structures better.

32
Q

Define Differential staining.

A

When multiple stains are used.

Each stain binds to a specific cell structure which stains each structure differently so that the structures can be easily identified.

33
Q

Stain: Acetic Orcein.

A

Binds to DNA and stains chromosomes dark red

34
Q

Stain: Eosin.

A

Stains cytoplasm dark red or pink

35
Q

Stain: Iodine.

A

Iodine stains starch blue-black (appears
violet under the microscope)

36
Q

Stain: Iodine in Potassium Iodide solution.

A

Stains cellulose yellow

37
Q

Stain Hematoxylin.

A

Stains RNA/DNA a purple/blue colour

38
Q

Stain: Methylene blue.

A

Is an all-purpose stain, used often to stain DNA blue

39
Q

What are the 4 sample preparations?

A

Wet Mount

Dry Mount

Smear slide

Squash slide

40
Q

Describe Dry Mount.

A
  1. Slice the specimen into a thin piece so that light can pass through.
  2. Use tweezers to pick it up and place it onto a clean slide.
  3. Put a cover slip on top of it.
41
Q

What specimens is Dry Mount used for?

A

Specimens such as hairs

parts of insects

pollen

and parts of flowers.

42
Q

Describe Wet mount.

A
  1. Use a pipette to put a drop of water onto the slide. Then use tweezers to place your specimen on top of the water drop.
  2. Put the coverslip on by standing it upright on the slide, next to the water droplet, then carefully tilt it down onto the specimen. (try to avoid air bubbles as it will obstruct your view of the specimen).
  3. Add a stain. Put one drop on one edge of the cover slip and put a paper towel on the opposite edge.

The paper will absorb the stain, drawing it under the cover slip, staining the specimen.

43
Q

Describe how to use a Compound Light Microscope.

A
  1. Clip the slide onto the stage and select the lowest objective lens.
  2. Use the Coarse adjustment knob to move the objective lens to just above the slide.
  3. Look down at the Eyepiece and adjust the focus by moving the lens away from the slide using the fine adjustment knob until a clear image appears.
  4. If a higher magnification is required, swap to a higher powered objective lens and refocus;.
44
Q

How do you determine the actual length of a Specimen?

A

Actual length = Number of divisions x length of 1 division.

Biology (17 decks)

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