Microscopy and its types

What is light?

• Light is electromagnetic radiation.

• What we usually describe as light
is only the visible spectrum of this
radiation with wavelengths
between 400nm and 700nm.
• The elementary particle that
defines light is the photon.

b) a)

There are 3 basic dimensions of light

a) Intensity (amplitude) which is related to the perception of brightness
b) Frequency (wavelength), perceived as colour
c) Polarization (angle of vibration) which is not or weakly perceptible to humans
What is a microscope?

The microscope has
become one of the
most recognizable
symbols of science.

Starting with use of a simple lens in ancient

times, to the first compound microscope
around 1590, and up to the microscopes you
are using in 7th grade life science, the
microscope has allowed scientists to make
discoveries about the “invisible world.”
 Micro- = “small”; -scope = “to look at”

Photographs of cells are taken using a microscope, and these pictures are
called micrographs.
 What is a microscope?
Theoretically a microscope is an array of two lenses.

Focal plane Image plane

Image plane

Objective Tube lens Eyepiece

lens lens

Classic compound microscope

Your friend - the objective
 What is magnification?
Magnification is a measure of how much larger a microscope (or set of lenses
within a microscope) causes an object to appear.

Magnification is defined by the

magnification by the objective

x
the magnification by eyepiece

BUT maximum magnification does not mean maximum resolution!

 What is resolution?
Resolution describes the minimal distance of two points that can be distinguished.
The resolution of a microscope or lens is the smallest distance by which two points can
be separated and still be distinguished as separate objects.
The smaller this value, the higher the resolving power of the microscope and the better
the clarity and detail of the image.

Picture taken from [Link]

 What is the numerical aperture?
NA is an estimate of how much light from the sample is collected by the objective

α2
α1

Oil (n = 1.5)
Air (n = 1.0) Objective lens
Coverslip (n = 1.5)
Glass slide (n = 1.5)

NA = n sin 
n = refractive index
α = angle of incident illumination
• The higher the numerical aperture of a
lens, the better the resolution of a
specimen will be which can be obtained
with that lens.
• d=0.5 λ/n sin Ɵ
– Where d= resolution
– Λ = wavelength of light used
Numerical aperture, NOT magnification determines resolution!

Increasing NA

A lens with a larger NA will be able to visualize finer details and will also
collect more light and give a brighter image than a lens with lower NA.
Microscopy
 Light (Optical) Microscopy
• Visible light is uses.
• Glass lens are used
• Advantage: It can often be performed on living
cells.
• so it’s possible to watch cells carrying out their
normal behaviors (e.g., migrating or dividing)
under the microscope.
 Principle
• When a ray of light passes from one
medium to another it bends by
phenomena called refraction.
• Bending of light slows the speed.
• The bending of light is determined by
refractive index of the medium.
 Types of Light Microscopes
[Link] field Light Microscope
[Link] Contrast Light Microscope
[Link]-Field Light Microscope
[Link] Light Microscope
Contrasting techniques
Fibroblast grown in culture

Brightfield Phase contrast

DIC Darkfield

Taken from: [Link]

 Brightfield
Principle: Light is transmitted through the sample and absorbed by it.

Application: Only useful for specimens that can be contrasted via dyes.
Very little contrast in unstained specimens.
With a bright background, the human eye requires local intensity fluctuations
of at least 10 to 20% to be able to recognize objects.

Cross section of sunflower root Piece of artificially grown skin

([Link] ([Link]/.../dt/PI_BioTechnica2001.[Link] )
Typical Classroom Microscope
Eyepiece
 Eyepiece
• Also known as the ocular
• Contains the first lens you look through
- usually a magnification of 10x
• Located on the top of the body tube
Objective Lenses
 Objective Lenses
• Used in combination with the eyepiece
to provide a range of magnification
• Magnification ranges from 40x to 400x
• Located on the nose-piece at the
bottom of the body tube
Nosepiece
 Nosepiece
• Holds the objective lenses
• Rotates to enable magnification
• Located at the bottom of the body tube
Arm
 Arm
• Supports the upper parts of the
microscope
• Used to carry the microscope
• When carrying a microscope, always
have one hand on the arm and one
hand on the base. Use two hands!!
Base
 Base
• Supports the whole microscope
• Used to carry the microscope
• When carrying a microscope, always
have one hand on the arm and one
hand on the base. Use two hands!!
Stage
 Stage
• Supports the slide
• The slide contains the specimen or
object that you are viewing with the
microscope.
Stage Clip
 Stage Clip
• Helps to hold the slide in place
• Usually one on each side of the hole
(stage opening) = 2 stage clips
• The stage opening allows light to pass
from the light source to the lenses.
Light Source
 Light Source
• Provides light necessary for viewing
the specimen
• Usually either a mirror or illuminator
• Sends light through the stage opening
to the diaphragm
Diaphragm
 Diaphragm
• Wheel or lever located below the stage
opening
• Regulates the amount of light that can
enter the lenses
• May need to be adjusted based on the
thickness of the specimen being
studied
Coarse Adjustment
Knob
 Coarse Adjustment Knob
• Raises and lowers the stage or
objective lenses
• Used only when focusing the low
power (4x) objective lens
Fine Adjustment Knob
 Fine Adjustment Knob
• Raises and lowers the stage or
objective lenses a small distance for
exact focusing
• Used when focusing the medium
power (10x) and high power (40x)
objective lenses
Let’s Review...
 Phase contrast
Principle: Incident light [Io] is out of phase with transmitted light [I] as it was slowed down while
passing through different parts of the sample and when the phases of the light are synchronized
by an interference lens, a new image with greater contrast is seen.

Phase ring

I0 not aligned aligned

Phase stops

[Link]
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 Phase contrast

Application: Phase contrast is the most commonly used contrasting technique All
tissue culture microscopes and the time-lapse microscopes are set up for phase.

brightfield wrong phase stop right phase stop

 Applications

• Determine morphologies of living

cells such as plant and animal cells
• Studying microbial motility and
structures of locomotion
• To detect certain microbial elements
such as the bacterial endospores
 Fluorescence

[Link]
 Applications
• Used in the visualization of bacterial agents such
as Mycobacterium tuberculosis.
• Used to identify specific antibodies produced
against bacterial antigens/pathogens in
immunofluorescence techniques by labeling the
antibodies with fluorochromes.
• Used in ecological studies to identify and observe
microorganisms labeled by the fluorochromes
• It can also be used to differentiate between dead
and live bacteria by the color they emit when
treated with special stains
 Confocal Microscope
• Confocal microscopy is a technique that uses
lasers and fluorescence to create a three-
dimensional image of a sample.
• A focused laser beam is used to excite the
molecules at one point of the sample.
• The molecules, called fluorophores, release
photons as they return to their unexcited
state, causing the fluorescence.
• By scanning across the sample, an image of it
can be created.
 Application
• Confocal microscopes are used in the
semiconductor industry, as well as in life
and material science labs. Confocal
microscopy is especially useful in
studying live cells.
 Darkfield
Principle: The illuminating rays of light are directed through the sample from the side by putting a
dark disk into the condenser that hinders the main light beam to enter the objective. Only light that
is scattered by structures in the sample enters the objective.

Application: People use it a lot to look at Diatoms and other unstained/colourless specimens

Darkfield

Symbiotic Diatom colony

([Link]/~t936927/[Link])
Brightfield
 Applications
• It is used to visualize the internal
organs of larger cells such as the
eukaryotic cells
• Identification of bacterial cells with
distinctive shapes such
as Treponema pallidum, a causative
agent of syphilis.
[Link]
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