MICROSCOPY.pptx
- 1. MICROSCOPY Name: Sneha Shinde Subject: Microbiology Course: Intg. MSc+PhD Biotechnology Section: B Roll No.: MSB/PH/2022/001 Enrollment No.: A005159222001 Submitted to: Dr. Manoranjan Nayak
- 2. Introduction Microorganisms are too small to be seen by our unaided eyes and the microscopes are of crucial importance as they help to view the microbes. A microscope is an optical instrument consisting of one or more lenses in order to magnify images of minute objects. Thus it is important to gain a preliminary knowledge about the principles of microscope and its types. Microscope, as evident from its name, is a device that enables us to see objects that are invisible to the naked eye. The name literally roughly translates to seeing small objects, where Micro means ‘small’ and scope means ‘to see’ (derived from Ancient Greek). Antony Van Leeuwenhoek invented the first simple microscope in 1683. In simple microscope, single lens is used for magnification whereas in compound microscope, multiple lenses are used. Monocular microscope has one eyepiece and binocular has two eyepieces. As research has advanced, we now have different types of microscopes that are commercially available.
- 3. The microscope works on three principles of physics: 1. Magnification 2. Resolving power 3. Numerical aperture Magnification: It is the ability of lenses to enlarge an object visually. If the magnifying power of lens is 10X it means that the given lens can enlarge the object up to 10 times. In compound microscope, the magnification is the product of magnifying power of both the lenses. The magnifying power of lens depends on focal length. Lower the focal length, higher is the magnifying power of lens. Resolution: The resolving power is the ability to distinguish two closely placed points. The resolution power of lens allows us to observe the details of an object. The resolution of microscope can find out by using Abbe’s equation. d – distance between two closely distant points λ – wavelength of light n sin θ – numerical aperture The microscope with higher magnification has small d value. λ is the wavelength of light, shorter is the wavelength; higher is the resolution. The wavelength of visible light is from 300 to 700 nm. The best resolution for light microscope is obtained in the range of 450 to 500 nm. ‘n’ is the refractive index of medium. Refractive index is the ability of the medium to bend the light. The angle of cone of light is affected by the refractive index of medium. The refractive index of air is 1. ‘θ’ is the half of the angle of the cone of light that enters the microscope. The value of ‘Sin θ’ cannot be more than 1 because angle of entering cone of light cannot be more than 90° and value of sin 90 is 1. d= 0.5 x 450 nm 1 d= 225 nm 0r 0.2 μm Hence, the resolution limit of light microscope is 0.2 μm.
- 4. Important parts of the microscope: Base: It gives support to the body. The illuminating source of microscope is placed at the base of microscope Arm: It is used to hold and carry the microscope. The coarse and fine focusing knobs are placed on the arm. Eyepiece: It is the part through which we observe the sample/object. The magnifying power of the eyepiece lens is generally 10X. Eyepiece tube or body tube: The function of eyepiece tube is to hold the eyepiece and hence it is names as eyepiece tube. Nosepiece: The flexibility of nosepiece allows switching the objective lenses. Objective lens: In general 3 objective lenses are placed. Their magnifying power is 10X, 40X and 100X respectively. Focusing mechanism (Adjacent knobs): It is used for right placing of sample and for focusing it. Stage: It is place where sample or object is kept for viewing. For accurate placement, it is provided with clips that hold the slide firmly. Aperture: It is present on the stage that allows the entry of light from illuminator to fall on sample or object. Source of Illumination: In non-electric microscope, the sunlight is used as source of illumination and hence mirror is placed to focus the sunlight. In electric microscope, lamp is placed of specific wavelength. Condenser: Its function is to focus the light on sample form illuminator. Diaphragm: It is generally associated with condenser. The diaphragm and condenser together produce hollow cone of light that strikes the sample and illuminate it.
- 5. Types of microscope: Light Microscope: Magnification is obtained by a system of optical lenses using light waves. It includes: (i) Bright field (ii) Dark field (iii) Fluorescence (iv) Phase contrast and (v) UV Microscope Electron Microscope: A system of electromagnetic lenses and a short beam of electrons are used to obtain magnification. It includes: (I) Transmission electron microscope (TEM) (II) Scanning electron microscope (SEM)
- 6. Light Microscope: Bright field This instrument contains two lens systems for magnifying specimens: the ocular lens in the eyepiece and the objective lens located in the nose-piece. The specimen is illuminated by a beam of tungsten light focused on it by a sub- stage lens called a condenser, and the result is that the specimen appears dark against a bright background. A major limitation of this system is the absence of contrast between the specimen and the surrounding medium, which makes it difficult to observe living cells. Therefore, most bright field observations are performed on nonviable, stained preparations. Dark field This is similar to the ordinary light microscope; however, the condenser system is modified so that the specimen is not illuminated directly. The condenser directs the light obliquely so that the light is deflected or scattered from the specimen, which then appears bright against a dark background. Living specimens may be observed more readily with dark field than with bright field microscopy. Maximum magnification of 1500x and resolution of 0.1 – 0.2 μm can be obtained. It is useful in studying the morphology and motility of microorganisms. Dark field is especially useful for finding cells in suspension. Dark field makes it easy to obtain the correct focal plane at low magnification for small, low contrast specimens.
- 7. Fluorescence This microscope is used most frequently to visualize specimens that are chemically tagged with a fluorescent dye. The source of illumination is an ultraviolet (UV) light obtained from a high-pressure mercury lamp or hydrogen quartz lamp. The ocular lens is fitted with a filter that permits the longer ultraviolet wavelengths to pass, while the shorter wavelengths are blocked or eliminated. Ultraviolet radiations are absorbed by the fluorescent label and the energy is re-emitted in the form of a different wavelength in the visible light range. The fluorescent dyes absorb at wavelengths between 230 and 350 nm and emit orange, yellow, or greenish light. This microscope is used primarily for the detection of antigen-antibody reactions. Phase contrast Observation of microorganisms in an unstained state is possible with this microscope. Its optics include special objectives and a condenser that make visible cellular components that differ only slightly in their refractive indexes. As light is transmitted through a specimen with a refractive index different from that of the surrounding medium, a portion of the light is refracted (bent) due to slight variations in density and thickness of the cellular components. The special optics convert the difference between transmitted light and refracted rays, resulting in a significant variation in the intensity of light and thereby producing a discernible image of the structure under study. The image appears dark against a light background.
- 8. UV Microscope Resolution of a microscope depends upon the wavelength of light used. If, longer the wavelength of light used, lower will be the resolving power while shorter the wavelength, more will be the resolution. With this principle, UV rays of shorter wavelength are used as light source. Since UV rays can’t penetrate the glass, quartz lenses are used. Since the UV rays are invisible, photographic plates should be used to record the image or special type of filters should be used to eliminate the UV rays from reaching the eyepiece. This is used in conjunction with fluorescent microscopy. Upon illumination with UV light certain fluorescent dyes emit light in visible range, which can be directly viewed.
- 9. Electron Microscope: Transmission electron microscope (TEM) TEM has a projector lens that project the image onto a fluorescent viewing screen or film plate, because the beam cannot be viewed directly. With TEM greater resolution and higher magnifications than light and Scanning Electron Microscope can be obtained. In TEM, the differential scattering of electrons by the specimen makes the contrast. Since most of the atoms of the biological material are of low mass, the contrast of the specimen is low. Staining with heavy metals such as platinum, uranium or tungsten can increase the contrast. They are of various types such as: a. Positive staining: The heavy metals are fixed on the specimen. b. Negative staining: It is used to increase the electron opacity of the surrounding area. Two techniques commonly employed for the observation of biological specimen are: a. Metal shadowing: The dried specimen is exposed at an acute angle to a stream of heavy metals like platinum, palladium or gold and thereby producing an image, that reveals the three-dimensional structure of the object. b. Freeze fracturing: The frozen specimen is fractured with a knife, and the exposed surface is coated with a heavy metal (Gold) at an acute angle. A supporting layer of carbon is evaporated on the metal surface. Then the specimen is destroyed and the replica is examined.
- 10. Scanning electron microscope (SEM) The specimen is coated with a thin layer of heavy metal and the specimen is subjected to a narrow beam of electrons, which rapidly moves and scans the surface of the specimen. The irradiated specimen depending upon its physical and chemical composition will release secondary electrons. These secondary electrons are then collected by anode detector, which generates an electronic signal. Then the electronic signal is scanned in TV system to produce an image on a cathode ray tube. Magnification on SEM is about 75,000 to 1,00,000 times. Limitations: a. Specimen is kept under high vacuum on the path of electron beam. So, living cells can’t be examined. b. Electrons have low penetration capacity, hence ultra thin section and staining should be done which is time consuming and also sometimes alter or distort the structures of microorganisms. c. High cost and specialized techniques prevent its use in all microbiology labs in spite of greater magnification and resolution.
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