cell structure - studying cells

light microscopes

beam of light rays with a long wavelength - results in lower resolution

simple preparation of specimen (unlikely to distort cell structure)

resolution: 0.2micro meters

image: viewed by light passing through specimen

scanning electron microscope (SEM)

uses a beam of electrons with short wavelengths which can be focused with electromagnets

resolution: 20nm

image: viewed by electrons being reflected off metal coated specimen

cannot be used on live specimen

transmission electron microscope (TEM)

uses a beam of electrons with short wavelengths (results in higher resolution) which can be focused with electromagnets

photomicrograph appears dark where electrons absorbed (denser appears darker)

resolution: 0.1nm

image: viewed by electrons passing through thin specimen sections

no live specimen

advantages of SEM

can be used to view a 3D image of specimen surface

higher resolution than light microscope

disadvantages of SEM

• lower resolution than TEM

• requires a vacuum (or electrons absorbed by air molecules)

• no live specimens (contain no water)

advantages of TEM

can view parts of the cell at high magnification

higher resolution than light microscope and SEM so can view inside organelles

disadvantages of TEM

needs a vacuum (or electrons absorbed by air molecules)

no living specimens (contain no water)

2D image

requires a very thin specimen

complex staining process

image may contain artefacts

magnification

the number of times an object is enlarged

resolution

the minimum distance between two points on a specimen that can still be distinguished by the observer or camera system as separate entities

contrast

the difference in shade between the lightest and darkest parts of an image

what is cell fractionation?

the process where cells are broken up and their organelles are separated

it occurs in two steps: homogenisation and ultracentrifugation

cell fractionation: why is the tissue first placed in a cold, buffered, isotonic solution?

cold: to reduce the enzyme activity, which may break down organelles

buffered: to maintain a constant pH, so enzymes are not denatured

isotonic: the water potential of the solution is the same as inside the cell to stop organelles bursting

describe what happens during homogenisation

cells are broken open by a homogeniser (blender), to release organelles from the cells

this creates a fluid ‘homogenate’ - fluid containing the organelles, cell membranes and cells

explain why the scientists homogenised the tissue before carrying out cell fractionation to isolate organelle G.

to break open the cells so that the organelles can be free floating in the tube

scientists homogenised pancreatic tissue before carrying out cell fractionation to isolate organelle G. Explain why the scientists filtered the resulting suspension.

to remove all of the whole cells so they can be placed back into the homogeniser

describe the process of ultracentrifugation

the filtered homogenate is spun in the ultracentrifuge to separate out the fragments at a very high speed creating a centrifuge force

the densest organelles form a sediment pellet at the bottom

the fluid above the pellet is called the supernatant

supernatant transferred into another tube where it can now be spun faster to create a new pellet

organelles in order of density (most to least)

1. nucleus

2. mitochondria / chloroplast

3. ribosomes

advantage of using light microscopes

can be used on live specimen

disadvantages of light microscopes

- lower resolution than SEM and TEM
- low magnification (max about x1500)
- can only see a little detail

preparing a slide

add a drop of water to a glass slide

obtain a thin section of the sample, or a single layer of cells, and place on the water droplet

add a stain to create contrast (specify which)

slowly lower the cover slip using a mounted needle, to avoid air bubbles

blot any excess stain/water using blotting paper or tissue

micrometry

the measuring of cells and structures seen with a light microscope

what two pieces of equipment are needed to make a measurement?

1. an eyepiece graticule

2. a stage micrometer

eyepiece graticule

a piece of glass inscribed with regular, numbered divisions

the eyepiece is unscrewed and the graticule is dropped into it

the divisions are arbitrary eye piece units (epu)

this scale can be lined up alongside cells to allow comparative lengths to be taken

stage micrometer

a precision scale, in micrometers, which is used to assign a value to the graticule scale

the scale is (usually) 1mm wide with 10 large divisions of 0.1mm

different magnifications

- at different magnifications the eyepiece graticule stays the same, but the stage (and stage micrometer) is enlarged
- therefore it needs to be calibrated

calibration

the micrometer is placed on the stage and its scale is lined up with the scale of the graticule

how would you find the average width of the nuclei in plant cells?

- calibrate the eyepiece graticule with the stage micrometer
- measure the width of a nucleus in eyepiece units
- convert this to micrometers using your calibration
- measure more nuclei
- calculate the mean average

name 3 structures in a eukaryotic cell that can’t be identified using an optical microscope

mitochondrion, ribosome, endoplasmic reticulum