The document describes fluorescence and phase contrast microscopy. Fluorescence microscopy uses fluorescent dyes and an external light source to excite samples, causing them to emit light of a longer wavelength. This technique allows visualization of cellular components. A fluorescence microscope contains a light source, excitation and emission filters, and detector. Phase contrast microscopy converts phase shifts in light passing through transparent samples to brightness changes, making highly transparent objects more visible without staining. It works using a phase annulus and plate to shift light rays passing through samples.

1. FLUORESCENCE &
PHASE CONTRAST
MICROSCOPY

2. What is Fluorescence?
Fluorescence is the property of some atoms and
molecules to absorb light at a particular wavelength (the
excitation: Ex) followed by a short-lived emission (Em) of
light at a longer wavelength.

3. Fluorescence involves an external light source to excite
the sample at a particular wavelength.
When excited at the appropriate wavelength, the
molecule is transformed from a ground to an excited state.
As the molecule returns to the ground state, energy is
released in the form of heat (loss of energy) and light at a
different longer wavelength of lower energy

4. What is Fluorescence Microscopy?
A fluorescence microscope is an optical microscope that
uses fluorescence and phosphorescence instead of, or in
addition to, reflection and absorption to study the
properties of organic or inorganic substances.
Fluorescence is the emission of light by a substance that
has absorbed light or other electromagnetic radiation
while phosphorescence is a specific type of
photoluminescence related to fluorescence.
Unlike fluorescence, a phosphorescent material does not
immediately re-emit the radiation it absorbs.
Most cellular components are colorless and cannot be
clearly distinguished under a microscope. The basic
premise of fluorescence microscopy is to stain the
components with dyes.

5. Fluorescent dyes, also known as fluorophores or
fluorochromes, are molecules that absorb excitation light
at a given wavelength (generally UV), and after a short
delay emit light at a longer wavelength. The delay
between absorption and emission is negligible, generally
on the order of nanoseconds.
The emission light can then be filtered from the
excitation light to reveal the location of the fluorophores.
Fluorescence microscopy uses a much higher intensity
light to illuminate the sample. This light excites
fluorescence species in the sample, which then emits light
of a longer wavelength.
The image produced is based on the second light source
or the emission wavelength of the fluorescent species
rather than from the light originally used to illuminate,

6. The image produced is based on the second light source
or the emission wavelength of the fluorescent species
rather than from the light originally used to illuminate,
and excite, the sample.
Magnification 2000 X, 3 D and colour image is
produced.
Both live and dead fluorescent dye labelled cells can be
observed.

9. Fig- Fluorescence Microscope

10. Construction of Fluorescence Microscope?
Fluorescent dyes (Fluorophore)
A fluorophore is a fluorescent chemical compound that
can re-emit light upon light excitation.
Fluorophores typically contain several combined
aromatic groups, or plane or cyclic molecules with several
π bonds.
Many fluorescent stains have been designed for a range
of biological molecules.
Some of these are small molecules that are intrinsically
fluorescent and bind a biological molecule of interest.
Major examples of these are nucleic acid stains like
DAPI and Hoechst, phalloidin which is used to stain actin
fibers in mammalian cells.

11. A light source
Four main types of light sources are used, including
xenon arc lamps or mercury-vapor lamps with an
excitation filter, lasers, and high- power LEDs.
Lasers are mostly used for complex fluorescence
microscopy techniques, while xenon lamps, and mercury
lamps, and LEDs with a dichroic excitation filter are
commonly used for wide-field epifluorescence
microscopes.
The excitation filter
The exciter is typically a bandpass filter that passes only
the wavelengths absorbed by the fluorophore, thus
minimizing the excitation of other sources of fluorescence
and blocking excitation light in the fluorescence emission
band.

12. The dichroic mirror
A dichroic filter or thin-film filter is a very accurate color
filter used to selectively pass light of a small range of
colors while reflecting other colors.
The emission filter.
The emitter is typically a bandpass filter that passes only
the wavelengths emitted by the fluorophore and blocks all
undesired light outside this band – especially the
excitation light.
By blocking unwanted excitation energy (including UV
and IR) or sample and system autofluorescence, optical
filters ensure the darkest background.
Detector
High sensitive photomultiplier tube is mostly used

13. Root tip of Maize plant
Neuron
Dividing cells

14. PHASE CONTRAST
MICROSCOPY

15. Phase Contrast Microscopy
Small,Unstained living cells can be seen.
 Poor light absorption results in extremely small
differences in the intensity distribution in the image.
This makes the cells barely, or not at all, visible in a
brightfield microscope.
Phase-contrast microscopy is an optical microscopy
technique that converts phase shifts in the light passing
through a transparent specimen to brightness changes in
the image.
It makes higly transparent objects more visible
Examining intracellular components of living cells at
relatively higher resolution.

17. 1. Phase Annulus in the Condenser
The Phase Annulus is a black disc with a transparent ring
or slit that resides in or underneath the condenser.
Its aim is to provide a focused cone of light on the
specimen. In contrast to black field lighting, phase
annulus light enters the objective.
2. Phase Plate in the Objective
The Phase Plate is a transparent plate with a circular ring.
Typically, the ring is a groove carved into the plate and
filled with a phase-advancing or phase-delaying material
(usually a dielectric).
Due to the presence of a substance that modifies the
light’s amplitude, the ring may look darker or brighter
than the remainder of the plate (a neutral density material
usually a thin film of evaporated metal).

18. 3. Phase Telescope
The eyepiece of the phase telescope focuses an image of
the back focal plane of the objective onto the retina of a
person.
Observing the back focal plane of the objective is required
for aligning the phase annulus to the phase ring. On some
microscopes, the phase telescope is replaced by an integral
Bertrand lens.
4. Green Filter
A phase contrast system is based on the use of green light
(Fraunhofer E line at 525 nm). The green line is used by
manufacturers to decide how much material to put in the
phase ring for phase advancement or retardation.

19. Principle of Phase Contrast Microscope
The condenser of a phase-contrast microscope has an
annular stop an opaque disk with a thin transparent ring
that produces a hollow cone of light.
As this cone passes through a cell some light rays are
bent due to variation in density and refractive index within
the specimen and are retarded by 1/4 wavelength. The
deviated light is focused to form an image of the object.
The undeviated light rays strike a phase ring in the phase
plate a special optical disks located in the objective, while
the deviated rays miss the ring and passed through the rest
of the plate. The undeviated light which strikes the phase
ring gets advance by 1/4 wavelength when passing
through this ring.

20. The deviated and undeviated waves become 1/2
wavelength to each other and will cancel each other to
come together to form an image. Therefore deviated and
undeviated lights from different image.
The background formed by undeviated light is bright
while the unstained object appears dark and well-defined.