Microscope Drawing skills

To be able to:

(d) the representation of cell structure as seen under

the light microscope using drawings and annotated
diagrams of whole cells or cells in sections of
tissue
(e) the use and manipulation of the magnification
Starter:
formula
What’sthe
(f) thedifference
differencebetween magnification and
resolution
Between these pictures?

Starter: What is the difference between

magnification and resolution?
 Magnification is -
the degree to which the size of an image Is larger than the
viewed object (specimen) itself, or,
•the increase in the size of an object (specimen) to produce a
magnified image (real or virtual)
Magnification can be calculated using a formula

Resolution (resolving power) is –

the degree to which it is possible to distinguish between two objects
(adjacent points) that are very close together - i.e. the smallest
distance apart that two separate objects can be seen clearly as two
objects - allows the observer to see Detail
 Optical and Electron Microscopes –
Feature Compared
Optical microscope Electron microscope
Radiation
Wavelength
Lens

Image formation

Magnification
Staining
Resolution
15 minutes

Vacuum in microscope?...
d
Present/Absent
i nate
Specimen is… e l a m he l p
se th for
U e et s
Dead/Alive Sh
how is it stained?

Other Information
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 Optical and Electron Microscopes - Compared
Feature Optical microscope Electron microscope
Radiation Light (bulb) Beam of electrons (tungsten filament – 50 kV)

Wavelength 400 – 700 nm (long) 0.5 – 1 nm (short)

Lens Glass (convex) Metal electromagnets (refract and focus electron
beam)
Image formation Retina of eye Projected (fluorescent screen / film)

Projection microscope Black and white (electron micrographs)

on screen
Magnification x 1500 TEM ≈ x 500 000; SEM – x100 000
Staining Chemicals Metal ions
Resolution 200 nm (0.2 um) 0.5 nm (0.005 um) - distinguish between objects
0.50 nm (0.005 um) apart
Eye – 100 um
Electrons - small wavelength
Higher resolution
Vacuum in Absent Present
microscope
Vacuum prevents electron scatter (e.g. by O2, H2O)
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Specimen is Alive or dead Dead – specimen in vacuum

Stained using coloured Stained using heavy metal ions – improves contrast
chemicals

Preparation is less Transmission microscope

harsh than for EM
Electrons are transmitted through
internal structure of specimen
2D image formed
Magnification ~ x500 000
Resolution – 0.2 nm
Scanning microscope:

Beams of electrons are scanned and

reflected off the surface of the
specimen
Lower resolution than TEM
3D image formed (increased depth of
field)
Magnification ~ x100 000
Resolution ~ 0.5 nm
Other Large; complex instrumentation; special room
temperature control required; trained personnel to
operate; restricted use; expensive; pumps required
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(vacuum); high voltage
Exam Questions /10 marks
light 50 - 200 nm / 0.05 - 0.2 µm ;

TEM 0.05 - 1.0 nm ;

3 dimensional / 3D, (image) ;
can see the surface (detail) ;
 120

ribosomes ;
Golgi ; endoplasmic reticulum / ER / RER / SER ;
cytoskeleton / microtubules / microfilaments / spindle fibres ;
centrioles ; vesicles / lysosomes ; mitochondria ;
 Cell Microscopy
Produce 1 biological drawing

Animal cells
• Using a cotton bud, GENTLY
swab the inside of your • Stain the cheek cell with
cheek. methylene blue.
• Rub the cotton bud on a
clean slide
• Add a drop of water to the
slide
• Put a drop of methylene blue
onto your sample.
• CAREFULLY lower a cover
slip onto the slide
• Use filter paper to gently
remove excess
Calculations
Work out the sizes of the
•Nucleus
•The entire cheek cell
RESOURC
ES
Light Microscope Radiation - visible light – 400 = ‫ ג‬nm (long)
Resolution = 200 nm (0.2 µm)
If points are closer than 200 nm (0.2 µm),they
cannot be distinguished
Wavelength of light is too large to pass between
points closer than 200 nm
Resolution of human eye ~ 100 µm
Magnification ~ x1500 to x2000

Light beam(radiation)

Image
Glass lens
+ Objects unresolved – 2 points seen as one
Light beam
Image is virtual – seen by eye through eyepiece
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lens., or projected onto screen
The resolution of a LM is improved by having: Advantages of LM Light Microscope
 An objective lens with a short focal length No vacuum
Specimen alive or dead
 A film of immersion oil (with a refractive
Large field of view
index)
Cheap; portable
between the objective lens and the specimen
No technical expertise required
on the glass slide – allows light to be refracted
– simple to use
(“directed”) from the specimen to the
Natural colours seen
objective lens – minimises scatter
Preparation of specimen –
Stains less harsh than for EM
Stains are coloured chemicals used to distinguish Less likely to produce artefacts
(highlight) specific structures
Disadvantages of LM
If an object is transparent it will allow light
waves to pass through it and therefore will still  Low magnification
not be visible  Low resolution
Therefore, many biological structures have to
be stained before they can be seen Ribosomes are smaller than 22
nm in diameter and can therefore
Nature of image in LM never be seen using a light
•2D – in colour microscope
•Virtual on retina of eye, viewed via A stained mitochondrion (~ 1000
eyepiece nm in diameter) interferes with
lens light waves and can therefore be
•Image can be viewed on a fluorescent 14
seen a using light microscope
screen – using a projection light
Staining for light microscopy Light Microscope
Stains (coloured chemicals) are used to see organelles
and tissues clearly
The first known stain was saffron - used to stain
muscle fibers by Leewenhoek, in the late 1600s. In
1883. Gram developed gram staining – to stain
bacterial cell walls
Cheek cell stained with
Coloured stains methylene blue (stains DNA)
Stains that bind to specific chemicals on, or, Shows nucleus and bacteria
in the specimen – allows specimen to be seen.
Some stains bind to specific cell structures, to
highlight specific structures – e.g.
- acetic orcein stains DNA dark red
- gentian violet stains bacterial cell walls
- methylene blue stains DNA (nuclei)

Differential staining
Involves the use of more than one stain on a A diagnostic pap smear
specimen Staining shows the cells
infected with the human
Used to contrast (differentiate) sub-cellular papilloma virus HPV) - cause15
parts against each other. of cervical cancer in females
Use of staining and microscopy in medical diagnosis - example

A 42-year-old teacher presented with a three week history of pulmonary disease

characterized by fever, dyspnoea, cough, yellowish sputum, shortness of breath, chest
pain and weight loss. Chest X ray (CXR) showed interstitial and diffuse bilateral infiltrates
and a fine needle biopsy was taken which showed the presence of fungal elements

Haematoxylin & Eosin (H & E) Culture Microscopy

stain of lung tissue
Shows abundant budding yeast
cells morphologically consistent
with Blastomyces dermatitidis.
(magnification = 1000X)
Colony of Blastomyces Septate hyphae bearing round
dermatitidis. or pear-shaped terminal conidia
(magnification = 600X)
H & E stain – the most widely
used stain in medical diagnosis

Diagnosis – Blastomycosis
A chronic lung disease – spreads to
other body parts
Treated by anti-fungal agents Ulcerated granuloma due16to
Fatal if not treated B. dermatitidis
Electron Microscope (TEM)
Radiation - electron beam
Wavelength ~ 1 nm (short) – free electrons behave
like electromagnetic radiation
Magnification – c. x 500 000 (TEM); x100 000
(SEM)
Resolution = 0.5 nm (0.0005 µm)
Wavelength of electron beam is small enough to
pass
betweenare
Electrons points which are very close (1.0 nm apart)
negatively charged
- they can be Electron Beam
focussed using
electromagnets
(“lenses”)

Fluorescent
screen – makes Object
image visible

Image

Resolved - 2 points seen separately

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Image seen on fluorescent screen
 Electron Microscopes (Scanning EM & Transmission EM)
Stains
Heavy positively charged metal ions are used to stain specimens (e.g. osmium, lead, or
uranium)
– make organelles distinct
– organelles and membranes absorb negatively charged electrons
differentially
Electrons are unable to pass through stained areas – produce electron shadows –
improves contrast
Image
•TEM – images are in 2D – internal structures can be seen; only electrons passing
through the specimen are seen
•SEM – images are in 3D (gives depth of field); surface structures seen; greater
depth of field – therefore, much of the specimen is in focus at the same time
SEM cannot achieve the same resolution as the TEM; cannot observe internal
structures of cells using the SEM
Reflected beam of electrons is observed
•Black and white images formed – images are colour enhanced using computer
Magnification TEM – x 500 000
SEM – x 100 000
Resolution TEM = 0.1 nm (0.0001 m)
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SEM = 0.5 nm
(0.0005 m)
Advantages of EM (compared to LM) Electron Microscopes (SEM &TEM)

Higher resolution and higher magnification – not possible with LM

Ultra structure (fine detail) can be observed using the TEM – not possible with LM
SEM gives depth of field - not possible with LM

Disadvantages of EM (compared to LM)

Specimen is dead – under vacuum
Specimen needs to be dehydrated – since water boils at room temperature under
vacuum
Vacuum pump required - vacuum prevents scatter of electrons caused by air,
water
vapour, etc.
Specimen, tungsten filament, and beam of electrons need to be contained within a
sealed chamber
Natural colours cannot be seen
Large; not portable; expensive; restricted use
Heat generated – requires room temperature control
Artefact
High voltage (safety + cost)
a structure which is not part of the
Artefacts likely specimen (object) – e.g. an air bubble 19
Technical expertise required
Preparing specimens for electron microscopy

Fix specimen to make it firm – by using a fixative (glutaraldehyde / osmium tetroxide)

Dehydrate using ethanol – since water boils at room temperature in the vacuum
Embed the dehydrated tissue in a solid (plastic/epoxy) resin (to allow sectioning)
Section using a microtome – cut thin sections using a glass / diamond knife – thin
specimen allows electrons to pass through
Stain – using heavy metals (improves contrast)
Place on a copper grid in a vacuum in the microscope
Damage and distortion may result, leading to artefacts – need to repeat to assess if
structure is permanent.

Stains (heavy metal compounds - e.g., osmic acid, lead nitrate) are opaque to
electrons – the stained area appears dark
For SEM the specimen is sprayed with atoms of heavy metal (e.g., gold) from a
particular angle – the sheltered areas remain uncoated forming a “shadow” – giving
depth. Useful in showing surface structures (viruses, cell walls, molecules)
Differential staining is used to enhance detail. More than one stain is used to treat
the
specimen. Allows different parts of a particular structure to be differentiated - e.g.
nucleolus in the nucleus
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