Microscope A Presentation ©Titas Mallick

A microscope (from the Ancient Greek: μικρός, mikrós, "small" and σκοπεῖν, skopeîn, "to look" or "see") is an instrument used to see objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using such an instrument. Microscopic means invisible to the eye unless aided by a microscope. -Wiki What is a Microscope? ©Titas Mallick

A look back to its history 2nd Century BC  Claudius Ptolemy described a stick appearing to bend in a pool of water, and accurately recorded the angles to within half a degree. 1st Century Romans were experimenting with glass and found objects appeared larger when viewed through this new material. 12th Century Salvino D'Armate from Italy made the first eye glass A look back to its history ©Titas Mallick

A look back to its history 1590  Two Dutch spectacle makers, Zacharias Jansen and his father Hans started experimenting by mounting two lenses in a tube, the first compound microscope. 1609 Galileo Galilei develops a compound microscope with a convex and a concave lens. 1665  Robert Hooke's book called Micrographia officially documented a wide range of observations through the microscope. A look back to its history ©Titas Mallick

A look back to its history 1674 Anton van Leeuwenhoek used his knowledge of grinding lenses to achieve greater magnification which he utilised to make a microscope, enabling detailed observations to be made of bacteria. 1826 Joseph Jackson Lister created an achromatic lens to eradicating the chromatic effect caused by different wavelengths of light. A look back to its history ©Titas Mallick

A look back to its history 1860 Ernst Abbe discovers the Abbe sine condition (a condition that must be fulfilled by a lens or other optical system in order for it to produce sharp images), a breakthrough in microscope design, which was until then largely based on trial and error. 1931 Ernst Ruska starts to build the first electron microscope. A look back to its history ©Titas Mallick

A look back to its history 1932 Transparent biological materials are studied for the first time using Frits Xernike's invention of the phase-contrast microscope. 1981 3-D specimen images possible with the invention of the scanning tunneling microscope by Gerd Binnig and Heinrich Rohrer. A look back to its history ©Titas Mallick

PTOLEMI WITH HIS BASIC CONCEPT 2ND CENTURY BC ©Titas Mallick

Salvino D'Armate AND HIS EYE GLASS 12ND CENTURY ©Titas Mallick

JANSEN AND HIS MICROSCOPE 1590 ©Titas Mallick

Galileo Galilei AND HIS MICROSCOPE 1609 ©Titas Mallick

Robert Hooke's AND HIS MICROSCOPE 1665 ©Titas Mallick

Anton van Leeuwenhoek AND HIS MICROSCOPE 1674 ©Titas Mallick

Joseph Jackson Lister AND HIS MICROSCOPE 1826 ©Titas Mallick

Ernst Abbe AND HIS MICROSCOPE 1860 ©Titas Mallick

Ernst Ruska AND HIS MICROSCOPE 1931 ©Titas Mallick

Frits Xernike's AND HIS MICROSCOPE 1932 ©Titas Mallick

Gerd Binnig AND HIS MICROSCOPE 1981 ©Titas Mallick

Two basics of microscopy Magnification Magnification is how much an image is enlarged under a microscope. Resolution Resolution is the amount of detail you can see in an image. You can enlarge a photograph indefinitely using more powerful lenses, but the image will blur together and be unreadable. Therefore, increasing the magnification will not improve the resolution. This is also known as the resolving power. Two basics of microscopy ©Titas Mallick

Increase of resolution over time What is Resolution? Resolution is the amount of detail you can see in an image. ©Titas Mallick

How It works Parts, Function & Types ©Titas Mallick

Parts of a Compound Light Microscope The microscope rests securely on a stand on a table. Daylight from the room (or from a bright lamp) shines in at the bottom. The light rays hit an angled mirror and change direction, traveling straight up toward the specimen. The mirror pivots. You can adjust it to capture more light and alter the brightness of the image you see. The light rays pass through a hole in an adjustable horizontal platform called the stage. The stage moves up and down when you turn a thumb wheel on the side of the microscope. By raising and lowering the stage, you move the lenses closer to or further away from the object you're examining, adjusting the focus of the image you see. Parts of a Compound Light Microscope ©Titas Mallick

Parts of a Compound Light Microscope To look at something under a microscope (such as a plant leaf), you prepare a specimen of it. The specimen has to be a very thin slice so light rays will pass through. You mount the specimen on a glass slide with a glass cover slip on top to keep it in place. The slide is held in place by two metal clips, one on either side. Light traveling up from the mirror passes through the glass slide, specimen, and cover slip to the objective lens (the one closest to the object). This makes the first magnification: it works by spreading out light rays from the specimen so they appear to come from a bigger object. The objective "lens" usually consists of more than one lens. Parts of a Compound Light Microscope ©Titas Mallick

Parts of a Compound Light Microscope A selection of other objective lenses can be used to magnify the specimen by more or less. The thumb wheel makes it easy to swing the other lenses into position. The eyepiece lens (the one closest to your eye) magnifies the image from the objective lens, rather like a magnifying glass. On some microscopes, you can move the eyepiece up and down by turning a wheel. This gives you fine control or "fine tuning" of the focus. You look down on a magnified image of the object. Parts of a Compound Light Microscope ©Titas Mallick

Optical Theory and Path of Light This simplified image shows the path of Light in a compound Light Microscope. And how it magnifies a object using visible light. To analyze it the light of Optical Theory we need to understand a few mathematical formulae. ©Titas Mallick

θ= one-half of the angular aperture (A) It is defined as the property of lens that decides the quantity of light that can enter. The angle of the cone of light entering an objective is known as theta NA=nsinθ ; NA=numerical aperture n=refractive index of the imaging medium between the front lens of the objective and the specimen cover glass, a value that ranges from 1.00 for air to 1.51 for specialized immersion oils. θ= one-half of the angular aperture (A) Numerical aperture ©Titas Mallick

It is defined as the shortest distance between two objects when they can be distinguished as two separate entities. Resolution is a subjective value in microscopy because at high magnification, an image may appear not very sharp but still it can be resolved to the maximum ability of the objective. Numerical aperture is defined as the resolving power of an objective, but the entire resolution of a microscope system is also depends on the numerical aperture of the sub stage condenser. For getting a better resolution, higher the numerical aperture of the total system. OIL EMERSION LENSE INCREASES THE nѳ, AND ICREASES THE RESOLUTION Limit of resolution ©Titas Mallick

Abbe equation is used to calculate the limit set on the resolution of light microscope.   The oil immersion objective gives the maximum theoretical resolving power of  the microscope , the numerical aperture of 1.25 and blue-green light is around  0.2μm.  Abbe equation ©Titas Mallick

Types Of Microscope Category Type Description Optical microscope Binocular stereoscopic microscope A microscope that allows easy observation of 3D objects at low magnification. Brightfield microscope A typical microscope that uses transmitted light to observe targets at high magnification. Polarizing microscope A microscope that uses different light transmission characteristics of materials, such as crystalline structures, to produce an image. Types Of Microscope ©Titas Mallick

Types Of Microscope Category Type Description Optical microscope Phase contrast microscope A microscope that visualizes minute surface irregularities by using light interference. It is commonly used to observe living cells without staining them. Differential interference contrast microscope (Nomarski interference microscope) This microscope, similar to the phase contrast, is used to observe minute surface irregularities but at a higher resolution. However, the use of polarized light limits the variety of observable specimen containers. Types Of Microscope ©Titas Mallick

Types Of Microscope Category Type Description Optical microscope Fluorescence microscope A biological microscope that observes fluorescence emitted by samples by using special light sources such as mercury lamps. When combined with additional equipment, brightfield microscopes can also perform fluorescence imaging. Total internal reflection fluorescence microscope A fluorescence microscope that uses an evanescent wave to only illuminate near the surface of a specimen. The region that is viewed is generally very thin compared to conventional microscopes. Observation is possible in molecular units due to reduced background light. Types Of Microscope ©Titas Mallick

Types Of Microscope Category Type Description Optical microscope Laser microscope (Laser scanning confocal microscope) This microscope uses laser beams for clear observation of thick samples with different focal distances. Multiphoton excitation microscope The use of multiple excitation lasers reduces damage to cells and allows high-resolution observation of deep areas. This type of microscope is used to observe nerve cells and blood flow in the brain. ©Titas Mallick

Types Of Microscope Structured illumination microscope Category Type Description Optical microscope Structured illumination microscope A high-resolution microscope with advanced technology to overcome limited resolution found in optical microscopes that is caused by the diffraction of light. Electron microscope Transmission electron microscope (TEM), scanning electron microscope (SEM), etc. These microscopes emit electron beams, not light beams, toward targets to magnify them.. ©Titas Mallick

Types Of Microscope Scanning probe microscope (SPM) Category Type Description Scanning probe microscope (SPM) Atomic force microscope (AFM), scanning near-field optical microscope (SNOM), etc. This microscope scans the surface of samples with a probe and this interaction is used to measure fine surface shapes or properties. Others X-ray microscope, ultrasonic microscope, etc. Atomic force microscope (AFM) Scanning ion-conductance microscopy (SICM) Scanning tunneling microscope (STM) Ultraviolet microscope X-ray microscope ©Titas Mallick

Thank You Presented by Titas Mallick Presentation Made for Universitty of Calcutta, Sem-I, M.Sc in Botany, 2017 Thank You ©Titas Mallick