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Uploaded byNistarini College, Purulia (W.B) India
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Comprehensive Principles and Applications of Various Microscopy Techniques

Explore different microscopy types including simple, compound, phase contrast, fluorescence, and electron microscopes, their principles, components, and scientific uses in biological and medical research.

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PRINCIPLES OF MICROSCOPY: DFFERENT TYPES AND USES Dr. Nandadulal Sannigrahi, Associate Professor, Department of Botany, Nistarini College, Purulia D.B. Road, Purulia, INDIA (W.B)
INTRODUCTION  Observation delicately in any scientific enquiries is very important for study and research,  The observation of biological objects in general and small one in particular needs much magnifications for the same,  Microscope is a device is used for getting the magnification of the object supposed to be observed before study and research,  Light microscopy is a general term used for any type of microscopy where light is being transmitted from a source which is on the opposite side of the sample, to the objective lens.  Generally, the light is passed through a condenser to focus it on the sample to have maximum brightness to form an image of the object,  After the light has passed through the sample, it goes through the objective lens to magnify the image of the sample & then to the oculars, where the enlarged image is viewed for the different purposes in scientific research,  The delicate observation through images can give deep insight about the objects under study.
SIMPLE MICROSCOPE • The most common type of microscope is simple microscope,  A simple microscope is a type of microscope that uses a single lens for magnification. It uses a single convex lens of a small focal length for magnification. In general, its magnification is about 10X. Its magnifying power (m) is given by the formulae; m=1+ D/F where, D = least distance of distinct vision F = focal length of the lens of a microscope.  when a sample is placed in the focus of the convex lens of a microscope, a virtual, erect, and magnified image is formed at the least distance of the distinct vision. Parts of a simple microscope; mirror as illuminator, convex lens for magnification, stage and metallic stand with base.  Used to study morphology of insects, algae, and fungi & Used in studying soil type and components,  Have very low magnification,  Up to 10X Mirror for illumination and lack of mechanical stage.
SIMPLE MICROSCOPE
COMPOUND MICROSCOPE  Compound microscope is compound being two lenses in operation simultaneously,  Compound Microscope is a type of microscope that used visible light for illumination and multiple lenses system for magnification of specimen. Generally, it consists of two lenses; objective lens and ocular lens.  It can magnify images up to 1000X. Its magnifying power is equal to the product of magnifying power of the objective lens in use and the ocular lens. Mathematically it is expressed as; m= D/f0 x L/fe where, m = magnifying power, D = least distance of distinct vision, L = length of the tube, fe = focal length of the ocular lens, f0 = focal length of objective lens. It is the most widely used microscope in biological fields like medicine, microbiology, life-sciences, pathology, hematology, anatomy, molecular biology, etc.
COMPOUND MICROSCOPE
COMPOUND MICROSCOPE  When light is focused through a condenser on a specimen placed on stage, the light transmitted by the specimen is picked by the objective lens.  A magnified image is formed at the body tube. This is called the primary image. The light bends in the body tube and passes through the ocular lens. When passing through the ocular lens, the image is magnified for the second time.  This is called the secondary image. Finally, a highly double magnified image is formed at a distance of distinct vision.  Consists of the following main parts-  Illuminator (Light Source), Diaphragm (Iris), Condenser, Condenser Focus Knob, Rack Stop Stage, Stage Control Knobs, Nose Piece, Objective Lens, Tube (Head), Eyepiece (Ocular Lens) ,Diopter Adjustment, Adjustment knobs-coarse and fine etc.  Used in microbiology to study the morphology of microorganisms,  Used in histopathology to study tissue,  Used in cytology to study cellular structure of different types of cells and to Use by biologist to observe slides of cells, tissues or segments of biological components during the different purposes.
PHASE CONTRAST MICROSCOPE  Phase Contrast Microscope is an optical microscope that converts small phase shifts in light into differences in light intensity developing more contrast in images that can be easily detected by human eyes.  When light passes through transparent specimens a small phase shift occurs which can’t be detected by our eyes.  Using phase plates, these small phase shifts are converted to changes in the amplitude of light. This change in amplitude can be observed as differences in image contrast.  It can be used for observing living cells in their natural state without staining or fixing. Transparent specimens and sub-cellular organelles can be clearly viewed with better contrast.  Due to the difference in thickness and refractive index of different parts of a specimen, a small phase shift in light rays occurs when the light passes through the specimens.  This phase shift can be changed into differences in light intensity (brightness) which will produce more contrast in the image.
PHASE CONTRAST MICROSCOPE
PHASE CONTRAST MICROSCOPE  Light from the illuminator is focused on the specimen through the condenser annulus. This light passes through different regions of the specimen having different refractive indexes and thicknesses.  The light rays that pass through an area of higher refractive index and thickness, will experience larger phase retardation than those rays passing through an area of lower refractive index and thickness.  These phase shifts are undetectable to the normal human eye. An optical device like a phase plate converts these phase shifts into brightness change which creates observable contrast differences in the final image.  It contains all the parts of a compound microscope, and additionally contains two optical parts, condenser annulus, and phase plate, for phase contrast.  Observing living cells in its natural form Used in microbiology to observe protozoan, diatoms, planktons, cysts, helminthes and larvae.  Used to study sub cellular structures and cellular processes  Used to study thin tissue slices Used to study lithographic pattern and latex dispersion, glass fragments and crystals.
FLUORESCENCE MICROSCOPE  Fluorescence Microscope is an optical microscope that uses fluorescence or phosphorescence to generate an enlarged image of a specimen. It is a modified light microscope.  This microscope can be used to study living cells and cell organelles, identify specific proteins, antigens and immunoglobulin. They have very high sensitivity.  It works on the principle of fluorescence. When monochromatic light is passed on an object stained with a fluorophore, it reemits the light. The emitted light is detected to form an enlarged image of the specimen.  The specimen is stained with a fluorophore and placed on the stage. High energy light is generated and passed through an excitation filter. This filter allows only the light of a particular short wavelength (UV region) capable of exciting the fluorescent molecule to pass through and block all other wavelength light.  The filtered light is reflected to the sample using a dichroic filter. The fluorophore absorbs the light rays which cause the electron to excite in a higher energy state.  The excited electrons return to the ground state releasing the excited energy in form of light rays with a longer wavelength.
FLUORESCENCE MICROSCOPE
FLUORESCENCE MICROSCOPE  The emitted light passes through the dichroic mirror and hits the emission filter. This filter blocks the short-wavelength light and allows longer wavelength light to pass through ocular lenses to a detector system.  In the detector, an enlarged image is formed. The background is observed as dark and the image appears as bright. • Fluorophore (Fluorescent Dye)  These are the chemical compounds that possess the property of fluorescence i.e. re-emit the light upon excitation by light. These are combinations of several aromatic or planar compounds with several pi (π) bonds. Most of them are organic compounds. They stain a wide range of bio-molecules and cellular structures.  Some common fluorophores used are fluorescein, rhodamine, cyanine, antraquinone, acridine orange, acridine yellow, auramine, malachite green, etc. • Light Source  Commonly mercury vapor lamp is used for generating UV light. Besides, xenon arc lamps, high-power LEDs, and lasers are also used. They emit the light of high energy.
ELECTRON MICROSCOPE  It is a microscope that uses accelerated electron, beams instead of light rays, to illuminate the specimen and get the highly magnified image. In this microscope, glass lenses are replaced by electromagnets.  Due to the very short wavelength of electrons, this microscope produces a very high-resolution image with magnification up to 10,000,000X. Very high-quality images with very high contrast, revealing detailed structures are produced.  Specimen up to 0.2 nm can be clearly viewed using an electron microscope.  An electron microscope uses accelerated electrons with a wavelength of about 100,000 times shorter than visible light to illuminate specimens and produce images.  The electron gun, usually a heated tungsten or field emission filament, is used to generate a stream of high voltage (100 – 1000 kV) electrons.  These electrons are accelerated using an anode plate in a vacuum system and focused on the specimen using aperture and electromagnetic lenses.
ELECTRON MICROSCOPE  The electron beam passes through the specimen and interacts with sample components. Upon striking the specimen, the electrons are scattered. The degree of scattering depends on the refractive index or thickness of the specimen.  The scattered electrons from the sample are collected and passed through objective and ocular electromagnetic lenses. These scattered beams are detected and transformed into highly magnified images by the magnetic lenses. • The Electron Microscope consists of the following parts for its high resolution and degree of accuracy:  Electron Gun- Source of electron,  Electromagnetic lens- for high magnifications by allowing electrons of specific energy to pass through and block other electrons with three type of lens-condenser lens, projector lens & objective lens,  Aperture system- pinholes to filter unwanted electrons.
ELECTRON MICROSCOPE
ELECTRON MICROSCOPE
ELECTRON MICROSCOPEs  Sample holder- mechanical arm for holding and positioning specimens in fixed position,  Vacuum system- Highly sensitive vacuum system contains to allow electrons to generate and travel without colliding and scattering from their path,  Imaging system- electromagnetic lenses used for refocusing, enlarging and projecting images of the specimen to form an electron micrograph on a phosphorescent plate of monitor. • There are different types of electron microscopes-  Transmission Electron Microscope ( TEM),  Scanning electron Microscope ( SEM),  Reflection Electron Microscope ( REM),  Scanning Transmission Electron Microscope ( STEM),  Scanning Tunneling Microscope ( STM).
ELECTRON MICROSCOPIC view of POLLEN IMAGES
ELECTRON MICROSCOPE-USES AND LIMITATIONS  Used in microbiology to study structure of viruses, flagella, pili, and bacterial cells.  Used in crystallography, and nanotechnology,  To study morphology of cellular organelles ,  Used in forensics for ballistic study of gunshots,  Used in geology for studying rocks, minerals and gems,  Used in quality control, detection of fracture and cracks, drug development and analysis of atomic structure. • LIMITATIONS  Highly expensive and complex system,  Images are in black and white,  TEM requires very thin specimen,  Need of vacuum system.
OTHER TYPES OF MICROSCOPES • Besides these microscopes, different other type of microscopes are used as follows:  Dark field microscope- uses light scattered by the specimen,  Dissecting microscope- to view 3D images during dissection by using reflected light  Digital Microscope-lack ocular lens, uses digital camera and screen for image observation,  Atomic Force Microscope ( ATM)- scanning probe electron microscope  Inverted Microscope- objective lens and turret below the stage to see upward  Acoustic Microscope-High frequency ultrasound wave instead of light,  X-ray Microscope-uses cathode ray tubes to produce electron beams,  Polarizing Microscope-used polarized light for illumination,  Metallurgical Microscope-uses reflected light instead of transmitted light for imaging  Pocket Microscope-Portable , LED light source, camera having 100X magnification,  USB Microscope-Digital camera with high power macro lens connected to USB port.
THANKS FOR YOUR VISIT • INFORMATION SOURCES:  https://microbenotes.com/types-of-microscopes/#compound-microscope- working –principle  Microscope- University of Edinburgh,  Microscope and types- braincart.com ,  Microbe online,  Microscope Clarity,  Microscopy- Khan Academy, DISCLAIMER: • This presentation has been designed to address the academic fraternity without any financial interest. This is absolutely free to use . The author does not claim any kind of financial benefits from this content.