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3.2 Cells > 3.2.1.3 Methods of studying cells > Flashcards

3.2.1.3 Methods of studying cells Flashcards

1
Q

Optical microscopes

Principles

A

-use light and several lenses to magnify a sample

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2
Q

Optical microscopes

Strengths

A
  • can use living specimens
  • cheaper
  • simpler preparation
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3
Q

Optical microscopes

Limitations

A

-lower resolution
└as light has longer wavelengths
-lower magnification

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4
Q

EQ: Name two structures in a eukaryotic cell that cannot be identified using an optical microscope.

A

Mitochondrion/ribosome/endoplasmic reticulum/lysosome/cell-surface membrane

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5
Q

EQ: Maximum magnification of a light microscope

A

1,500

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6
Q

Optical microscope

Resolution

A

0.2 μm (micrometre)

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7
Q

Electron microscope

Resolution

A

0.0002 μm (micrometre)

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8
Q

Transmission electron microscopes

Principles

A

-electrons pass through a (thin) specimen
-denser parts absorb more electrons
└so appear darker
-electrons have a short wavelength
└=give high resolution

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9
Q

Transmission electron microscopes

Strengths

A

-higher resolution (than SEM and optical)
└as electrons have shorter wavelengths
└so can see organelles/internal structure
-allows cross section to be given

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10
Q

Transmission electron microscopes

Limitations

A 
-cannot look at living material
└as must be in a vacuum
 -specimen must be very thin
-artefacts are present
-long, complex staining method and preparation time 
-only 2D images are produced
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11
Q

EQ: Maximum magnification of a transmission electron microscope

A

500,000

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12
Q

Scanning electron microscopes

Principles

A
  • they scan a beam of electrons across the specimen
  • this knocks off electrons from the specimen, which are gathered in a cathode ray tube to form an image
  • shows surface of the specimen and can be 3D
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13
Q

Scanning electron microscopes

Strengths

A

-higher resolution (than optical)
└as electrons have shorter wavelengths
-can have 3D images
-can be used on thicker specimens

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14
Q

Scanning electron microscopes

Limitations

A
  • can only see external structure
  • lower resolution (than TEM)
  • specimens must be non-living
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15
Q

Magnification comparison

A

Highest

TEM
SEM
Light

Lowest

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16
Q

Resolution comparison

A

Highest

TEM
SEM
Light

Lowest

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17
Q

Why can’t the maximum resolution always be achieved?

A
  • complex preparation process which can be difficult and affect the resolution possible
  • high energy electron beams can sometimes damage the specimen
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18
Q

How temporary mounts are made

A
  • add water, a solution or a stain (e.g. iodine for starch, eosin for cytoplasm) to the slide
  • take a thin slice
  • put on a slide
  • lower cover slip using mounted needle
19
Q

Temporary mount

definition

A

Where the specimen is suspended in a drop of liquid (e.g. water, oil) on the slide

20
Q

Why is it important that sections being studied are thin

A
  • so there is a single layer of cells

- so light can pass through

21
Q

How would you use light microscope to calculate size of cells?

A
  • eyepiece graticule fitted to microscope eyepiece
  • stage micrometre placed on the stage
  • used to work out the divisions of the eyepiece
  • use graticule divisions to work out length of the cell
22
Q

What is the eyepiece graticule?

A
  • glass disk with scale etched on
  • placed in the eyepiece of a microscope
  • can calibrate with particular objective lens

-scale typically 100mm one and divided into 100 sections

23
Q

How to calibrate eyepiece graticule

A
  • calculate length of each division on eyepiece graticule with stage micrometre
  • divide difference in magnification for scale
24
Q

What is a stage micrometre?

A
  • scale etched onto it

- 2mm long and smallest sub-divisions are 0.01mm (10um)

25
Magnification | Definition
How much bigger the image is than the specimen | -relates to size
26
Resolution | Definition
The ability to distinguish two objects that are close together as separate objects -to see detail
27
Magnification formula
magnification = image size /actual size | I/AM
28
Put these in order: nanometres, micrometres and metres | How you convert between them
1 metre = 1000mm 1 mm = 1000 micrometres 1 micrometre = 1000 nanometres nm
29
Cell fractionation | Stages
- preparation - homogenation - filtration - ultracentrifugation
30
Cell fractionation | Process
The process where cells are broken up and the different organelles they contain are separated out
31
Filtration | Process
to remove large debris and whole cells
32
Cell fractionation | Principles
-cell homogenisation- break open cells and release contents -filter to remove large debris and whole cells -keep in cold, isotonic, buffered solution └cold: to reduce damage by enzymes └isotonic: to prevent damage to organelles (e.g. mitochondria) by osmosis as there is no movement of water └buffered: to prevent proteins denaturing (e.g. enzymes)
33
Cell homogenisation | Definition
cells are broken up by a homogeniser to release the organelles from the cell
34
Methods of homogenisation
- break cells with high frequency sound - mild detergent makes holes in plasma membrane - force cells through small hole at high pressure - blending
35
Cell fractionation | Why the solution must be cold
to reduce damage to organelles by enzymes
36
Cell fractionation | Why the solution must be isotonic
to prevent damage to organelles (e.g. mitochondria) as there is no net movement of water by osmosis as the water potential is the same
37
Cell fractionation | Why the solution must be buffered
to prevent proteins denaturing (e.g. enzymes) within organelles
38
Cell ultracentrifugation | Principles
-centrifuge at lower speed └separates heavy organelles (e.g. nuclei) -remove supernatant -re-spin supernatant at higher speed -repeat until all organelles are separated out
39
Ultracentrifugation separation order | heaviest to lightest
nuclei → mitochondria/chloroplast → lysosomes → endoplasmic reticulum → ribosomes
40
Pellet | Definition
Solid at bottom (heaviest organelles)
41
Supernatant | Definition
Liquid left over (lighter organelles)
42
Uses of cell fractionation and ultracentrifugation
Detailed study of structure and function of organelles | -shows what isolated components do
43
Artefact | Definition
Things you can see down the microscope that aren't part of the specimen e.g. dust, air bubbles
44
How did the first scientists using electron microscopes distinguish between artefacts and cell organelles?
- by repeatedly preparing specimens in different ways | - if an object could be seen with one preparation technique, but not another, then it was likely an artefact

3.2 Cells (8 decks)

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