MATERIALS

SCIENCE AND
TECHNOLOGY-1
 THE FLUORESCENCE
MICROSCOPE

Sema Asena Deniz Semih

AŞIK DEMİRCAN SANCAR
20622459 20622608 20622852
 INDEX
• History……………………………………………………..…………….4
• What is a fluorescence microscope?.........................5
• Basic requirements …………………………………………………7
• Parts of fluorescence microscope……………………........8
• What is fluorescence microscopy used for?.............17
• How does fluorescent microscopy works?...............18
• Advantages of fluorescence microscopy……………....19
• Disadvantages of fluorescence microscopy…………..20
• Applications………………………………………………………….21
• Summary…………………………………………………………..….23
• References…………………………………………………………….24
 HISTORY
The termofluorescence was coined by Sir George
Gabriel Stokes (1819–1903). In 1843, he
described the luminescence in calcium fluoride
as fluorescence. In 1904, August Köhler working
in the Jena Zeiss factory invented the ultraviolet
absorption microscope that preceded the
fluorescence microscope. Together with
Siedentopf, they made a fluorescence
microscope. Several later developments improved
the fluorescence microscope.
 WHAT IS A FLUORESCENCE MICROSCOPE?

• A fluorescence microscope is basically a conventional light

microscope with added features and components
that extend its capabilities.
• Fluorescence microscopy is the most popular
method for studying the dynamic behavior
exhibited in live cell imaging.
• Different molecules can now be stained
with different colors, allowing multiple
types of molecule to be tracked simultaneously.
 WHAT IS A FLUORESCENCE MICROSCOPE?

• A conventional microscope uses light to illuminate

the sample and produce a magnified image of the
sample.

• A fluorescence microscope uses a much higher

intensity light to illuminate the sample. This light
excites fluorescence species in the sample, which
then emit light of a longer wavelength.
BASIC REQUIREMENTS OF FLUORESCENCE
MICROSCOPE OPTICS

Nearly all fluorescence microscopes use the

objective lens to perform two functions:

• Focus the illumination (excitation) light on the

sample.
• Collect the emitted fluorescence.
PARTS OF FLUORESCENCE MICROSCOPE
 PARTS OF FLUORESCENCE MICROSCOPE

Main parts of a fluoresence microscobe are;

dichroic mirror,
emission and excitation filter
detector
the light source.
 Light Source
• Direct chemical is being used as light source.
• The conventional microscope uses visible light (400-
700 nanometers)
• A fluorescence microscope uses a much higher
intensity light source
which excites a fluorescent
species in a sample of
interest.
 The Dichroic Mirror
In a fluorescence microscope, the dichroic mirror
separates the light paths.

• The excitation light reflects off the surface of the

dichroic mirror into the objective.

• The fluorescence emission passes through the

dichroic to the eyepiece or detection system.
 The Dichroic Mirror
The mirror reflects
wavelengths of light
below thetransition
wavelength value and
transmits wavelengths
above this value. This
property accounts for
the name given to this
mirror.
Excitation and Emission Filters
 Excitation and Emission Filters

• Excitation filter :In order to select the excitation

wavelength, an excitation filter is placed in the
excitation path just prior to the dichroic mirror.

• Emission filter: An emission filter is placed beneath

the dichroic mirror. In this position, the filter
functions to both select the emission wavelength
and to eliminate any trace of the wavelengths used
for excitation.
The Filter Cube
The dichroic mirror is
mounted on an optical
block commonly referred
to as a filter cube.

This cube provides a

convenient means to
change the dichroic mirror
without direct handling of
either the mirror or filters.
 Detector

• The fluorescence detector is used almost exclusively

in liquid chromatography and is a specific detector
that senses only those substances that fluoresce.

• A flow cell is used as the sensor through which the

excitation light passes axially.
 WHAT IS FLUORESCENCE MICROSCOPY
USED FOR?
• It is often used to image
specific features of small
specimens such as microbes.
• And also used to visually
enhance 3-D features at small
HUMAN LUNG
scales.
• This is achieved by using powerful light sources, such
as lasers, that can be focused to a pinpoint.
HOW DOES FLUORESCENCE MICROSCOPE
WORK?
• The microscope has a filter that only lets through
radiation with the specific wavelength .
• The radiation collides with the atoms in the
specimen.
• Electrons are excited to a higher energy level.
• When they relax to a lower level, they emit light.
• To become detectable, the fluorescence emitted
from the sample is separated from the much brighter
excitation light in a second filter.
 ADVANTAGES OF FLUORESCENCE
MICROSCOPY
 Image clarity is higher.
 The modern fluorescence microscope combines the
power of high performance optical components to
achieve a level of sophistication that far exceeds that
of simple observation by the human eye.
 Microscopy now depends heavily on electronic
imaging to rapidly acquire information at low light
levels or at visually undetectable wavelengths.
 DISADVANTAGES OF FLUORESCENCE
MICROSCOPY
 The relatively high costs of a microscopy unit.
 The fluorescence microscope is less robust than
conventional instruments.
 Component parts,particularly bulbs, have to be
replace from time to time,and may be expensive and
difficult to procure;repairs are occasionally
necessary.
 A continuous supply of standard electrical power
with minimal voltage fluctuations is needed and it s
difficult to meet these requirements in developing
countries.
 APPLICATIONS
A fluorescence microscope is
used to study properties of
organicor inorganic substances
using the phenomena of
MAUSE KİDNEY

fluorescence and
phosphorescence
instead of, or in
addition to, reflection
SHEEP TONGUE and absorption.
 These microscopes are often used for;

• Imaging structural components of small specimens,

such as cells
• Conducting viability studies on cell populations (are
they alive or dead?)
• Imaging the genetic material within a cell (DNA and
RNA)
• Viewing specific cells within a larger population with
many techniques.
 SUMMARY
• The modern fluorescence microscope has emerged
as an important tool in the life sciences.
• The concomitant developments in instrumentation,
specifically light sources and detectors, as well as
probe development have resulted in fluorescence
with unprecedented sensitivity and specificity.
• Modern fluorescence microscopes have the
capability of single-molecule imaging, subdiffraction
limited resolution and the specificity of imaging
single proteins in living cells and organisms.
 REFERENCES
• [Link]
• [Link]
• [Link]
620/Week2_assignment/Lichtman_05_01.pdf
• [Link]
ml
• [Link]
• [Link]
[Link]/topics/fluorescence/[Link]
• ENCYCLOPEDIA OF LIFE SCIENCES & 2010, John Wiley & Sons, Ltd.
[Link]