Microscopy

Light Electron

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Microscopy

Microscopy is the technical field of using microscopes to view samples &

objects that cannot be seen with the unaided eye (objects that are not
within the resolution range of the normal eye).

Light microscopy is a general term used for any type of microscopy

where light is being transmitted from a source which is on the
opposite side of the sample, to the objective lens.

Generally, the light is passed through a condenser to focus it on the

sample to have maximum brightness. After the light has passed through
the sample, it goes through the objective lens to magnify the image of
the sample & then to the oculars, where the enlarged image is viewed.

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Light (Optical) Microscopy

• Visible light is used.

• 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.

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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.

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Microscopy

• General Principles of Microscopy:

– Wavelength of radiation
– Magnification
– Resolution
– Contrast

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The electromagnetic spectrum

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 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.

If two bacterial cells were very close together on a slide, they might look like a
single, blurry dot on a microscope with low resolving power, but could be told apart
as separate on a microscope with high resolving power.
 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
 Numerical aperture
• 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.
The limits of resolution

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Microscopy

• General Principles of Microscopy

– Contrast
– Differences in intensity between two objects, or between an
object and background
– Important in determining resolution
– Staining increases contrast
– Use of light that is in phase increases contrast

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Light refraction and image magnification

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Four kinds of light microscopy

DIC
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Microscopy

• Light Microscopy
– Bright-field microscopes
– Simple
– Contain a single magnifying lens
– Similar to magnifying glass
– Leeuwenhoek used simple microscope to observe
microorganisms

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Microscopy

• Light Microscopy
– Bright-field microscopes
– Compound
– Series of lenses for magnification
– Light passes through specimen into objective lens
– Oil immersion lens increases resolution
– Have one or two ocular lenses
– Total magnification = magnification of objective lens X
magnification of ocular lens
– Most have condenser lens (direct light through specimen)

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A bright-field, compound light microscope

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The effects of immersion oil on resolution

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Microscopy

• Light Microscopy
– Dark-field microscopes
– Best for observing pale objects
– Only light rays scattered by specimen enter objective lens
– Specimen appears light against dark background
– Increases contrast and enables observation of more details

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The light path in a dark-field microscope

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PHASE CONTRAST MICROSCOPY
Microscopy

• Light Microscopy
– Phase contrast microscopy-

– Light rays in phase produce brighter image, while light

rays out of phase produce darker image
– Contrast is created because light waves are out of
phase

Rely on phase difference between the sample and

background.

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Principles of phase microscopy

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 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

I
Microscopy

• Phase contrast yields image intensity values as a function of

specimen optical path length magnitude, with very dense regions
(those having large path lengths) appearing darker than the
background.

• Alternatively, specimen features that have relatively low thickness, or

a refractive index less than the surrounding medium, are rendered
much lighter when superimposed on the medium gray background.

• Good for thin samples.

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 Phase Contrast Microscopy
Phase contrast is an excellent method to increase contrast when viewing or
imaging living cells in culture, but typically results in halos surrounding the
outlines of edge features.

The technique is ideal for thin

unstained specimens such as
culture cells on glass (which are
approximately 5 to 10 micrometers
thick above the nucleus, but less
than a micrometer thick at the
periphery), thick specimens (such
as plant and animal tissue
sections).
The amount of the phase shift
depends on what media
(refractive index) the waves
have passed through on their
paths, and how long the paths
Slight differences in phase are translated into
were through these media.
differences in intensity
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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

Contrasting techniques
Fibroblast grown in culture

Brightfield Phase contrast

DIC Darkfield

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 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 MICROSCOPY
 Sir George G. Stokes
The phenomenon of
fluorescence was known by
the middle of the nineteenth
century. British scientist Sir
George G. Stokes first made
the observation that the
mineral fluorspar exhibits
fluorescence when
illuminated with ultraviolet
light, and he coined the word
"fluorescence"
 What is Fluorescence?
The absorption and subsequent re-
radiation of light by organic and
inorganic specimens is typically the
result of well-established physical
phenomena described as being either
fluorescence.
Principle of Fluorescence Microscopy

The basic function of a fluorescence microscope is to irradiate

the specimen with a desired and specific band of wavelengths,
and then to separate the much weaker emitted fluorescence
from the excitation light.
Principle of Fluorescence Microscopy
The underlying process of fluorescence involves the absorption
of light energy (a photon) by an indicator followed by the
emission of some of this light energy (as another photon) a few
nanoseconds later.

Because some energy is lost in this process, the emitted photon

has less energy than the absorbed photon.

Light with a short wavelength (toward the blue) has higher

energy than light with a long wavelength (toward the red).

Therefore, light emitted from an indicator usually has a longer

wavelength than that of the absorbed (excitation) light. This
change is called the Stokes shift.
 Fluorescence – Jablonski Diagram

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Microscopy

• Light Microscopy
– Fluorescence microscopy
– Direct UV light source at specimen
– Specimen radiates energy back as a longer, visible
wavelength
– UV light increases resolution and contrast
– Some cells are naturally fluorescent; others must be stained
– Used in immunofluorescence to identify pathogens and to
make visible a variety of proteins

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 Fluorescence Microscope
• Specimens usually stained • shows a bright image of the
with fluorochromes object resulting from the
fluorescent light emitted by
• exposes specimen to the specimen.
ultraviolet, violet, or blue
light.
• Shows the locations of
• These fluorescent specific `molecules in the
substances absorb cell by tagging the
ultraviolet radiation and molecules with fluorescent
emit visible light. dyes or antibodies.

•
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 Differences between Conventional and
Fluorescent Microscope

T h e Conventional A fluorescence microscope,

uses a much higher
microscope uses intensity light source
visible light (400-700 which excites a
fluorescent species in a
nanometers) to sample of interest.
illuminate and This fluorescent species in
produce a magnified turn emits a lower energy
light of a longer
image of a sample. wavelength that produces
the magnified image
instead of the original
light source.
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 Fluorescence Microscope

• There are two main features that sets fluorescent

microscope apart from the traditional microscope.

• One is the type of light source and the other is the use of
specialized filter elements.

• A fluorescence microscope uses a higher intensity light to

illuminate the samples.

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Fluorescence Microscope
 Parts of a Fluorescence Microscope
A powerful light source (xenon or mercury arch lamp): The light
emitted from the mercury arc lamp is 10-100 times brighter than
most incandescent lamps and provides light in a wide range of
wavelengths, from ultra-violet to the infrared.

Excitation filter: The purpose of the excitation filter is to filter out

all wavelengths of the light source, except for the excitation range
of the fluorophore under inspection. The brightness and brilliance
of images are dictated by the minimum transmission percentage of
the filter. The ideal transmission being >85%.
 Parts of a Fluorescence Microscope
The Dichroic mirror (beam splitter): The Dichroic mirror or beam
splitter is placed at an angle of 45° between the excitation filter
and emission filter. The function of a dichroic filter is to reflect the
excitation signal towards the fluorophore and to transmit the
emission signal towards the detector.

Objective/ Condenser lens: The light is then directed through a

system of optics that serves as both a condenser, gathering the
light into a narrow beam on the sample, and a focusing objective
for the light emitted back by the specimen.
 Parts of a Fluorescence Microscope
Emission filter: The emission filter is located within the imaging
path of a fluorescence microscope. Its job is to filter out the entire
excitation range and to transmit the emission range of the
fluorophore under inspection.

Eyepieces and detector: The light after passing through an

emission filter is directed toward a set of eyepieces to facilitate the
user to see or to a camera. The fluorescence images can then be
quantitatively analysed using a combination of digital imaging and
image processing.
Fluophores

These are reactive fluorescent dyes which form a fluorescent

image by generating highly contrasted visible range light after
being activated by highly illuminating UV radiation.

• Fluorescein/DAPI/Texas Red/Acridine orange

• GFP and related proteins (YFP/RFP)

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 Images seen through Fluorescence Microscope

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Fluorescent microscopy

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Immunofluorescence

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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

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Fluorescence Resonance Energy Transfer

Occurs between donor

molecule and acceptor
molecule
Can be used to measure
distance between two sites on
a macromolecule
Also used to detect protein-
protein interactions
Membrane fusion and lipid
exchange

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Types of Fluorescence Microscopy

• Epifluorescence

• Confocal

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 Upright Scope

Epi-
illumination
Source

Brightfield
Source

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Inverted Microscope

Brightfield
Source

Epi-
illumination
Source

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