Remember, Efficiency

of a
MAGNIFICATION
→What factors determine the magnification of a
microscope?
ocpm
erci i s assessed b y
ehT magnification of a microscope si determined by several
its M agnification Rdna
,soractf ngudinlci het type of enls used, het distance between
esolution
Magnification
het lens and het object, and het ezis fohet eyepiece.
Additionally, eht type fomicroscope being used, such as a Magnification
impact
compound microscope
eht magnification.
ro a electron microscope, can also
86 fpeci me n o f Drawing
To calculate magnification

Eyepiece at 10x

WHERE: | - M
I AGE / D
R
AW
NIG E
ZIS
A - ACTUAL Z
SIE FO M
IA
G
E
Objective lens at 4x M- MAGNIFICATION
A
eyepiece x objective lens = magnification
M =
(10x) x(4x) = 40x
Calculate the onitacm
ifgani of t h e esanvsert section o f t h e leaf edalb
Using a scale bar
• fI the calculation required includes a scale bar on hte micrograph or drawing neht
follow these steps:
.1 Use aruler to measure the length of the scale bar ni millimetres m
( m)
.2 Convert this measurement into the same units as the number no eht scale bar
.3 Insert these numbers into the magnification formula above (note: eht ezis o f
the image is the measured length of the scale bar and the actual sizeis the
number on the sceal bar)

Before doing any calculations make suer that lla the measurements have the same
units. When doing the calculations ti si easier to write the formula, then rearrange ,ti
before you add any measurements, as this helps avoid any possible errors. Note that
002 pm
when you do calculations using a scale bar, the number on the scale bar is informing
o) Calculate the lenghest
m e s o p palisade
hull you how many mm/um or m
n het line actually represents ge(. if the scael bar has 20
nm above it and het neil si 10 mm, then every 01 mm on the diagram si actually 02nm)
cauletl the magnification fo these scale bars:

67um
Diagram to show conversion of units
ujm
TO CONNVERT m →
100nm 100um
× 1000 × 1000 × 1000

1 m = 1000 m 1 mm 1000 m
u 1um = 1000nm
m
H05
+1000) + 1000 + 1000

1500m Remember -rof

the image is
ca'clualet eht
irrelevant sa
magconiafnti'
gnol a s you have
on,siqutes
ascale bar
TO CONNVERT um → m

1
Resolution Resolution =
Resolution of an image is a measure of detail. Resolution is a
numerical measure of how detailed and clear an image s.i

Resolution of a microscope is its ability to distinguish two

•

close points. Resolution si a measure of the microscope's

ability to distinguish between two points which are close
together on an object. fI a microscope can clearly distinguish
between the two points, ti has a good or high resolution.

• Resolution is half of the wavelength of radiation being used for example the wavelength of light
microscope ranges from 400 um to 700 um so the resolution of light microscope is 200 um

> Floatma M i a u m e r a
 V I R O P A
Light microscope TEM SEM

Light TEM SEM

Microscope Electron scanning
Microscope electron
Transmission
Source of Radiation Visible light Beam of electrons Beam of electrons

How it Works Light passes through Electrons pass through Electrons scan tools
or reflects from an ultra-thin specimen

Wavelength -400-700 nm 0.005 nm 0 . 0 6 nm

Resolution ~200 nm 0•5 nm 20 nm

斧
Lenses Glass lenses Electromagnetic lenses Electromagnetic lenses
Specimen Living or fixed, thir Ultra-thin, dead, fixed Usually coated with
Requirement sections optional specimen metal, dead
True Color Yes, natural color seen No, black and white: No, black and white;
false color can be added false color can be added
Stains/Dyes Required ot enhance
contrast Heavy metal stains Conductive coating

Type of Image Up to ~2500x Up to -2500,00 x

dl orplatinume
usUupalyto go~100,000x

Methods of studying cells

There are many methods for studying cells and one way ni which this is done si through the use
of microscopes of which there are two main types.
Light Microscopes
Light microscopes use a pair of convex glass lenses that can resolve images that arem
u2.0
apart. The reason for this si that this si wavelnght of ghtil and therefore restricts the
resolution that a light microscope resolve to. This si compared ot electron microscopes which
can distinguish between items 0.5nm apart.
The magnification of an image as seen through a microscope can be calculated using the
following equation:
Magnification = size o f m
i agezsile o f ealr object
Resolution si defined as the minimum distance apart that two objects can be distinguished as
separate objects ni an image. The greater the resolution the more clear the image will be.

Electron Microscopes
The limitation of light microscopes only resolving to a resolution of 0.2um means that electron
microscopes can be used to look at objects that are closer than 0.2um apart. There are two
nain types of electron microscope, these are transmission onrtcele microscopes (TEM) anc
gninacs electron m
ocspresi (SEM). Electron microscopes work ni a similar way to ligh
microscopes, but instead use a beam of electrons that are focused by electromagnets inside a
vacuum environment. Them
uucav environment is needed so that particles ni the air do not
deflect the electrons out of the beam alignment.
The following details how each type of electron microscope works:
.1 Transmission oE
rnectl Microscope - a beam of electrons passes through a thin section of a
specimen. Areas that absorb the electrons appear darker on the electron micrograph that is
produced.

.2 Scangi Electron Microscope - ni a scanning electron microscope a beam of electrons passes

3 image depending on
across the surface and scatter. The pattern of scattering builds up a D
the contours of the specimen.
There are some limitations though when using electron microscopes, the limitations for SEM and
TEM a r e :

- The whole system must be in a vacuum so living specimens cannot b e [Link]

- Ac o m p l e x s t a i n i n g p r o c e s s is required which may introduce a r t e f a c t s into the image.

- Specimens have to be very n,hit particularly for TEM so that the electrons can pass through.
• SEM has a lower resolving power than TEM, but both have greater resolving power than a light
microscope.

Disadvantages of Electron Microscope: Advantages of Light Microscope:

(1) High Running Cost (1) Living specimen
(2) Expensive (2) True Colors
(3) Only trained professionals can operate.
1 . 1.
(3)、Easy
.
to use
 14/ u n y nenn s p e c e n c u n de viewen (4) cheaper
( 5 ) T r u e C o l o r c a n n o t be seen. (5) Low Running Costs
'Advantages of Electron Microscope: Disadvantages of Light Microscope:
(L) Hign Resolution and High Magnification Lower Resolution + Magnification.

A few questions:
Q:What happens ot the resolution and magnification wavelength being used ni hte microscope changes
from 500 u m to 400 nm?
A: resolution increases, magnification remains unaffected.
Rils there any effect of wavelength no resolution?
: yes resolution si half of the wavelength.
A
RiDoes magnification afect resolution?
Aino there is no effect ofmagnification on resolution until the magnification remains working (the
working magnification of light microscope is X1500. Beyond X1500 the clarity of the image or
resolution decreases, resolution gets lower.
Q:Does resolution affect magnification?

Cel Fractionation and Ultracentrifugation unit for rate of sedimentatio = SvedbergS eg

Cell fractionation is the process in which different parts and organelles of a cell a separated so 705 ribosomes,
that they can be studied in detail. The most common method of cell fractionation is differential
centrifugation.
805 ribosomes
The process of homogenation is detailed below: The largeet
.1 The cells are first blended in an homogeniser forming the resultant fluid called the
organelle has
tnaogeThis tube of homogenate is then placed in a
.ehom cenufirtge and spun at a slow the highest
speed.
sadimentation;
2. The heaviest organelles, the nuclei, are forced to the bottom of the tube where a thin h e n c e t. h e
sediment or pellet forms. highest ' S '
3. The fluid at the top, called the supernatant, is removed which leaves just the sediment of value.
the nuclei. The supernatant is then transferred to another tube and spun at a slightly faster
speed. This time the pellet that forms contains the next heaviest organelle, the

4. This process continues so that each time the speed is increased the next heaviest organelle is
sedimented and separated out.
The homogenate at the beginning is placed ni a cold, buffered solution o f t h e s a m e water
potential as the cells. This is to prevent the organelles from bursting under osmotic pressure,
t o inactivate any enzymesm orf breaking down organelles a n d s o t h a t t h e p H does n o t
fluctuate.

Homogenization Differential centrifugation

800 g
10 min
200,00 g ,
15 min
/100,600009
min
, 1500,300hr g,
Supernatant
Pellet rich ni peleronie
nuclei and
cellular debris it cells an membranes and ribo
Tissue Homogenate somes
cells t r o m n i n n i

m e m o r a n e s

VIRUSES
Structure a n d Classification epolE
colE
evn protein /caps omore
evn

of Vi r u s e s Vral m
oenegB
/NA/ RNA
 S t r u c t u r e of V i r u s e s cleocapsil

SIZE: 20-300 m

Fig 1. G e n e r a l i s e d S t r u c t u r e of a Virus. The genetia

maetrail fohte virus si surrounded yb a proetni capsid
• Viruses invade host cells. Viruses ear continuously invading which

m
p r o t e c t s

i mune sy em
st. The dispac
atrom t h e c h i v e o n m e n t a n d

(envelope) sah "spikes",

h o s

host cells, inside which they reproduce. Before het host can hcw
ih represent atachment (envelope) proetnis,
detect hte usrvisa foreign and respond, hte usrvi mya have sticking out of .ti These attachment proteins help het
usrvi atch ot and tcenfi a tsoh .lcel
reproduced and spread.
• Viruses have no cell organelles. yT
eh have on ,uesnulc on
ribosomes, and none fo hte membrane bound organelles. Structure of Viruses

• Viruses have a genome. Viruses carry genetic material. Vriuses

have almost 8 different types fogenomes, and each genome si
made pu of different variations fo D
N
A and RNA.
Classification o f Viruses
• Viruses have a protein coat called a capsid. The capsid
protects the viral genome from het external environment. Some All viruses h a v e a c a p s i d a n d a nucleic acid core, a n d the
seurvi can also have a lipid coat containing glycoproteins.
structure and type of each can be used when classifying them.
• Viruses have attachment proteins. Atachment proteins ar
present no the surface of the capsid. The virus uses het This has been done in Table 1 using four example viruses.
atachment proteins ot bind tohost cesl ni order to infect
them.

• Viruses are extremely smal. Viruses structure are even smaller

naht eht smallest prokaryote. Their sizes are typicaly in the
nanometer range.
Host ti infects Capsid Structure Nucleic Acid Type

in
elgnSdaerts RoUA

ingle-stranded N
R
IA

Feunrte Coneshaped elgnS

i naderts V
A
R
I

Prolate M e l i c a
Fullerene/
Cone-Shaped

Fig.2 Diferent sepyT foCapsid Srutcutres.

m e a su re m e n t 0 8

Micrometey

Measuring cells using a graticule

• Eyepiece graticule is a little scale bar placed in the eyepiece of light microscope.
• The graticule is marked off ni 'graticule units'.
• Turn the eyepiece so that the graticule scale lies over the object: the width of one cell is 23
graticule units.

Field fov i e w # 1
(specimen + EPG,
Calibration: the conversion of graticule units into real units (mm, um).
• use a special slide called a stage micrometer that is marked off in a tiny scale. The smallest
markings are often 0.01 mm (10 ug) apart.
• Take the specimen off the stage or the microscope and replace ti with the stage micrometer. Use
the same objective lens.
• Line up the micrometer scale and the eyepiece graticule scale (by turning the eyepiece and
moving the micrometer on the stage). Make sure that 2 large markings on each scale are lined
up.
 Eyepiece graticule scale

60.

• Stage micrometer scale Field o f view # 2

stage micrometer+ EAG

• The 50 mark (stage micrometer) si lined up with the 1.0 mark (eyepiece graticule).
• Work towards the right until you see another two lines lined up.
• The 68 mark (stage micrometer) si lined up with the 9.0 mark (eyepiece graticule). So you can
say that:
30 small eyepiece graticule markings = 18 stage micrometer markings
= 18 × 0.01 mm = 0.18 mm
= 180 um
so 1 small eyepiece graticule marking = 180: 80 = 2.25 pm

• The plant cell was 23 eyepiece graticule units long >-- its real width is: 23 x 2.25 = 51.75 um

VE :
SOL 1.
The diagram shows a stage micrometer scale viewed with an eyepiece graticule.
magnification of ×200.

1 02 0 50 60 70

Using the same magnification, a chloroplast si measured as 4 se graticule divisions long

How long si the chloroplast?

A 10. ×10' mu
B 40. x 10° um
2.5 x 10-'um
D 2.5 ×107um

benig ueds ot calibrate

2. i 21.. shows a diagram fo aegats
.gF
eyepiece graticule.
micrometer elacs that si

One division, no either the stage mciromeetr scale rohet eyepeice graticule, si het distance
between w to adjacent niles.

The length of one division no shit egats m

orceite is 01. m
m

Fig. 2.1

(b) i ) Using htsi stage micrometer, where enodivision is 01. mm, calculate eht actual
length ofeno eyepiece eculiag
tr unti using Fig. 21. yb completing Fig. 22..
Setp 1

1 eyepiece graticule tuni = divided yb .... m

m

Setp 2

Convert eht answerot a measurement w

hti the tuni tm
so suitable rof esuin gilht microscopy

multiplied yb

Fig. 2. 13[
 3. The diagram shows a stage micrometer viewed hti an
w
eyepiece graticule scael, usnig a magnification fo ×400.

m
1m
.0-

Using the same magnification, a chloroplast is measured and

found ot be 8 eyepiece gracituel divisions long. How long is eht
chlor oplas t?

ik
al
aM
id
r. N
D