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Different types of microscopes
 

Different types of microscopes

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microscopy in pathology

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    Different types of microscopes Different types of microscopes Presentation Transcript

    • DIFFERENT VARIANTS OF MICROSCOPE Dr Uttam Kumar Das PGT-1 Dept Of Pathology BSMC Bankura
    • BASICS  Visible Light: Electromagnetic spectrum detected by human eye- 400nm(deep violet) to 800nm(Far red). Human eye doesn’t see much beyond 700nm(deep red).  White light: Mixture of light containing some % of wavelength from all visible portion.
    • BASICS  Color Temperature: Measure of mixture of light given off by light source. Higher the color temp closer is to natural daylight. Natural daylight color temperature~5200kelvin Incandescent light from Tungsten Bulb~3200k Higher color temp- more blue/white to eye. Lower color temp- more red to yellow to eye & regarded as being ‘warmer’ in color.
    • BASICS  The Amplitude (i.e. brightness): Decreases as light gets further from source because of absorption in media it passes.  Energy Level: Energy content of light, expressed as electron volt per photon (the particle representation of light). Visible light-1electron volt/photon. Soft x-ray portion-50-100ev/photon Energy increases moving towards violet & ultraviolet range of spectrum.
    • BASICS  Spherical aberration: Light rays hitting periphery will be more refracted than the rays hitting centre of lens.  Chromatic Aberrations: White light of passing through simple lens, each wavelength will be refracted to different extent. Blue brought to a shorter focus than red, results in a un-sharp image with color fringes. Corrected with ‘Achromat’ & ‘Apochromat’ lens.
    • BASICS  Magnification: For variable tube length=From 160-250mm Optical tube length X Magnification of eye piece Focal length of the objective  Resolving Power: Capacity of the optical system to produce separate images of objects very close to each other. Resolving power of std LM=200nm
    • BASICS  Illumination: Critical illumination-when the light source is focused by condensor in the same plane as the object, when the object is in focus. The illuminating light consists of rays or waves that are nearly parallel to one another.  This quality of light is called partially coherent light. The ultimate resolving power of the microscope depends on the use of partially coherent illumination.  The condenser in Köhler illumination is used to create and focus this light onto the specimen
    • BASICS  Real Image: The real image forms on the side of the lens opposite the object. A slide projector produces a real image on a projection screen. The objective lens produces a real image in the intermediate image plane.
    • BASICS  Virtual Image: A virtual image cannot be seen on a screen. A second lens is required to see a virtual image and this would cause the image to be inverted relative to the specimen. To a human, the virtual image appears to be on the same side of the lens as the object. The image we see in a compound microscope is a virtual image. In upright microscopes the virtual image is inverted. In inverted microscopes the image is made to be upright by optics in the binocular.
    • BASICS  Back Focal Plane: The side of a lens where an image is formed is called the image side or back side of the lens. The plane at one focal length on this side is the back focal plane. The back focal plane of the objective lens is on the inside of the microscope tube.  Coma: Resulting in the images of structures out from the center being smeared outwards.  Astigmatism: Resulting in light in the X plane being focused differently to light in the Y plane.
    • •NUMERICAL APARTURE: • Is a dimensionless number that characterizes the range of angles over which the system can accept or emit light. •It is the acceptance cone of an objective (and hence its light-gathering ability and resolution). The numerical aperture with respect to a point P depends on the half-angle θ of the maximum cone of light that can enter or exit the lens. where n is the index of refraction of the medium in which the lens is working, and θ is the half- angle of the maximum cone of light that can enter or exit the lens BASICS
    • BASICS  Compound Lenses: There are four basic lens shapes: double convex, double concave, plano convex and plano concave. In the 1830’s, the solution to chromatic aberrations was found by cementing two lenses made of different glasses (one Double convex and one Plano-concave) together into an Achromatic Doublet to make a compound lens. Today, compound lens consists of many lens elements. These elements are necessary for magnification and correction of various lens aberrations.
    • REFRACTIVE INDEX •Refractive index is the light-bending ability of a medium. • The light may bend in air so much that it misses the small high-magnification lens. • Immersion oil is used to keep light from bending. RI of Air- 1.0 RI of Water -1.3 RI of Glycerol- 1.47 RI of Glass(avg)- 1.5 RI of Oil- 1.52
    • UNITS OF MEASUREMENT  1 µm micrometer = 1/1000 mm  1 nm nanometer = 1/1000 µm  Parfocal distance: Distance between objective shoulder and specimen(45 mm for most microscopes).  Working Distance Focal Length Scanning- 17-20mm 40mm Low power- 4-8mm 16mm High power- 0.5-0.7mm 4mm Oil immersion- 0.1mm 1.8-2.0mm
    • INFINITY CORRECTED MICROSCOPES  A majority of modern research microscopes are equipped with infinity-corrected objectives that no longer project the intermediate image directly into the intermediate image plane. Light emerging from these objectives is instead focused to infinity, and a second lens, known as a tube lens, forms the image at its focal plane.
    • INFINITY CORRECTED MICROSCOPES  Wavetrains of light leaving the infinity-focused objective are collimated, allowing beam-splitters, polarizers, Wollaston prisms, etc., to be introduced into the space between the objective and tube lens.
    • INFINITY CORRECTED MICROSCOPES  In a microscope with infinity-corrected optics, magnification of the intermediate image is determined by the ratio of the focal lengths of the tube lens and objective lens.  Because the focal length of the tube lens varies between 160 and 250 millimeters (depending upon the manufacturer and model), the focal length of the objective can no longer be assumed to be 160 millimeters divided by its magnification.  Thus, an objective having a focal length of 8 millimeters in an infinity-correct microscope with a tube lens focal length of 200 millimeters would have a lateral magnification of 25x (200/8).
    • MODERN TECHNOLOGY IMPROVING MICROSCOPY  The invention of the microscope allowed scientists and scholars to study the microscopic creatures in the world around them.  When learning about the history of the microscope it is important to understand that until these microscopic creatures were discovered, the causes of illness and disease were theorized but still a mystery.  The microscope allowed human beings to step out of the world controlled by things unseen and into a world where the agents that caused disease were visible, named and, over time, prevented.
    • MICROSCOPES CAN BE SEPARATED INTO SEVERAL DIFFERENT CLASSES  One grouping is based on what interacts with the sample to generate the image: -Light or photons(optical microscopes) -Electrons (electron microscopes) -Probe (scanning probe microscopes).  Whether they analyze the sample via: -Scanning point (confocal optical microscopes, scanning electron microscopes and scanning probe microscopes) -Analyze the sample all at once (wide field optical microscope and transmission electron microscopes).
    • BRIGHT-FIELD MICROSCOPY  The useful magnification of Light microscope is limited by its resolving power.  The resolving power in limited by wavelength of illuminating beam.  Resolution is determine by certain physical parameters like wave length of light and light generating power of the objective & condenser lens.  Higher N.A Better light generation Better Resolution Shorter the Wavelength Better Resolution.  The ordinary microscope is called as a bright field microscope. It forms dark image against bright background.
    • Dark objects are visible against a bright background. Light reflected off the specimen does not enter the objective lens.
    • ADVANTAGES  Bright field compound microscopes are commonly used to view live and immobile specimens such as bacteria, cells, and tissues.  For transparent or colorless specimens, however, it is important that they be stained first so that they can be properly viewed under this type of a microscope.  Staining is achieved with the use of a chemical dye. By applying it, the specimen would be able to adapt the color of the dye. Therefore, the light won’t simply pass through the body of the specimen showing nothing on the microscope’s view field
    • DARK FIELD MICROSCOPY
    • PRINCIPLE  When observing unstained/living cells, such specimens & their components have refractive indices close to that of the medium in which they are suspended & are thus difficult to see by bright field techniques, due to their lack of contrast.  Dark field microscopy overcomes these problems by preventing direct light from entering the front of the objective & the only light gathered is the reflected or diffracted by structures within the specimen. This causes the specimen to appear as a bright image on a dark background, the contrast being reversed & increased.  Dark field permits the detection of particles smaller than the optical resolution that would be obtained in bright field, due to high contrast of scattered light.  In the microscope, oblique light is created by using a modified or special condensor that form a hollow cone of direct light which will pass through the specimen but outside the objective.
    • DARK FIELD MICROSCOPY  Light enters the microscope for illumination of the sample.  A specially sized disc, the patch stop blocks some light from the light source, leaving an outer ring of illumination. A wide phase annulus can also be reasonably substituted at low magnification.  The condenser lens focuses the light towards the sample.
    • DARK FIELD MICROSCOPY  The light enters the sample. Most is directly transmitted, while some is scattered from the sample.  The scattered light enters the objective lens, while the directly transmitted light simply misses the lens and is not collected due to a direct illumination block.  Only the scattered light goes on to produce the image, while the directly transmitted light is omitted.
    • DARK FIELD MICROSCOPY  Dark field microscopy is a very simple yet effective technique and well suited for uses involving live and unstained biological samples: -Spirochetes -Flagellates -Cell Suspension -Flow Cell Techniques -Parasites -Autoradiographic grain counting -Once used in Fluorescence Microscopy
    • DARK FIELD MICROSCOPY  In Dark Field Illumination the objective must have a lower Numerical Aperture than the condensor.  In Bright Field Illumination optimum efficiency is obtained when the Numerical Aperture of both the Objective and the Condensor are matched.  Dark Field Condensor may be: -Dry, low power objective -Oil immersion, high power objective
    • ADVANTAGES  The advantage of darkfield microscopy also becomes its disadvantage: not only the specimen, but dust and other particles scatter the light and are easily observed For example, not only the cheek cells but the bacteria in saliva are evident. The dark field microscopes divert illumination and light rays thus, making the details of the specimen appear luminous.  Dark field light microscopes provide good results, especially through the examination of live blood samples. It can yield high magnifications of living bacteria and low magnifications of the tissues and cells of certain organisms. Certain bacteria and fungi can be studied with the use of dark field microscopes.
    • THANKYOU
    • PHASE CONTRAST MICROSCOPY
    • DEFINITION  Phase contrast microscopy is an optical illumination technique in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image. The technique was invented by Frits Zernite in the 1930s for which he received the Nobel prize in physics in 1953 .
    • Accentuates diffraction of the light that passes through a specimen. Direct and reflected light rays are combined at the eye. Increasing contrast PHASE CONTRAST MICROSCOPY
    • APPLICATION  Applications for phase contrast microscopy equipment range from the study of living biological specimens, medical applications, study of live blood cells, and other biological and science applications.  Most commonly used to provide contrast of transparent specimens such as living cells or small organisms.  Useful in observing cells cultured in vitro during mitosis.
    • PHASE CONTRAST MICROSCOPY  Unstained/living biological specimens have little contrast with their surrounding medium. To see them clearly involves: A-Closing down the iris diaphragm of condensor which decreases the Numerical Aperture producing diffraction effects & destroy the Resolving Power of the Objective. B-Or when using dark field illumination, which enhances contrast by reversal, but often fails to reveal internal details.  The Phase Contrast microscopy overcomes these problems by controlled illumination using the full aperture & thus improving the Resolution. The high the refractive index of the structure, the darker it will appear against the light background, i.e. with more contrast.
    • PHASE CONTRAST MICROSCOPY  If a diffraction grating is examined under the microscope, diffraction spectra are formed in the Back Focal Plane of the objective due to interference between the direct & diffracted rays of light.  The grating consists of alternate strips of material with slightly different refractive indices, through which light acquire small phase difference & these form the images.
    • PHASE CONTRAST MICROSCOPY  By converting the phase differences, between light passing through a specimen and that passing through the surrounding medium, into amplitude (brightness) differences, phase contrast microscopy provides a difference in brightness between the object and the background, which the eye can then see.
    • PHASE CONTRAST MICROSCOPY  Phase contrast microscopy uses an annular stop in the condenser and a phase plate within the objective lens, which is aligned with the annular stop. In this configuration, the light path can be split and each of the separated beams will pass through the same transparent medium at the specimen stage. The light passes through the annular stop and forms a cone of light, which comes to its vertex at the focal point of the specimen. Any background light, which is un-deviated by the specimen, will go through the phase ring in the phase plate and is advanced by a quarter of a wavelength. Deviated light passing through the specimen is retarded by a quarter of a wavelength and passes through the phase plate without going through the ring.
    • PHASE CONTRAST MICROSCOPY  When the beams are recombined further along the light path, the differences in the phase of the deviated and un-deviated light beams become additive and subtractive.  The resultant wave is the sum of the two waves which have their crests and troughs opposite each other. The difference in amplitude can be seen as a change in brightness, since brightness is proportional to the square of the amplitude. The net result is that features of the object are either lighter or darker than the surrounding field.
    • Plane Light Polarized Light High Relief
    • HOW IS LIGHT POLARIZED
    • PRINCIPLE  Light is a series of pulse of energy radiating away from a source & shown diagrammatically as sine curve, with wavelength & amplitude.  Light can also be described as electromagnetic vibration, which travels outwards from source of its propagation, much in the same way as vibration along a rope.  Natural light vibrates in many directions but polarized light in only one direction.
    • POLARIZED LIGHT MICROSCOPY  Polarization can be achieved with the use of substance/crystals which allow vibration only in one plane & called birefringent.  Light entering a birefringent crystal such as calcite is split into two light paths, each determined by a different refractive index & each vibrating in one direction only, but at right angle to each other.  The higher the RI, the greater the retardation of the ray, so that each ray leaves the crystal at a different velocity.  The high RI ray is called slow and low RI ray is called fast.  There is also a phase difference between the rays, so that if they are recombined interference occurs & various spectral colors are seen.
    • POLARIZED LIGHT MICROSCOPY  Two discs made up of prism are placed in a path of light, one below the objective known as Polarizer & another placed in the body tube which is known as Analyzer.  Polarizer sieves out ordinary light rays vibrating in all directions allowing light waves of one orientation to pass through.  The lower disc (polarizer) is rotated to make the light plane polarized. During rotation, when analyzer comes perpendicular to polarizer, all light rays are cancelled or extinguished.  Birefringent objects rotate the light rays & therefore appear bright in a dark background.
    •  Analyzer (upper polarizer) -- a polarizing prism located above the microscope stage, between the objective lens and the eyepiece. This restricts the transmission of light vibrating perpendicular to the polarizer. The analyzer can be slipped in or out of the light path or rotated for partially crossed polarized light. Light passing through the polarizer will not pass through the analyzer unless the vibration direction of the light is changed between the two prisms. Anisotropic minerals can perform this deed.  Polarizer (lower polarizer) -- a polarizing prism located beneath the microscope stage (between the light source and the object of study). This restricts transmission of light to that vibrating in only one (N-S) direction. Some microscopes have a different orientation direction. In effect, it plane polarizes the incident light beam.
    • POLARIZED LIGHT MICROSCOPY  Numerous crystals, fibrous structures (both natural & artificial), pigment, lipid, protein, bone & amyloid deposits exhibit birefringence.  Originally, polarizer's, made from calcite & known as Nicol Prism, were cemented together with Canada balsam in such away that the slow rays was deflected away from the optical path & into the mount of the prism, leaving only the polarized fast rays to pass through (optic axis).  Some substances & crystals can produce plane polarized light by differential absorption & give rise to phenomenon of Dichorism. These absorb the slow rays & are pleochoric (absorbing all colors equally) and are the most useful in microscopy. Such crystals suspended in thin plastic films & oriented in one direction have replaced bulky Nicol Prism.
    • POLARIZED LIGHT MICROSCOPY  Two phenomenon detected in polarized light- Birefringence & Dichroism.  When a birefringent substance rotated between two crossed polarizers, the image appears & disappears four times each in 360 deg rotation (at 45̊ each).  Dichroism- Only the polarizer is used &, if no rotatory stage is available the polarizer itself can be rotated. Changes in intensity & color is seen during rotation at 90 deg. This is due to differential absorption of light, depending upon the vibration direction of the two rays in a birefringent substance.  Weak birefringence in biological specimen is increased by addition of dyes or impregnating metals, in a orderly linear alignment e.g. amyloid fibrils.
    • DIFFERENTIAL INTERFERENCE CONTRAST
    • DIFFERENTIAL INTERFERENCE CONTRAST  In this complex form of polarised light microscopy two slightly separate, plane polarised beams of light are used to create a 3D-like image with shades of grey.  Wollaston prisms situated in the condenser and above the objective produce the effect, and additional elements add color to the image.  Care must be taken to interpreting DIC images as the apparent hills and valleys in the specimen can be misleading. The height of a "hill" (e.g. the nucleus) is a product of both the actual thickness of the feature (i.e. ray path length) and its refractive index.  Variations of the DIC system are named after their originators, Nomarski and de Senarmont. Options can be selected to maximize either resolution or contrast.
    • Accentuates diffraction of the light that passes through a specimen; uses two beams of light. Adding color Differential Interference Contrast Microscopy
    • ELECTRON MICROSCOPY
    • INTRODUCTION  Electron microscope is a type of microscope that uses a particle beam of electrons to illuminate a specimen & create a highly-magnified image. Co- invented by Germans, Max Knoll and Ernst Ruska in 1931
    • ELECTRON GUN  2 types of guns are used in electron microscopy- Thermionic Emission Gun & Field Emission Gun.  Thermionic: Electrons emitted from heated filament ( tungsten, Lanthanum Hexaboride). Most common, cheap & ultra high vacuum not required.  Field Emission: Strong electron field used to extract electrons from filament. High vacuum needed.
    • ELECTROMAGNETIC LENS  An electromagnet designed to produce a suitably shaped magnetic field for focusing & deflection of electrons in electron optical instruments.  A strong magnetic field is generated by passing a current through a set of windings.  This field acts as a convex lens in case of electron microscope.
    • CONDENSER LENS  The first lens(controlled by "spot size knob") largely determines the "spot size"; the general size range of the final spot that strikes the sample.  Second condenser lens: The second lens(controlled by the "intensity/ brightness knob" changes the size of the spot on the sample; changing it from a wide dispersed spot to a pinpoint beam.
    • OTHER PARTS  The other parts include: -Condenser aperture, -Objective lens, -Objective aperture, -Selected area aperture(to examine diffraction patterns), -Intermediate lens(magnifies initial image formed by objective lens) & -Projector lens.
    • TYPES OF ELECTRON MICROSCOPES  There are 2 types of electron microscopes:  Transmission Electron Microscope: Process is carried out under vacuum to avoid friction. The "Virtual Source" at the top represents the electron gun, producing a stream of monochromatic electrons. The usual potential is around 10000 – 15000V.  SCANNING ELECTRON MICROSCOPE
    • TRASMISSION ELECTRON MICROSCOPY
    • •Ultrathin sections of specimens. •Light passes through specimen, then an electromagnetic lens, to a screen or film. •Specimens may be stained with heavy metal salts. Transmission Electron Microscopy (TEM)
    • TRANSMISSON ELECTRON MICROSCOPE  This transmitted portion is focused by the objective lens into an image.  The Objective & Selected Area metal apertures restrict the beam.  The image is passed down the column through the intermediate and projector lenses, being enlarged all the way.  The image strikes the phosphor image screen & light is generated, allowing the user to see the image.  The darker areas represent areas that fewer electrons were transmitted (thicker or denser). The lighter areas represent areas that more electrons were transmitted (thinner or less dense)
    • TRANSMISSION ELECTRON MICROSCOPE  Transmission electron microscopy (TEM) involves a high voltage electron beam emitted by a cathode and formed by magnetic lenses. The electron beam that has been partially transmitted through the very thin (and so semitransparent for electrons) specimen carries information about the structure of the specimen.  The spatial variation in this information (the "image") is then magnified by a series of magnetic lenses until it is recorded by hitting a fluorescent screen, photographic plate, or light sensitive sensor such as a CCD (charge-coupled device) camera. The image detected by the CCD may be displayed in real time on a monitor or computer.  Transmission electron microscopes produce two-dimensional, black and white images.  Resolution of the TEM is also limited by spherical and chromatic aberration, but a new generation of aberration correctors has been able to overcome or limit these aberrations.
    • SCANNING ELECTRON MICROSCOPE
    • •An electron gun produces a beam of electrons that scans the surface of a whole specimen. •Secondary electrons emitted from the specimen produce the image. Scanning Electron Microscopy (SEM)
    • SCANNING ELECTRON MICROSCOPE  Unlike the TEM, where the electrons in the primary beam are transmitted through the sample, the Scanning Electron Microscope (SEM) produces images by detecting secondary electrons which are emitted from the surface due to excitation by the primary electron beam.  In the SEM, the electron beam is scanned across the surface of the sample in a raster pattern, with detectors building up an image by mapping the detected signals with beam position.
    • SCANNING ELECTRON MICROSCOPE  The 1st scanning electron microscope (SEM) debuted in 1938 by Von Ardenne with the first commercial instruments out around 1965.  In this case electrons are not used to directly image the specimen, but to excite it in such a way that it gives out secondary electrons which are collected by detectors & used to form the image.  The first scanning electron microscope (SEM) debuted in 1938 ( Von Ardenne) with the first commercial instruments around 1965.
    • SPECIMEN  For electron microscopy, tissue is fixed in 4% glutaraldehyde at 4°C for 4 hours.  Fixation - In chemical fixation for electron microscopy, glutaraldehyde is often used to crosslink protein molecules and osmium tetroxide to preserve lipids.  Dehydration - Organic solvents such as ethanol or acetone for SEM specimens or infiltration with resin and subsequent embedding for TEM specimens.  Embedding - Resin (for electron microscopy) such as araldite or LR White, which can then be polymerised into a hardened block for subsequent sectioning.
    • SPECIMEN  Sectioning - Typically around 90nm cut on an ultramicrotome with a glass or diamond knife. Glass knives can easily be made in the laboratory and are much cheaper than diamond, but they blunt very quickly and therefore need replacing frequently.  Staining - uses heavy metals such as lead and uranium to scatter imaging electrons and thus give contrast between different structures, since many (especially biological) materials are nearly "transparent" to the electron beam.  By staining the samples with heavy metals, electron density is added to it which results in there being more interactions between the electrons in the primary beam and those of the sample, which in turn provides us with contrast in the resultant image.
    • ARTIFACTS  It must be emphasized from the outset that every electron micrograph is, in a sense, an artifact.  Artifacts present themselves in many ways: -There could be loss of continuity in the membranes, -Distortion or disorganization of organelles, -Empty spaces in the cytoplasm of cells or sharp bends or curves in filamentous structures that are usually straight, such as microtubules and so on.  With experience, microscopists learn to recognize the difference between an artifact of preparation and true structure.
    • DISADVANTAGES OF ELECTRON MICROSCOPY  Electron microscopes are very expensive to buy and maintain.  They are dynamic rather than static in their operation: requiring extremely stable high voltage supplies, extremely stable currents to each electromagnetic coil/lens, continuously-pumped high/ultra-high vacuum systems and a cooling water supply circulation through the lenses and pumps.  As they are very sensitive to vibration and external magnetic fields, microscopes aimed at achieving high resolutions must be housed in buildings with special services.  A significant amount of training is required in order to operate an electron microscope successfully and electron microscopy is considered a specialised skill.  The samples have to be viewed in a vacuum, as the molecules that make up air would scatter the electrons. This means that the samples need to be specially prepared by sometimes lengthy and difficult techniques to withstand the environment inside an electron microscope.  Recent advances have allowed some hydrated samples to be imaged using an environmental scanning electron microscope, but the applications for this type of imaging are still limited.
    • CONFOCAL MICROSCOPY
    • CONFOCAL MICROSCOPY
    • PRINCIPLE  Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane.  It enables the reconstruction of three- dimensional structures from the obtained images.
    • •Uses fluorochromes and a laser light. •The laser illuminates each plane in a specimen to produce a 3-D image. Confocal Microscopy
    • INTRODUCTION  Although conventional light and fluorescence microscopy allow the examination of both living and fixed specimens, certain problems exist with these techniques. -One of the main problems is out-of-focus blur degrading the image by obscuring important structures of interest, particularly in thick specimens.  In conventional microscopy, not only is the plane of focus illuminated, but much of the specimen above and below this point is also illuminated resulting in out-of-focus blur from these areas.  This out-of-focus light leads to a reduction in image contrast and a decrease in resolution.
    • APPLICATIONS IN CONFOCAL MICROSCOPY  The broad range of applications available to laser scanning confocal microscopy includes a wide variety of studies in neuroanatomy and neurophysiology, as well as morphological studies of a wide spectrum of cells and tissues.  In addition, the growing use of new fluorescent proteins is rapidly expanding the number of original research reports coupling these useful tools to modern microscopic investigations.
    • OTHER APPLICATIONS  Include- -Resonance energy transfer, -Stem cell research, -Photobleaching studies, -Lifetime imaging, -Multiphoton microscopy, -Total internal reflection, -DNA hybridization, -Membrane and ion probes, -Bioluminescent proteins, and -Epitope tagging.
    •  Confocal reflection microscopy can be utilized to gather additional information from a specimen with relatively little extra effort, since the technique requires minimum specimen preparation and instrument re-configuration.  In addition, information from unstained tissues is readily available with confocal reflection microscopy, as is data from tissues labeled with probes that reflect light.  The method can also be utilized in combination with more common classical fluorescence techniques.  Examples of the latter application are detection of unlabeled cells in a population of fluorescently labeled cells and for imaging the interactions between fluorescently labeled cells growing on opaque, patterned substrata
    • FLUORESCENCE MICROSCOPY
    • FLUORESCENT MICROSCOPY  This method is used for demonstration of naturally occurring fluorescent material and other non-fluorescent substances or micro- organisms after staining with some fluorescent dyes e.g. Mycobacterium tuberculosis, amyloid, lipids, elastic fibres etc.  UV light is used for illumination.
    • •Uses UV light. • Fluorescent Substances absorb UV light and emit visible light. • Cells may be stained with fluorescent dyes (fluorochromes). Fluorescence Microscopy
    • WHY FLUORESCENCE MICROSCOPY?  In all types of microscopes, cell constituents are not distinguishable, although staining dose, but not totally.  In fluorescent microscopy, various fluorescent dyes are used which gives property of fluorescence to only specific part of the cell and hence it can be focused.  Fluorescent microscopy depends upon illumination of a substance with a specific wavelength (UV region i.e. invisible region) which then emits light at a lower wavelength (visible region).
    • FLUORESCENCE PRINCIPLE  When certain compounds are illuminated with high energy light, they then emit light of a different, lower frequency. This effect is known as fluorescence.  Often specimens show their own characteristic autofluorescence image, based on their chemical makeup.  The key feature of fluorescence microscopy is that it employs reflected rather than transmitted light, which means transmitted light techniques such as phase contrast and DIC can be combined with fluorescence microscopy.
    • COMPONENTS  Typical components of a fluorescence microscope are:  the light source (xenon arc lamp or mercury- vapor lamp),  the excitation filter,  the dichroic mirror and  the emission filter.
    • STAINING  Many different fluorescent dyes can be used to stain different structures or chemical compounds.  One particularly powerful method is the combination of antibodies coupled to a fluorochrome as in immunostaining.  Examples of commonly used fluorochromes are fluorescein or rhodamine.
    • FUNCTIONING  A component of interest in the specimen is specifically labeled with a fluorescent molecule called a fluorophore.  The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit longer wavelengths of light (of a different color than the absorbed light).
    • APPLICATIONS  Fluorescence microscopy is a critical tool for academic and pharmaceutical research, pathology, and clinical medicine.
    • The distance between the focal plane of the objective (f1) and the focal plane of the eyepiece (f2) is called the tube length (l). The object to be viewed is placed just outside the focal point at the left side of the objective lens. The enlarged image is formed at a distance l + f1 from the objective.
    • X-RAY MICROSCOPY
    • X-RAY MICROSCOPY  less common,  developed since the late 1940s,  resolution of X-ray microscopy lies between that of light microscopy and the electron microscopy.  X-rays are a form of electromagnetic radiation with a wavelength in the range of 10 to 0.01 nanometers, corresponding to frequencies in the range 30 PHz to 30 EHz.
    • RECENT ADVANCES IN MICROSCOPY
    • RECENT ADVANCES IN MICROSCOPY  In the recent times, computers and chip technology have helped in developing following advances in microscopy.
    • IMAGE ANALYSERS AND MORPHOMETRY  In these techniques, microscopes are attached to video monitors and computers with dedicated software systems.  Microscopic images are converted into digital images and various cellular parameters (e.g. nuclear area, cell size etc) can be measured. This quantitative measurement introduces objectivity to microscopic analysis.
    • TELEPATHOLOGY (VIRTUAL MICROSCOPY)  It is the examination of slides under microscope set up at a distance. This can be done by using a remote control device to move the stage of the microscope or change the microscope field or magnification called as robobic telepathology.  Alternatively and more commonly, it can be used by scanning the images and using the highspeed internet server to transmit the images to another station termed as static telepathology. Telepathology is employed for consultation for another expert opinion or for primary examination
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