different types of Microscopy..... .docx

Bright field microscopy
 Bright field Microscopy is the simplest of all the optical microscopy illumination
techniques. Sample illumination is transmitted white light and contrast in the sample is
caused by absorbance of some of the transmitted light in dense areas of the sample.
 Bright field microscopy is the simplest of a range of techniques used for illumination of
samples in light microscopes and its simplicity makes it a popular technique. The typical
appearance of a bright field microscopy image is a dark sample on a bright background,
hence the name BFM.
Light path of bright field microscope
 When a ray of light passes through one medium into another, the ray bends at the
interface causing refraction. The bending of light is determined by the refractive index,
which is a measure of how great a substance slows the speed of light. The direction and
magnitude of the bending of the light are determined by the refractive indexes of the
two mediums that form the interface.
 Light path - The light path of a bright field microscope is extremely simple, no additional
components are required beyond the normal light microscope setup (Fig 1). The light
path therefore consists of:
 Transillumination light source, commonly a halogen lamp in the microscope stand; A
halogen lamp, also known as a tungsten halogen lamp or quartz iodine lamp, is an
incandescent lamp that has a small amount of a halogen such as iodine or bromine
added.
 The combination of the halogen gas and the tungsten filament produces a halogen cycle
chemical reaction which redeposits evaporated tungsten back onto the filament,
Tanvi Raulaji
Department of biosciences

increasing its life and maintaining the clarity of the envelope Condenser lens which
focuses light from the light source onto the sample.
 condenser is one of the main components of the optical system of many transmitted
light compound microscopes. A condenser is a lens that serves to concentrate light from
the illumination source that is in turn focused through the object and magnified by the
objective lens.
 Objective lens: In an optical instrument, the objective is the optical element that gathers
light from the object being observed and focuses the light rays to produce a real image.
Objectives can be single lenses or mirrors, or combinations of several optical elements.
Microscope objectives are characterized by two parameters: magnification and
numerical aperture. The typically ranges are 4× , 10x , 40x and 100×. 4. oculars to view
the sample image. An eyepiece, or ocular lens, is a type of lens that is attached to a
variety of optical devices such as microscopes. It is so named because it is usually the
lens that is closest to the eye when someone looks through the device.
 The objective lens or mirror collects light and brings it to focus creating an image. The
eyepiece is placed near the focal point of the objective to magnify this image. The
amount of magnification depends on the focal length of the eyepiece.
 Magnification is the process of enlarging something only in appearance, not in physical
size. typically magnification is related to scaling up visuals or images to be able to see
more detail, increasing resolution.
 Resolving power is the ability of an imaging device to separate points of an object that
are located at a small angular distance.
If an object is put between these two mediums i.e between water and air, in this case, a
prism, the prism will bend the light at an angle. This is how the microscopic lenses work,
they bend the light at an angle. The lens (convex) on receiving the light rays, focuses the
rays at a specific point known as the focal point (F-point). The measure of distance from
the center of the lens and the focal point is known as the focal length.
 In optics, the numerical aperture (NA) of an optical system is a dimensionless number
that characterizes the range of angles over which the system can accept or emit light. In
most areas of optics, and especially in microscopy, the numerical aperture of an optical
system such as an objective lens is defined by
NA = n Sin
where n is the index of refraction of the medium in which the lens is working (1.0 for air,
1.33 for pure water, and up to 1.56 for oils; see also list of refractive indices), and θ is the
half-angle of the maximum cone of light that can enter or exit the lens. In general, this is
the angle of the real marginal ray in the system
Parts of a bright-field microscope (Compound light microscope)
It is composed of:
 Two lenses which include the objective lens and the eyepiece or ocular lens.
 Objective lens is made up of six or more glasses, which make the image clear from
the object
 The condenser is mounted below the stage which focuses a beam of light onto the
specimen. It can be fixed or movable, to adjust the quality of light, but this entirely
depends on the microscope.

 They are held together by a sturdy metallic curved back used as an arm and a stand
at the bottom, known as the base, of the microscope. The arm and the base hold all
the parts of the microscope.
 The stage where the specimen is placed, allowing movement of the specimen around
for better viewing with the flexible knobs and it is where the light is focused on.
 Two focusing knobs i.e the fine adjustment knob and the coarse adjustment knob,
found on the microscopes’ arm, which can move the stage or the nosepiece to focus
on the image. the sharpen the image clarity.
 It has a light illuminator or a mirror found at the base or on the microbes of the
nosepiece.
 The nosepiece has about three to five objective lenses with different magnifying
power. It can move round to any position depending on the objective lens to focus on
the image.
 An aperture diaphragm also is known as the contrast, which controls the diameter of
the beam of light that passes through the condenser, in that, when the condenser is
almost closed, the light comes through to the center of the condenser creating high
contrast. But when the condenser is widely open, the image is very bright with very
low contrast.
Working Performance

 Bright field microscopy typically has low contrast with most biological samples as few
absorb light to a great extent. Staining is often required to increase contrast, which
prevents use on live cells in many situations.
 Bright field illumination is useful for samples which have an intrinsic colour, for example
chloroplasts in plant cells.
 Light is first emitted by the light source and is directed by the condenser lens on to the
specimen, which might be a loose object, a prepared plate or almost anything.
 A microscope can even be applied to small parts of larger objects, though with a bit
more difficulty. (The light does not absolutely need to originate below the specimen.)
The light from the specimen then passes through the objective lens.
 This lens is often selected from among three or four and is the main determinant for the
level of magnification.
 It bends the light rays and in the case of this example sends them to a projector lens,
which reverses their direction so that when the image reaches the eye it will not appear
"upside-down". Not all microscopes have a projector lens, so the viewer may be seeing a
reverse image.
 In these cases, when the slide is moved, it will appear to be moving in the opposite
direction to the viewer. The light rays then travel to the oracular lens or "eye piece". This
is often a 10X magnification lens, meaning it magnifies the magnified image an
additional ten times.
 The image is then projected into the eye. It is very seldom that a specimen is in focus the
moment it is placed beneath a microscope. This means that some adjustment will have
to be made.
 Unlike in telescopes, the focal length between lenses remains constant when adjusting
the focus. The lens apparatus is brought closer to or further from the object. The focus
adjustment is often along the neck of the tube containing the lenses, but it might just as
well move the slide up and down.
 The best way to make this adjustment is to make a course adjustment so that it is too
close to the object and then back off with the fine adjustment2. This helps to ensure that
the specimen is not inadvertently smashed by the lens.
Advantages
 The name "brightfield" is derived from the fact that the specimen is dark and contrasted
by the surrounding bright viewing field. Simple light microscopes are sometimes referred
to as bright field microscopes.
 Brightfield microscopy is very simple to use with fewer adjustments needed to be made
to view specimens.
 Some specimens can be viewed without staining and the optics used in the brightfield
technique don’t alter the color of the specimen.
 It is adaptable with new technology and optional pieces of equipment can be
implemented with brightfield illumination to give versatility in the tasks it can perform.
Disadvantages
Certain disadvantages are inherent in any optical imaging technique.
 By using an aperture diaphragm for contrast, past a certain point, greater contrast adds
distortion. However, employing an iris diaphragm will help compensate for this problem.

 Brightfield microscopy can’t be used to observe living specimens of bacteria, although
when using fixed specimens, bacteria have an optimum viewing magnification of 1000x.
 Brightfield microscopy has very low contrast and most cells absolutely have to be stained
to be seen; staining may introduce extraneous details into the specimen that should not
be present.
 Also, the user will need to be knowledgeable in proper staining techniques.
 Lastly, this method requires a strong light source for high magnification applications and
intense lighting can produce heat that will damage specimens or kill living
microorganisms.
Phase contrast microscopy
 Phase contrast microscopy is an optical microscopy technique that converts phase shifts
in light passing through a transparent specimen to brightness changes in the image.
Phase shifts themselves are invisible, but become visible when shown as brightness
variations.
 causes the wave amplitude and phase to change in a manner dependent on properties
of the medium.
 Changes in amplitude (brightness) arise from the scattering and absorption of light,
which is often wavelength dependent and may give rise to colors. Photographic
equipment and the human eye are only sensitive to amplitude variations. Without
special arrangements, phase changes are therefore invisible. Yet, often these changes in
phase carry important information. History and Background Information
 Frits Zernike, a Dutch physicist and mathematician, built the first phase contrast
microscope in 1938.
 It took some time before the scientific community recognized the potential of Zernike’s
discovery; he won the Nobel Prize in 1953 and the German-based company Zeiss began
manufacturing his phase contrast microscope during World War II.
Working Principle

 The basic principle to make phase changes visible in phase contrast microscopy is to
separate the illuminating background light from the specimen scattered light, which
make up the foreground details, and to manipulate these differently.
 The ring shaped illuminating light (green) that passes the condenser annulus is focused
on the specimen by the condenser. Some of the illuminating light is scattered by the
specimen (yellow).
The remaining light is unaffected by the specimen and form the background light (red).
 When observing unstained biological specimen, the scattered light is weak and typically
phase shifted by -90° — relative to the background light. This leads to that the
foreground (blue vector) and the background (red vector) nearly have the same intensity,
resulting in a low image contrast (a).
 In a phase contrast microscope, the image contrast is improved in two steps. The
background light is phase shifted -90° by passing it through a phase shift ring. This
eliminates the phase difference between the background and the scattered light, leading
to an increased intensity difference between foreground and background (b).
 To further increase contrast, the background is dimmed by a gray filter ring (c).
 Some of the scattered light will be phase shifted and dimmed by the rings. However, the
background light is affected to a much greater extent, which creates the phase contrast
effect (Fig 2). The above describes negative phase contrast.
 In its positive form, the background light is instead phase shifted by +90°. The
background light will thus be 180° out of phase relative to the scattered light. This results
in that the scattered light will be subtracted from the background light in (b) to form an
image where the foreground is darker than the background.
Applications in Microscopy
 The possible applications of Zernike’s phase contrast microscope in microscopy are
evident in the fields of molecular and cellular biology, microbiology and medical
research.
 Specimens that can be observed and studied include live microorganisms such as
protozoa, erythrocytes, bacteria, molds and sperm, thin tissue slices, lithographic
patterns, fibers, glass fragments and sub-cellular particles such as nuclei and organelles.

Advantages
 The capacity to observe living cells and, as such, the ability to examine cells in a natural
state
 Observing a living organism in its natural state and/or environment can provide far more
information than specimens that need to be killed, fixed or stain to view under a
microscope
 High-contrast, high-resolution images
 Ideal for studying and interpreting thin specimens
 Ability to combine with other means of observation, such as fluorescence
 Modern phase contrast microscopes, with CCD or CMOS computer devices, can capture
photo and/or video images
 In addition, advances to the phase contrast microscope, especially those that
incorporate technology, enable a scientist to hone in on minute internal structures of a
particle and can even detect a mere small number of protein molecules.
Disadvantages
 Annuli or rings limit the aperture to some extent, which decreases resolution
 This method of observation is not ideal for thick organisms or particles
 Thick specimens can appear distorted
 Images may appear grey or green, if white or green lights are used, respectively,
resulting in poor photomicrography
 Shade-off and halo effect, referred to a phase artifacts
 Shade-off occurs with larger particles, results in a steady reduction of contrast moving
from the center of the object toward its edges
 Halo effect, where images are often surrounded by bright areas, which obscure details
along the perimeter of the specimen
 Modern advances and techniques provide solutions to some of these confines, such as
the halo effect.
 The pros that phase contrast has brought to the field of microscopy far exceed its
limitations. This is easily seen with the myriad of advances in the fields of cellular and
microbiology as well as in medical and veterinary sciences.
Application
 Ideal for examination of live cells and tissues.
 The nucleus in a cell for example shows up darkly against the surrounding cytoplasm.
 Suitable to examine the cells grown in artificial media by tissue culture technique.
 The changes caused by chemical and physics factors on live cells can be studied by using
PCM.
 The changes that occur during cell division can be studied.
 The nature and development of membranes and ultra-outgrowths can be studied.
 The empty spaces that arise in the cells and their nature can be studied.
Fluorescence microscope

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. 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.
George G. Stokes was first to describe this phenomenon.
Stokes Shift: the mineral Fluorspar emitted red light when illuminated with UV light. Such a
substance is known as fluorophore and the shift in wavelength is called Stokes Shift.
Principle
 The specimen is illuminated with light of a specific wavelength (or wavelengths) which is
absorbed by the fluorophores, causing them to emit light of longer wavelengths (i.e., of a
different colour than the absorbed light).
 The illumination light is separated from the much weaker emitted fluorescence through
the use of a spectral emission filter. Typical components of a fluorescence microscope
are a light source (xenon arc lamp or mercury-vapor lamp are common; more advanced
forms are high-power LEDs and lasers), the excitation filter, the dichroic mirror (or
dichroic beamsplitter), and the emission filter (see figure below).
 The filters and the dichroic are chosen to match the spectral excitation and emission
characteristics of the fluorophore used to label the specimen.
 In this manner, the distribution of a single fluorophore (colour) is imaged at a time.
Multi-colour images of several types of fluorophores must be composed by combining
several single-color images.
Fluorescence Light Microscope has the same basic construction of light compound
microscopes with the following additional parts.

Lamp house unit
Here uses a xenon or mercury vapour arc lamp as a light source. The lamp house unit
includers a condenser lens that controls the path of light.
The lamps produce short wavelength light of very high intensity. There is provision to
vary the intensity from low to very high levels.
Complementary Filters
Fluorescence Light Microscope differ from the conventional microscopes in having a pair
of complementary filters called Excitation & emission Filter.
Excitation filter
one filter is inserted in the pathway of illumination to give a monochromatic light for the
excitation of Fluorescence.is called Excitation filter.
Emission filter
The second filter is used in the body tube of microscope to prevent the excitation light
from reaching the observer’s eyes while permitting the longer wavelengths emitted by
the fluorescent object, so that the fluorescence will be seen against a dark background.
This filter is called emission filter or barrier filter.
Dichroic Mirror
It’s place in body tube between the objective below and the emission filter above in the
path of light. It is oriented at 45 so that it can deflect the excitation light down through
the objective on to specimen to reach ocular through the emission filter.
Digital Camera System
Expensive models are provide with digital camera system to document the image
obtained as digital data.
There are two types of fluorescence microscopes based on the positioning of the lamp
house unit
1} Trans illumination Fluorescence Microscope

 In this type, the lamp house unit is provided as a substage arrangement. The excited light
is transmitted through the specimen. So this type is called trans illumination
fluorescence microscope.
 The fluorochromes present in the specimen emit fluorescence. Therefore, in trans
illumination fluorescence microscope both the excitation light and emission light travel
through the objective and reach the emission filter.
 The emission filter allows only the fluorescence to pass through and prevent the
excitation light from reaching the ocular.
2} Epi illumination Fluorescence Microscope
 In this type, the lamp house unit is located above the stage.it uses a mercury vapour
lamp as light source.
 The excitation filter attached with the light source lets out the excited light to fall on the
dichroic mirror placed at a 45 angle in the body tube. The mirror deflects the excitation
light downwards through the objective lens system.
 This emission light travels upwards through the objective .The dichroic mirror allows the
emission to traverse it and reach the emission filter.
 Thus, in epi illumination fluorescence microscope the separation of excitation light from
the emission light is fully achieved.
Working
 Light of the excitation wavelength is focused on the specimen through the objective lens.
The fluorescence emitted by the specimen is focused to the detector by the same
objective that is used for the excitation which for greatest sensitivity will have a very high
numerical aperture.
 Since most of the excitation light is transmitted through the specimen, only reflected
excitatory light reaches the objective together with the emitted light and the
epifluorescence method therefore gives a high signal to noise ratio.
 An additional barrier filter between the objective and the detector can filter out the
remaining excitation light from fluorescent light.
 Many different fluorescent dyes can be used to stain different structures.one particularly
powerful method is the combination of antibodies coupled to a fluorochrome as in
immunostaining. Example of fluorochromes are fluorescin or Rhodamine.
Application
 It’s useful in studying the location of molecules in the cell & their movements.
 To study the auto fluorescent cell components.
 Fluorophores are used as markers to locate and study the cell components that lack
autofluorescence.
 In recent years, antibodies labelled with fluorescent markers are used to locate specific
proteins in the cells. Thus, fluorescence microscopy has lead to the development of a
new technique called immunofluorescence.
 In recent work, highly efficient fluorescent proteins such as the Green Fluorescent
Protein (GFP) Have been specifically fused on the DNA level to the protein of interest.
Darkfield Microscope

 Dark field microscopy is used to examine live microorganisms that either invisible in the
ordinary light microscope, cannot be stained by standard method. So distorted by
staining that their characteristics then cannot be identified.
 In Dark field microscope uses a dark fild condenser that contain a opaque disc. The disc
blocks light that would enter the lens directly, only the light is reflected off the specimen
enters the objective lens. Because there is no background light the specimen appears
light against black background the dark field.
Principle
 The dark ground microscope creates a contrast between the object and the surrounding
field such that the background is dark and object is bright.
 The objective and ocular lenses used in the dark ground microscope are the same as in
the ordinary light microscope. However a special condenser is used, which prevent the
transmitted light from directly illuminating the specimen.
 Only oblique scattered light reaches the specimen and passes onto the lens system
causing the object to appear bright against a dark background.
The clarity of the image depends on the contrast between the image and field of vision.
the contrast can be enhanced by combination of following ways.
1] Staining the specimen if it does not have colour of its own
2]controlling intensity of illumination
 When light hits an object, rays are scattered in all directions. The design of the dark field
microscope is such that it removes the dispersed light. So that only the scattered beams
hit the sample.
 The introduction of a condenser and stop below the stage ensures that these light rays
will hit the specimen at different angles, rather than a as direct light source above/below
the object.
 The result is a “cone of light” where rays are diffracted reflected and refracted off the
object, ultimately allowing the individual to view a specimen in dark field.
Working
 Light reaches the object mounted on the stage through the condenser.
 The condenser lens focuses the light towards the sample.

 The patch stop provided in the condenser lens blocks light around the central region
allowing light to pass only the periphery of the lens.
 The light that enters the specimen, most id directly transmitted while some is scattered
from the sample
 The scattered light alone enters the objective lens and produce the image.
 The directly transmitted light is not collected and is omitted.
 Thus, the field of vison is rendered dark and the image created is by the scattered light.
therefore cell components that reflect light are clear in the image.
Rheinberg Illuminationis is a special variant of dark field illumination and is named after
its inventor, Julius Rheinberg. In this variant, Trasparent coloured filters are inserted just
before the condenser. So that light rays at high aperture are differently coloured than
those at low aperture. other colour combination are possible but their effectiveness is
quite variable.
Advantages
 Resolution by dark field microscopy is better than BFM.
 Improves image contrast without the use of staining process.
 Direct detection of nonculturable bacteria present in patient samples.
 No sample preparation is required.
 Requires no special set up, even a light microscope can be converted to dark field.
 DFM is very simple yet effective technique.
 It is well suited for uses involving live and unstained biological samples. Such as a smear
from a tissue culture or single celled organisms.
 Considering the simplicity of the setup, the quality of image obtained from this
technique is impressive.
Disadvantages
 Low light intensity in final image of many biological samples.
 Low apparent resolution due to the blur of out of focus objects.
Application
 It is useful for the demonstration of very thin bacteria not visible under ordinary
illumination.
 Useful for the demon clinical of motility of flagellated bacteria and protozoa.
 Used to study mounted cells and tissues.
 It is more useful in examining external details such as outlines, edges, grain boundaries
and surface defects than internal structure.
Confocal Microscope

 Confocal microscopes produce sharp images of an object which would appear blurred in
the conventional microscopes. Confocal microscopes only include the light from the
object which is from microscope’s focal plane, thereby excluding most of the light
coming from the object.
 The obtained images have less haze along with better contrast than achievable by the
normal microscopes and represent a fine cross section of the object.
 Therefore, in addition to better observation of fine details, this technique also allows to
build three-dimensional (3D) reconstructions of a volume of the object by accumulating
a series of thin slices taken along the vertical axis.
 Marvin Minsky discovered the basic idea of confocal microscopy in mid 1950s while
working at Harvard University. Approximately 10 years later, Egger and Petran developed
a spinning disk, multiple-beam confocal microscope. They employed this technique to
examine unstained brain sections and ganglion cells.
 Working in this field, Egger developed earliest mechanically scanned confocal laser
microscope and took first recognizable images of cells in 1973.
 Scanning confocal microscope was devised by G. Fred Brakenhoff in 1979. By providing
explanation for image formation, Colin Sheppard greatly contributed to the development
of the technique. Confocal Microscopy equipment was first commercialized in 1987.
 In laser scanning confocal microscopy(LSCFM), the image of an extended specimen is
generated by scanning the focused beam across a defined area in a raster pattern
controlled by two high-speed oscillating mirrors driven by galvanometer motors.

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