SOUTHEAST

UNIVERSITY
Department Of Pharmacy
Assignment
Course Code : BPH 125.2
Course Tittle : Pharmaceutical Microbiology
Assignment On : Fluorescence Microscope

Submitted To : MD. Mosiqur

Rahman( Lecturer)

Submitted By
Name : Nusrat Jaman Rhidita
ID : 2020000300046
Batch : 35th
Section :B
Submission Date :15/11/2020
Fluorescence Microscope
Fluorescence microscope definition

 A fluorescence microscope is an optical microscope that uses fluorescence

and phosphorescence instead of, or in addition to, reflection and absorption
to study 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.
 The fluorescence microscope was devised in the early part of the twentieth
century by August Köhler, Carl Reichert, and Heinrich Lehmann, among
others.

Types of Fluorescence Microscope

There are various types of fluorescence microscopes. Some of the common types
are:
 1. Epifluorescence microscopes: The most common type of fluorescence
microscope in which, excitation of the fluorophore and detection of the
fluorescence are done through the same light path (i.e. through the
objective).
2. Confocal microscope: In this type of fluorescence microscope, high‐
resolution imaging of thick specimens (without physical sectioning)
can be analyzed using fluorescent-labeled dye.
3. Multiphoton microscope: In this type of microscope, multiphoton
fluorescence excitation results in the capture of high-resolution three-
dimensional images of specimen tagged with highly specific
fluorophores.
4. Total internal reflection fluorescence (TIRF) microscope: Total
internal reflection fluorescence microscopy (TIRFM) exploits the
unique properties of an induced evanescent wave or field in a limited
specimen region immediately adjacent to the interface between two
media having different refractive indices. 

Principle of Fluorescence Microscopy

 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.
 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
emit 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, and excite, the sample.

Working
Light of the excitation wavelength is focused on the specimen through the
objective lens. The fluorescence emitted by the specimen is focused on the detector
by the objective. Since most of the excitation light is transmitted through the
specimen, only reflected excitatory light reaches the objective together with the
emitted light.

Forms
The “fluorescence microscope” refers to any microscope that uses fluorescence to
generate an image, whether it is a more simple set up like an epifluorescence
microscope, or a more complicated design such as a confocal microscope, which
uses optical sectioning to get better resolution of the fluorescent image.
Most fluorescence microscopes in use are epifluorescence microscopes, where
excitation of the fluorophore and detection of the fluorescence are done through
the same light path (i.e. through the objective).

Parts of Fluorescence Microscope

Typical components of a fluorescence microscope are:

 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. 
 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. 
 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. 
Inverted Fluorescence Microscope Design
A similar version of the vertical fluorescence illuminator is available for inverted
(tissue culture) microscope stands. The inverted stands also permit combining or
alternating between reflected light fluorescence and the various contrast enhancing
techniques of transmitted light microscopy. Research-level inverted microscopes
feature multiple (up to six) input/output ports, usually with single ports on each
side of the frame, as well as one or two ports (upper and lower) at the rear and a
bottom port underneath the base of the microscope. In some models, primary
images can be obtained simultaneously from three or more ports without the use of
relay lenses. This level of connectivity enables the use of multiple light sources,
filter wheels, and camera systems for complex fluorescence analysis. Mercury and
xenon lamphouses for inverted microscopes are available with the standard multi-
element or aspherical collector lenses to improve performance and reduce
aberration in the ultraviolet and infrared spectral regions. In addition, a wide
spectrum of lamphouse adapters can be utilized to attach several illumination
sources, similar to the accessories available for upright microscopes.
Presented in Figure 10 is a cut-away schematic diagram of a modern inverted
(tissue culture) fluorescence microscope equipped with both a Peltier-cooled CCD
image sensor and a traditional 35-millimeter film camera system. Although film
cameras are of limited use, most inverted microscopes still include a port for these
attachments in the lower portion of the front base. The microscope illustrated in
Figure 10 can perform traditional brightfield transmitted illumination (with or
without contrast enhancement) using the tungsten-halogen lamphouse mounted on
the pillar. A mercury or xenon arc-discharge lamp is employed for fluorescence
microscopy using a reflected light illuminator attached to a specially configured
rear port. Aperture and field diaphragms, along with neutral density filters, are
accessed near the port at the rear of the microscope. In some inverted microscope
models (not illustrated), an L-shaped reflected light illuminator containing
centerable diaphragms is available to improve access to the rear auxiliary ports for
additional accessories. The light path through the microscope in Figure 10 is
depicted in yellow for transmitted light, violet for unfiltered arc lamp illumination,
green for filtered fluorescence excitation, and red for fluorescence emission.
Modern inverted microscope frames, like their upright counterparts, are computer
engineered and fabricated with composite materials for structural and thermal
stability. In addition, the mechanical stage components and circuit structures are
designed with short travel distances and high rigidity to avoid pitch and yaw when
the nosepiece is manipulated during routine operations such as DIC prism insertion
or adjustment of objective correction collars. Advanced nosepiece stages are able
to virtually eliminate focus drift during time-lapse and prolonged fluorescence
observations. Other stage options not available for standard upright microscopes
include gliding and 360-degree rotating stages, glass stage insert plates, heating
plates, Petri dish and plate holders, and carbon dioxide incubator chambers.
Inverted microscopes having a modular design can easily be configured for
investigations in electrophysiology, in vitro fertilization, micromanipulation, high-
resolution DIC, video-enhanced observations, and a variety of advanced
fluorescence techniques. The instruments are also readily adapted for confocal and
multiphoton microscopy. Motorized accessories include shutters, filter wheels,
revolving nosepieces, fluorescence block turrets, focus drives, and condensers.
When coupled to the advanced objectives available in long working distance, water
immersion, ultraviolet excitation, and phase contrast, all having a high degree of
optical correction, inverted microscopes are ideal instruments for conducting
fluorescence investigations on living cells and tissues.

Applications of Fluorescence Microscope

 To identify structures in fixed and live biological samples. 
 Fluorescence microscopy is a common tool for today’s life science research
because it allows the use of multicolor staining, labeling of structures within
cells, and the measurement of the physiological state of a cell.

Advantages of Fluorescence Microscope

1. Fluorescence microscopy is the most popular method for studying the
dynamic behavior exhibited in live-cell imaging.
2. This stems from its ability to isolate individual proteins with a high
degree of specificity amidst non-fluorescing material.
3. The sensitivity is high enough to detect as few as 50 molecules per
cubic micrometer.
4. Different molecules can now be stained with different colors, allowing
multiple types of the molecule to be tracked simultaneously.
5. These factors combine to give fluorescence microscopy a clear
advantage over other optical imaging techniques, for both in vitro and in
vivo imaging.

Limitations of Fluorescence Microscope

 Fluorophores lose their ability to fluoresce as they are illuminated in a
process called photobleaching. Photobleaching occurs as the fluorescent
molecules accumulate chemical damage from the electrons excited
during fluorescence.
  Cells are susceptible to phototoxicity, particularly with short-
wavelength light. Furthermore, fluorescent molecules have a tendency
to generate reactive chemical species when under illumination which
enhances the phototoxic effect.
 Unlike transmitted and reflected light microscopy techniques
fluorescence microscopy only allows observation of the specific
structures which have been labeled for fluorescence. 

Fluorescence Light Sources

An unfortunate consequence of low emission levels in most fluorescence
microscopy applications is that the number of photons that reach the eye or camera
detector is also very low. In most cases, the collection efficiency of optical
microscopes is less than 30 percent and the concentration of many fluorophores in
the optical path ranges in the micromolar or nanomolar regions. In order to
generate sufficient excitation light intensity to produce detectable emission,
powerful compact light sources, such as high-energy short arc-discharge lamps, are
necessary. The most common lamps are mercury burners, ranging in wattage from
50 to 200 Watts, and the xenon burners that range from 75 to 150 Watts
(see Figure 5). These light sources are usually powered by an external direct
current supply, furnishing enough start-up power to ignite the burner through
ionization of the gaseous vapor and to keep it burning with a minimum of flicker.
The microscope arc-discharge lamp external power supply is usually equipped
with a timer to track the number of hours the burner has been in operation. Arc
lamps lose efficiency and are more likely to shatter if used beyond their rated
lifetime (200-300 hours). The mercury burners do not provide even intensity across
the spectrum from ultraviolet to infrared, and much of the intensity of the lamp is
expended in the near ultraviolet. Prominent peaks of intensity occur at 313, 334,
365, 406, 435, 546, and 578 nanometers. At other wavelengths in the visible light
region, the intensity is steady although not nearly so bright (but still useable in
most applications).
 In considering illumination efficiency, mere lamp wattage is not the prime
consideration. Instead, the critical parameter is the mean luminance must be
considered, taking into account the source brightness, arc geometry, and the
angular spread of emission.

In the past few years, optical microscopy has experienced an increase in the
application of laser light sources, particularly the argon-ion and argon-krypton
(ion) lasers. These lasers have the virtues of small source size, low divergence,
near-monochromicity, and high mean luminance. They have become essential in
scanning confocal microscopy, a technique that has proven to be a powerful tool in
rendering very sharp fluorescence images through rejection of non-focused light
removed from the specimen focal plane. Confocal microscopes accomplish this
task through point or line scanning with coincident imaging through a conjugate
aperture. Optical sections of the specimens can be stored in a host computer and
reconstructed into the final image, which is then displayed on the monitor.

Conclusion

The modern fluorescence microscope combines the power of high-performance

optical components with computerized control of the instrument and digital image
acquisition 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. These technical improvements are not mere window
dressing, but are essential components of the light microscope as a system.

The era when optical microscopy was purely a descriptive instrument or an

intellectual toy is past. At present, optical image formation is only the first step
toward data analysis. The microscope accomplishes this first step in conjunction
with electronic detectors, image processors, and display devices that can be viewed
as extensions of the imaging system. Computerized control of focus, stage
position, optical components, shutters, filters, and detectors is in widespread use
and enables experimental manipulations that were not humanly possible with
mechanical microscopes. The increasing application of electro-optics in
fluorescence microscopy has led to the development of optical tweezers capable of
manipulating sub-cellular structures or particles, the imaging of single molecules,
and a wide range of sophisticated spectroscopic applications.