Microscope PDF

Uma Sharma Assistant professor College of Biotechnology,DUVASU,Mathura Introduction to the Mi...

Uma Sharma Assistant professor College of Biotechnology,DUVASU,Mathura Introduction to the Microscope Microscope – an instrument that produces an enlarged image of an object. It is an instrument which deals with too small organisms that they cannot be seen distinctly with the naked eye. Microscopes provide the observer with enhanced resolution (ability to observe two nearby objects as distinct objects), contrast (ability to detect different regions of the specimen on the basis of intensity or color) and magnification (ability to make small objects visible). The human eye can resolve objects of the order of 0.1 mm, while the light microscope can resolve objects on the order of 0.2 µm (200 nm) with a magnification of 1000.The transmission electron microscope, can resolve objects on the order of 0.1nm (100 A ˚ units). Animal cell (20–30 µm), a red blood cell (7.8 µm ), a mitochondrion (2–5µm), a nucleus (3-10 µm), microvilli (1 µm), a cell membrane (10 nm), a microfilament (8– 10 nm), a bacterium ) 0.5-5 µm) and a virus (10–100 nm) Credit for the first microscope is usually given to Zacharias Jansen, in Middleburg, Holland, around the year 1595. Biologists use microscopes to study cells, cell parts, and organisms that are too small to be seen with the naked eye. Microscopes magnify and show details of the image. History of microscopes: Zacharias Janssen – Dutch spectacle maker who tested several lenses in a tube and discovered thatnearby objects appeared significantly enlarged. Robert Hooke – used a microscope to observe a thin slice of cork. The spaces he saw reminded him of the small rooms where monks lived, so he called them “cells,” which he used to describe the smallest units of life. He used microscopes with two and three lenses, but they didn’t produce very detailed images. Anton van Leeuwenhoek - Dutch merchant who learned how to grind lenses to make simple microscopes (have only one lens). These produced clearer and more enlarged images than Hooke’s. He is considered “The Father of Microscopy” and built over 500 different microscopes. He was the first to discover microorganisms under a microscope by observing a drop of pond water filled with life. He called them “tiny animalcules.” 1932 – Frits Zernike invented the phase-contrast microscope that allowed for the study of colorless and transparent biological materials for which he won the Nobel Prize in Physics in 1953. 1931 – Ernst Ruska co-invented the electron microscope for which he won the Nobel Prize in Physics in 1986. An electron microscope depends on electrons rather than light to view an object, electrons are speeded up in a vacuum until their wavelength is extremely short, only one hundred thousandth that of white light. Electron microscopes make it possible to view objects as small as the diameter of an atom.  Uma Sharma Assistant professor College of Biotechnology,DUVASU,Mathura 1981 – Gerd Binnig and Heinrich Rohrer invented the scanning tunneling microscope that gives three-dimensional images of objects down to the atomic level. Binnig and Rohrer won the Nobel Prize in Physics in 1986. Parts of Microscope: Body tube: Keeps the two sets of lenses a set distance Rotating nosepiece: Allows one to change between the objective lenses. Objective lens: The second set of lenses in a compound microscope (usually 4x, 10x, 40x). Stage clips: Hold the slide in place. Diaphragm: Adjusts the amount of light that hits the slide from the light source. Light source: Where light comes from to see image. Ocular lens: The first lens in a compound microscope (usually 10x). Arm Stage: Where one puts the slide to view. Coarse Adjustment Knob: Moves the stage up and down very quickly. Fine Adjustment Knob: Moves the stage up and down very slowly. Base  Uma Sharma Assistant professor College of Biotechnology,DUVASU,Mathura Light passes through the specimen on the slide and uses two lenses to form its image. Capable of two things (below) that vary in different microscopes: Magnification: a measure of how much the image is enlarged Total magnification = (ocular lens)(objective lens being used) [The ocular lens usually has a 10x magnification, but that can vary.] 4x objective lens = (10x)(4x) = 40 times total magnification 10x objective lens = (10x)(10x) = 100 times total magnification 40x objective lens = (10x)(40x) = 400 times total magnification Resolution: a measure of the clarity of an image; how clear the details are. Two types of microscopes: 1. Light Microscope – light passes through one or more lenses to produce an enlarged image of a specimen Electron Microscope – forms image of a specimen using a beam of electrons rather than light Types of Light Microscope There are a variety of light microscopes mostly employed in Microbiology: a) Bright-field b) Dark-field c) Phase-contrast d) Fluorescence a.The Bright-field Microscope: Called the ordinary microscope because it forms a dark image against a brighter background. Image should remain in focus when objectives are changed. The objective lens forms an enlarged real image within the microscope and the eyepiece lens further magnifies this primarily image. Upon looking in the microscope, the enlarged specimen image. Virtual image( appears to lie just beyond the stage about 25 cm away.  Uma Sharma Assistant professor College of Biotechnology,DUVASU,Mathura Total magnification is calculated by multiplying the objective and eyepiece magnification together. b. Dark-field microscopy : is one such technique that is often used to observe living, unstained cells and organisms. Dark field microscopes illuminate the sample in such a way that unreflected, and unrefracted light does not enter the objective, only light that has been reflected/refracted by the specimen passes through the objective and forms the image. This results in a specimen that is brilliantly illuminated on a dark field, The field surrounding the specimen appears black. c.Phase-contrast microscopes Convert slight differences in refractive index and density into variations in light intensity. d. Fluorescence microscopy Specimens are treated with dye molecules called fluorochromes which brightly fluoresce when exposed to light of a specific wavelength.  Uma Sharma Assistant professor College of Biotechnology,DUVASU,Mathura 2. The Electron Microscope Resolution is the limiting factor to a light microscope since the greater the magnification is, the less it is able to resolve the image. At magnifications beyond 2,000x, the image becomes blurry, but electron microscopes can be used at greater magnifications. Characteristics of the Electron Microscope  A beam of electrons is used to produce an enlarged image of the specimen (it does not use light).  This electron beam and the specimen must be placed inside a vacuum chamber so that the electrons in the beam do not bounce off gas molecules in the air.  Since living things cannot survive in a vacuum, the electron microscope cannot be used to view living cells.  Much more powerful than light microscopes. There are two types of electron microscopes: 1. Transmission Electron Microscope (TEM): Uses a beam of electrons transmitted through a very thinly sliced specimen. Magnets guide the beam of electrons toward the specimen, and the image is produced to view. Magnification to 200,000 times. 2. Scanning Electron Microscope (SEM): Provides detailed 3-D images. The specimen is sprayed with a fine metal coating (it is not sliced to view as in the TEM).  Uma Sharma Assistant professor College of Biotechnology,DUVASU,Mathura As the beam of electrons is passed over the specimen’s surface, the metal coating emits a shower of electrons, and a 3-D image of the specimen’s surface is produced to view. Rules for Using the Microscope ALWAYS carry the microscope with one hand holding the arm and your other hand supporting under the base.  Plug in the cord and turn on the light source of the microscope.  Place your slide on the stage and arrange the stage clips to hold the slide in place.  Keep the stage dry and ALWAYS make sure that your slide is dry, especially the bottom,  before putting it on the microscope.  Always start with the 4x objective lens (should already be on this from when the microscope was put away). Focus this objective lens using the coarse adjustment knob.  Once the image is in focus, carefully swing the 10x objective lens in place and focus this objective lens using the coarse adjustment knob.  Once the image is in focus, VERY carefully swing the 40x objective lens into place – BE SURE TO NOT TOUCH THE SLIDE. Focus this objective lens using ONLY the fine adjustment knob. o NEVER use the coarse adjustment knob while using the high power objective lens (40x).  Make observations and take notes as needed before preparing to put the microscope away.  Lower the stage using the coarse adjustment knob.  Swing the objective lens back to low power (4x).  Turn off light source, unplug and neatly wrap cord around microscope (NOT around lenses or light source).  Place protective cover over microscope before you put the microscope away. Preparation of Slide Wet mount slides are used to view living organisms, such as ones that need to be kept moist, and any liquid substances. Using the appropriate tool, put your specimen in the center of a clean and dry slide. Add one large solid drop of water over your specimen (water should not move on slide). Hold a clean and dry coverslip at a 45° angle over your specimen/water drop. Let one edge of the coverslip touch an edge of the water drop.  Uma Sharma Assistant professor College of Biotechnology,DUVASU,Mathura Gently drop the rest of the coverslip into place – want to avoid getting air bubbles (the whole slip should touch water). *Remember to keep the rest of the slide [and microscope stage] dry. 8