2. Definition
Microscopy is the technical field of using
microscopes (instruments) to view samples
and objects that cannot be seen with the naked
eyes (objects that are not within the resolution
range of the normal eye).
Resolution is the term used to describe the
number of dots, or pixels, splitting after
passage of light or electron beam through an
object to display an image.
3. Microscopy ……….
Resolution power is the ability of an
instrument (in our case a microscope)
to differentiate two nearby points or
dots or pixels
Other instruments which work on
different ways using resolution power
are:
11. To advance resolution need
the following factors
1. short wavelength
2. higher index immersion liquid to increase
.….numerical aperture (N)
3. confocal improve resolution squares
4. if you can study single spots (ie single
…..molecules or particles in a space field) can
…..find their centre at arbitrarily high resolution.
5. multicolour resolution not limited by
….diffraction.
6. super resolution.
15. Categories of microscopy
There are three well-known
branches of microscopy:
- optical microscopy
- electonic microscopy,
- scanning probe microscopy.
16. Types ……..
Optical and electron microscopy
involve the diffraction, reflection,
or refraction of light or
electomagnetic radiation /electron
beams interacting with the
specimen, and the subsequent
collection of this scattered radiation
or another signal in order to create
an image.
17. Types…….
This process may be carried out by
wide-field irradiation of the
sample (for example standard light
microscopy and transmission
electronic microscopy) or by
scanning of a fine beam over the
sample (for example conforcal
laser scanning microscopy and
scanning electron microscopy).
18. Types……
Diffraction – this phenomenon is
described as the apparent bending of
waves around small obstacles and the
spreading out of waves past small
openings (eg light travels as waves but
not in rays)
Reflection is the change in direction of
a wave front at an interface between two
different media so that the wave front
returns into the medium from which it
originated.
19. Types……
Refraction - Due to change of medium, the
phase velocity of the wave is changed but its
frequency remains constant.
The development of microscopy
revolutionized biology and remains an
essential technique in the life and physical
science.
20. MICROSCOPES
These are instruments commonly used in the
technique of microscopy.
They are of different types but are all basically
containing the following parts.
single lens with its attachments, or the system
of lenses and imaging equipment
Body tube or component
the appropriate (lighting) irradiation or
scanning equipment
sample stage or sub stage
Support or basement.
23. LIGHT OR OPTICAL MICROSCOPE
Optical or light microscopy involves
passing visible light transmitted through
or reflected from the sample through a
single or multiple lenses to allow a
magnified view of the sample
The resulting image can be detected
directly by the eye, imaged on a
photographic plates or captured
digitally.
24. Limitations of light
microscope
Limitations of standard optical
microscopy (bright field microscope)
lie in three areas;
This technique can only image dark or
strongly refracting objects effectively.
Diffraction limits resolution to
approximately 0.2 micrometers
Out of focus light from points outside the
focal plane reduces image clarity.
25. Limitations ……
Live cells do not give sufficient contrast to be
studied successfully, due to their colourless
and transparent internal structures.
The most common way to increase contrast is
to stain different structures with selective dyes,
this often involves killing and fixing the
sample.
This can introduce artifacts (apparent
structural details that are caused by the
processing of the specimen and are thus not a
legitimate feature of the specimen).
26. ELECTRONIC MICROSCOPE
Until the invention of sub-diffraction
microscopy, the wavelength of the light limited
the resolution of traditional microscopy to
around 0.2 micrometers.
higher resolution is achieved by the use of an
electron beam with a far smaller wavelength by
electron microscope.
Two types are
Transmission electron microscopy (TEM)
Scanning electron microscope (SEM)
27. EM……
Transmission electron microscopy (TEM)
is a microscopy technique in which a beam of
electrons is transmitted through an ultra-thin
specimen, interacting with the specimen as it
passes through.
An image is formed from the interaction of
the electrons transmitted through the
specimen;
the image is magnified and focused onto an
imaging device, such as a fluorescent screen,
on a layer of photographic film
Also may be detected by a sensor or camera.
28. EM…….
A scanning electron microscope (SEM) is a type of
electron microscope that produces images of a sample
by scanning it with a focused beam of electrons.
The electrons interact with atoms in the sample,
producing various signals that can be detected and that
contain information about the sample's surface
topography and composition.
The electron beam is generally scanned in a raster scan
pattern, and the beam's position is combined with the
detected signal to produce an image.
SEM can achieve resolution better than 1 nanometer.
Specimens can be observed in high vacuum, in low
vacuum, and (in environmental SEM) in wet
conditions.
29. How to Use a Microscope
Compound
Microscopes:
The compound
microscope consists of
two optical components
(thus the term
compound): the objective
lens system, which has a
very short focal distance
and is placed very close
to the object; and the
ocular or eyepiece
system, which has a
longer focal length,
lower magnification; and
which further magnifies
and projects the image
onto the retina of the eye.
30. How to use a microscope……
Turn the revolving turret (2) so that the lowest
power objective lens (eg. 4x) is clicked into
position.
Place the microscope slide on the stage (6) and
fasten it with the stage clips. Make sure that the
cover slip of the slide is on upper side.
Look at the objective lens (3) and the stage from the
side and turn the focus knob (4) so the stage moves
upward. Move it up as far as it will go without
letting the objective touch the coverslip.
Look through the eyepiece (1) and move the focus
knob until the image comes into focus.
Adjust the condenser (7) and light intensity for the
greatest amount of light.
31. How to use a microscope………
Move the microscope slide around until the sample
is in the centre of the field of view (what you see).
Use the focus knob (4) to place the sample into
focus and readjust the condenser (7) and light
intensity for the clearest image (with low power
objectives you might need to reduce the light
intensity or shut the condenser).
When you have a clear image of your sample with
the lowest power objective, you can change to the
next objective lenses. You might need to readjust
the sample into focus and/or readjust the condenser
and light intensity.
If you cannot focus on your specimen, repeat steps
3 through 5 with the higher power objective lens in
place. Do not let the objective lens touch the slide!
32. How to use a microscope……..
When finished, lower the stage, click the low power
lens into position and remove the slide.
NOTES:
Your microscope slide should be prepared with a
coverslip over the sample to protect the objective
lenses if they touch the slide.
Do not touch the glass part of the lenses with your
fingers. Use only special lens paper to clean the
lenses.
Always keep your microscope covered when not in
use.
Always carry a microscope with both hands. Grasp
the arm with one hand and place the other hand
under the base for support.
33. How to use a microscope……..
Stereomicroscopes
34. How to use a microscope……..
Place your sample on the stage (3) and turn
on the LED light (2).
Look through the eyepieces (4) and move
the focus knob (1) until the image comes
into focus.
Adjust the distance between the eyepieces
(4) until you can see the sample clearly with
both eyes simultaneously (you should see
the sample in 3D).
NOTES:
When you move the sample, you will have
to focus again by moving the focus knob.
