3.2.1.3 Methods of studying cells Flashcards
Optical microscopes
Principles
-use light and several lenses to magnify a sample
Optical microscopes
Strengths
- can use living specimens
- cheaper
- simpler preparation
Optical microscopes
Limitations
-lower resolution
└as light has longer wavelengths
-lower magnification
EQ: Name two structures in a eukaryotic cell that cannot be identified using an optical microscope.
Mitochondrion/ribosome/endoplasmic reticulum/lysosome/cell-surface membrane
EQ: Maximum magnification of a light microscope
1,500
Optical microscope
Resolution
0.2 μm (micrometre)
Electron microscope
Resolution
0.0002 μm (micrometre)
Transmission electron microscopes
Principles
-electrons pass through a (thin) specimen
-denser parts absorb more electrons
└so appear darker
-electrons have a short wavelength
└=give high resolution
Transmission electron microscopes
Strengths
-higher resolution (than SEM and optical)
└as electrons have shorter wavelengths
└so can see organelles/internal structure
-allows cross section to be given
Transmission electron microscopes
Limitations
-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
EQ: Maximum magnification of a transmission electron microscope
500,000
Scanning electron microscopes
Principles
- 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
Scanning electron microscopes
Strengths
-higher resolution (than optical)
└as electrons have shorter wavelengths
-can have 3D images
-can be used on thicker specimens
Scanning electron microscopes
Limitations
- can only see external structure
- lower resolution (than TEM)
- specimens must be non-living
Magnification comparison
Highest
TEM
SEM
Light
Lowest
Resolution comparison
Highest
TEM
SEM
Light
Lowest
Why can’t the maximum resolution always be achieved?
- complex preparation process which can be difficult and affect the resolution possible
- high energy electron beams can sometimes damage the specimen
How temporary mounts are made
- 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
Temporary mount
definition
Where the specimen is suspended in a drop of liquid (e.g. water, oil) on the slide
Why is it important that sections being studied are thin
- so there is a single layer of cells
- so light can pass through
How would you use light microscope to calculate size of cells?
- 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
What is the eyepiece graticule?
- 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
How to calibrate eyepiece graticule
- calculate length of each division on eyepiece graticule with stage micrometre
- divide difference in magnification for scale
What is a stage micrometre?
- scale etched onto it
- 2mm long and smallest sub-divisions are 0.01mm (10um)
Magnification
Definition
How much bigger the image is than the specimen
-relates to size
Resolution
Definition
The ability to distinguish two objects that are close together as separate objects
-to see detail
Magnification formula
magnification = image size /actual size
I/AM
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
Cell fractionation
Stages
- preparation
- homogenation
- filtration
- ultracentrifugation
Cell fractionation
Process
The process where cells are broken up and the different organelles they contain are separated out
Filtration
Process
to remove large debris and whole cells
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)
Cell homogenisation
Definition
cells are broken up by a homogeniser to release the organelles from the cell
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
Cell fractionation
Why the solution must be cold
to reduce damage to organelles by enzymes
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
Cell fractionation
Why the solution must be buffered
to prevent proteins denaturing (e.g. enzymes) within organelles
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
Ultracentrifugation separation order
heaviest to lightest
nuclei → mitochondria/chloroplast → lysosomes → endoplasmic reticulum → ribosomes
Pellet
Definition
Solid at bottom (heaviest organelles)
Supernatant
Definition
Liquid left over (lighter organelles)
Uses of cell fractionation and ultracentrifugation
Detailed study of structure and function of organelles
-shows what isolated components do
Artefact
Definition
Things you can see down the microscope that aren’t part of the specimen e.g. dust, air bubbles
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
