MICROSCOPY 2

Science toolkit: Life Science

EDNS111
2022
Benita Kalicharan
Lecturer, School of Education

Kalicharanb@ukzn.ac.za
Microscopes

microscopes

The Light The Electron

Microscope Microscope

Dissecting/ Transmission Scanning

Compound
Stereo Electron Electron
Microscope
Microscope Microscope Microscope
 Dissecting/ stereo microscope

• Used for viewing live specimens or three-dimensional objects too large or thick to be accommodated by compound
microscopes and too small to be viewed by the naked eye.
• Can be monocular or binocular
• Objects are viewed under low magnification, ranging from about 10x to 80x magnification
• Eyepiece/ocular lens: Lenses at the top that the
viewer looks through; they are usually 10X
• Diopter ring: adjustment of ocular lens
• Arm: structural element that connects the head of
the microscope to the base.
• Stage: the flat platform that supports the slides.
Stage clips hold the slides in place.
• Coarse adjustment knob: can be moved up and
down, allowing the image to be sharpened.
• Lighting: may both top and bottom lighting. Top
lighting shines down on the stage to light up solid
specimens with direct illumination, and bottom
lighting is transmitted up through the stage to
highlight translucent objects.
• Objective lens: The second lens of the microscope.
The objective lens together with the lens of the
eyepiece makes up the microscope’s total
magnification (mag = objective x eyepiece).
Compound microscope

• Ocular lens, arm, on/off switch, stage, stage clips, light source, base.
Compound microscope
• Revolving nosepiece: movement of
objective lens into place
• Objectives: multiple lens for image
enhancement (4x, 10x, 40x)
Compound microscope
• Condenser: focuses light onto
slide.
• Iris diaphragm lever: controls the
amount of light (size & intensity)
reaching the specimen.
 Compound microscope
• Stage manipulator knobs: move the
stage up/down and left/right
• Course adjustment : usually a large
knob on the side of the microscope
that moves the objective lens towards
or away from the slide. It allows you to
find your specimen and roughly focus
on it.
• Fine adjustment: a smaller knob that is
used to focus specifically on the
specimen. It allows you to fine tune
what you’re looking at under the
microscope.
Microscope use
Procedure
1. Plug in and switch on microscope
2. Adjust brightness ( can use the dimmer
switch at the side of the microscope)
3. Use revolving nosepiece to rotate the
4x objective into position. Make sure
the stage is at the lowest point.
Always start using the 4x objective
Procedure
4. Place prepared slide (e.g. slide containing
human blood) onto stage. Secure using stage
clips and align it at the centre using the stage
manipulator knobs.
5. Bring sample into focus (use coarse
adjustment and look through the ocular lens)
Procedure
6. Adjust ocular lens until you see a single image
➢If you see two images when you look
through the eyepieces, you need to
continue to adjust the distance.
➢Move the eyepieces closer together or
further apart until you see a single circle of
light.
➢Remove your glasses, if you wear them.
You can use the microscope's settings to
focus the object according to your sight.

8. Open iris diaphragm until you see the entire

field of view and the image is clear.
Before any adjustment After adjustment

Microscopy simulation – viewing human blood samples

Procedure
9. Rotate the 10x objective into position
10.Focus using coarse and fine adjustment
11.Increase/decrease contrast by
opening/closing iris diaphragm
12. View specimen
Visit http://www.ncbionetwork.org/iet/microscope/
Procedure
13. Rotate the 40x objective into place.
Only use Fine adjustment

NEVER USE THE 100X OBJECTIVE without

oil immersion !!!
Visit http://www.ncbionetwork.org/iet/microscope/
 Removal of slide
• Make sure the stage is lowered
• Move down to the lowest power
objective lens before removing
slide (use slide clips).
• Switch off microscope, unplug
and carefully put the microscope
away.
• Discard slides in appropriate
manner.
 Slide preparation: Wet mount
1.Collect a thin slice of your
sample and place it on a
clean, dry slide.
2.Place one drop of water
over your sample.
3.Place the coverslip at a 45-
degree angle with one edge
touching the water and let
go.
4.Your slide is ready to be
viewed.
Slide preparation: irrigation
1. Prepare wet mount – add drop
of stain/dye at the edge of one
side of the coverslip. Place a
small piece of blotting paper
on the opposite side – “draw”
stain through
2. Some stains may be added
directly to the specimen before
being covered with a coverslip
Rules for Biological drawings
• Drawn and labelled in pencil on unlined (Used to capture prey)
paper
• Covers ½ blank page
• Drawings must have label lines ONLY on
the right hand side.
• Label lines must terminate at the same
point and should not cross each other.
• Annotations can be included*. These can
be structural or functional features of the
label. *Depends if it is asked of you
Biological drawings
• Caption must be descriptive with species
and genus name, if available. Scientific
names must be underlined.
• Steatoda capensis
• Drawings must have a figure caption
(below) and magnification at the end in
brackets.
• Figure 1: Body parts of the brown house
spider, Steatoda capensis (x185)
• Do not shade or colour in.
 How to calculate magnification of drawings
Magnification = drawing size/image size
actual size

Actual size = Diameter of field of view_____________

Number of times the specimen fits across the screen
A 1. Measure the scale bar image (beside
drawing) in mm/cm.
2. Convert to µm (multiply by 1000).
3. Magnification = scale bar image divided by
actual scale bar length (written on the scale
bar).
Scale: 1 cm/10 mm = 10 000 μm
M= 10 000/50
Magnification= 200x

1 m = 1000 mm Actual size of one cell

1 cm = 10000 μm Measure the drawing = 5 cm (red line)
1mm = 1000 μm Therefore Actual size of one cell =
1μm = 1000 nm Cell actual size = 50 000/200
Cell actual size = 250 μm
MICROMETRY
• Width of each square = 100 µm
• Width of each grid line = 40 µm

(Number of squares x100) + (number of lines x 40)

= Diameter of field of view for that objective

• Work out the diameter of field of view for the 4x,

10x and 40x objective.
 Micrometry: Field of view
Field of view is different for each objective lens.
A B c

4x 10 x 40 x

Line =40 µm Grid/block = 100 µm

 Micrometry
Table 1: Diameter of field of view for each microscope objective lens
Objective Diameter of field of view (µm)

4x 2250

10x 980

40x 230

• Magnification = Drawing size /Actual size

• Actual size = Diameter of field of view for specific
objective (µm)/Number of times the specimen fits
across field of view
 Actual size =
Diameter of field of view
Number of times the specimen fits across the screen

= 230/1,5
=153 µm

Image size
Magification = Actual size
200 = x /153
Onion cells Image size =30600 µm
under 40x
objective ~ 3,06cm
Magnified 200x
 Examples for you to try: Example 1

1. A mitochondrion has a length of 12 µm. It is drawn

8.4 cm long. What is the magnification?
 Examples for you to try: Example 2

The image shows a virus and

viral head. Using the
information provided,

1. Calculate magnification.
2. Calculate the size of the
virus head
Type of microscopes
Transmission Electron microscope

• TEM is electron illuminated. This

gives a 2-D view. Thin slices of
specimen are obtained.

• The electron beams pass through

this. It has high magnification and
high resolution.

Maximum magnification:
50 0000 000 x
Type of microscopes
Scanning Electron microscope
• All components of a SEM are housed
in one unit
• A SEM can be used to observe surface
structure

• This applicable to many branches of

science and technology including life
sciences.
• Specimens can be brought into the
chamber with minimal preparation
Maximum magnification:
1 000 000 x
Type of microscopes
Which microscope
could be used to
visualise the items?
Light microscope, transmission electron microscope or scanning
electron microscope
Coronavirus

Frog blood cells Cockroach antenna Moth head