methods of studying cells

Definition of magnification + equation

number of times larger an image appears to be compared to its actual size

magnification = image size / actual size

Definition of resolution

the ability to distinguish between 2 separate points

the minimum distance apart 2 objects can be in order for them to appear as seperate objects.

What are the 2 types of microscopes

1. optical microscope (light microscope)

2. electron microscope

Compare optical and electron microscopes

optical microscopes	electron microscope
image formed by light	image formed by electron beams
lower resolution than electron microscope (200nm/0.2μm)	higher resolution than optical microscope
lower magnification than electron microscopes (X1500)	higher magnification than optical microscopes (X1500,000)
colour image	cant produce colour images
can view living cells	can’t view living cells
observe nucleus (eukaryotic cells) + mitochondria + chloroplast (plants)	observe small organelles and virus cells eg ribosomes, endoplasmic reticulum, golgi apparatus, lysosomes, cell membranes
use glass lenses to focus light on the specimen	use condenser lenses to focus electron beams on the specimen

Student cut thin sections of tissue to view with an optical microscope.

Explain why it was important that the sections were thin

Allow more light through and better stain penetration

Explain why electron microscopes are able to resolve objects better than light microscopes

resolution in optical microscope determined by wavelength of light

resolution of electron microscope determined by wavelength of beam of electrons

electrons have a shorter wavelength than light → high resolving power

How do transmission electron microscopes (TEM) work

use electron gun that produces a beam of electrons focused on the specimen by a condenser electromagnet

this beam of electrons is transmitted through the specimen

• denser parts of specimen absorb more electrons

• denser part → darker image

How do Scanning electron microscopes (SEM) work

beams of electrons scattered across specimen

electrons knocked off the specimen are gathered in the cathode ray tube to form image

Compare SEM and TEM

TEM	SEM
higher resolution than SEM	lower resolution than TEM
no colour image	artificial colour can be added
2 dimensional image	3 dimensional image (shows surfaces of specimen)
very thin specimen	can be used w thick specimen

• both cannot observe live specimen → vacuum inside TEM and SEM → specimen must be dead

• lengthy preperation of specimen (including staining) means artefacts can be introduced

Definition of artefacts

visible details that arent part of the specimen eg: dust, fingerprints, air bubbles

results of preparing

occurance of artefacts decreased by more careful preparation of specimen

Explain why specimens have to be kept in a near-vacuum in order to be viewed effectively using an electron microscope

live specimens cannot be viewed under electron microscope because the entire process takes place in a vacuum

This is because electrons are absorbed or deflected by molecules in air and this would prevent them from reaching the specimen

Explain why an electron beam can be focused more precisely than a light beam

Electrons are negatively charged so can be focused using condenser electromagnets, whereas light has no charge

In practice, the theoretical resolving power of an electron microscope cannot always be achieved. Explain why not

1. complex preparation of specimen may result in artefacts eg air bubbles, fingerprints and dust particles.

2. a high energy electron beam is required → may destroy the specimen.

Definition of cell fractionation + steps

sepearting organelles from rest of cell

• homogenisation

• filteration

• ultracentrifugation

What happens in homogenisation

breaking up of cells

• blend/vibrate cells (in homogeniser)

• breaks the plasma membrane of the cells and releases the organelles in a solution called homogenate

How must the solution be before putting the cells in a homogeniser

• ice cold to reduce the acticity of enzymes that breaks down/ digest organelles

• isotonic (same water potential as cells being broken up) to prevnt cells bursting due to movement of water by osmosis

• buffered to maintain constant pH to prevent proteins eg: enzymes from becoming denatures

ice-cold isotonic buffered solution

What happens in filteration

• homogenate is filtered w sieve/gauze to remove the large cell or tissue debris that were not broken up

• organelles are small → pass through sieve/gauze

• leaves filterate containing mixture of organelles

What happens in ultracentrifugation

stage where organelles are seperated

• filterate placed into tube → tube placed in a centrifuge

• filterate spun low speed

• heaviest organelles (eg nuclei) settle to bottom →form thick sediment called pellet

• rest of organelles stay suspended in solution above pellet called supernatant (liquid)

• supernatant drained off + placed into another tube + spun at higher speed

• process repeated at increasing speeds until diff organelles/ desired organelle is seperated out

What is the order of mass of organelles

• nuclei

• (chloroplast) → plant tissue

• mitochondria

• lysosomes

• endoplasmic reticulum

• ribosomes