Microscopy and Micrometry PDF

MICROSCOPY AND MICROMETRY BY DR. ABU A. RAHAMANI 1  A PERSON IS PAID BASED ON WHICH OF THE FOLLOWING? 1. TIME 2. VALUE 2 OBJECTIVES Microscopy and micrometry: Light, Fluor...

MICROSCOPY AND MICROMETRY BY DR. ABU A. RAHAMANI 1  A PERSON IS PAID BASED ON WHICH OF THE FOLLOWING? 1. TIME 2. VALUE 2 OBJECTIVES Microscopy and micrometry: Light, Fluorescent, phase contrast and electron microscopy 3  MICROSCOPY Microscopy is a technical field that involves the use of Microscopical components such as microscopes or microscope objectives to obtain greater detail of examined samples 4  TERMS AND DEFINITIONS Principle Microscopy is to get a magnified image, in which structures may be resolved which could not be resolved with the help of an unaided eye. Magnification It is the ratio of the size of an object seen under a microscope to the actual size observed with an unaided eye. The total magnification of microscope is calculated by multiplying the magnifying power of the objective lens by that of eye piece. BUT maximum magnification does not mean maximum resolution! 5  Resolving power It is the ability to differentiate two close points as separate. The resolving power of human eye is 0.25 mm The light microscope can separate dots that are 0.25µm apart. The electron microscope can separate dots that are 0.5nm apart The smaller this value, the higher the resolving power of the microscope and the better the clarity and detail of the image. 6  TERMS AND DEFINITIONS Limit of resolution It is the minimum distance between two points to identify them separately. It is calculated by Abbé equation. Working distance It is the distance between the objective and the objective slide. The working distance decreases with increasing magnification. 7  TERMS AND DEFINITIONS Numerical aperture(NA) The numerical aperture of a lens is the ratio of the diameter of the lens to its focal length. NA can be decreased by decreasing the amount of light that passes through a lens. Diameter of the lens 8  What is the numerical aperture? NA is an estimate of how much light from the sample is collected by the objective α2 α1 Oil (n = 1.5) Air (n = 1.0) Objective lens Coverslip (n = 1.5) Glass slide (n = 1.5) NA = n sin  n = refractive index α = angle of incident illumination 9  The higher the numerical aperture of a lens, the better the resolution of a specimen will be which can be obtained with that lens. d=0.5 λ/n sin Ɵ Where d= resolution Λ = wavelength of light used 10  Numerical aperture, NOT magnification determines resolution! Increasing NA A lens with a larger NA will be able to visualize finer details and will also collect more light and give a brighter image than a lens with lower NA. 11 Imaging Techniques Technique Image Formed By Optical Microscopy Light Rays Confocal Microscopy Coherent Light Source (Laser) Transmission Electrons Electron Microscopy (TEM) Scanning Electron Microscopy Electrons (SEM) Atomic Force & Scanning Tunneling Microscopies Molecular Mechanical Probes (AFM/STM) 12 What is a microscope? 13  Definition-A microscope is a high precision optical instrument that uses a lens or a combination of lenses to produce highly magnified images of small specimens or objects especially when they are too small to be seen by the naked (unaided) eye. A light source is used (either by mirrors or lamps) to make it easier to see the subject matter. Microscope is used to view objects or specimens that are too small to be seen with just the human eye. Micro- = “small”; -scope = “to look at” Photographs of cells are taken using a microscope, and these pictures are called micrographs. 14 What is a microscope? Theoretically a microscope is an array of two lenses. Focal plane Image plane Image plane Objective Tube lens Eyepiece lens lens Classic compound microscope 15  History of microscope In 1590 F.H Janssen & Z.Janssen constructed the first simple compound light microscope -10x to 30x. In 1665 Robert Hooke developed a first laboratory compound microscope. Later, Kepler and Galileo developed a modern class room microscope. In 1672 Anton Von Leeuwenhoek developed a first simple microscope with a magnification of 200x – 300x. In 1674, Anton was the first to see and describe bacteria, yeast, plants, and life in a drop of water- He is called as Father of microscopy. The term microscope was coined by Faber in 1623. In the early 1930’s the first electron beam microscopes were developed which were a breakthrough in technology as they increased the magnification from about 1000x or so up to 250,000x or more. 16 Microscopy Light Electron 17  There are two types of microscopes: Light microscopes (4 types) Electron microscope (2 types) 18 Light (Optical) Microscopy Visible light is used. Glass lens are used Advantage: It can often be performed on living cells, so it’s possible to watch cells carrying out their normal behaviors (e.g., migrating or dividing) under the microscope. 19 Principle When a ray of light passes from one medium to another it bends by phenomena called refraction. Bending of light slows the speed. The bending of light is determined by refractive index of the medium. 20 Types of Light Microscopes 1.Bright field Light Microscope 2.Phase Contrast Light Microscope 3.Dark-Field Light Microscope 4.Fluorescence Light Microscope 21 Contrasting techniques Fibroblast grown in culture Brightfield Phase contrast DIC Darkfield Differential interference contrast(DIC) Taken from: http://fig.cox.miami.edu/~cmallery/150/Fallsyll.htm Introduces contrast to images of specimens which have little or no contrast when viewed under Brightfield microscopy 22  Brightfield Principle: Light is transmitted through the sample and absorbed by it. Application: Only useful for specimens that can be contrasted via dyes. Very little contrast in unstained specimens. With a bright background, the human eye requires local intensity fluctuations of at least 10 to 20% to be able to recognize objects. Cross section of sunflower root Piece of artificially grown skin (http://www.zum.de/Faecher/Materialien/beck/12/bs12-5.htm) (www.igb.fhg.de/.../dt/PI_BioTechnica2001.dt.html ) 23 Compound Microscope Common type of microscope. High power microscope- The magnification (power) 40x to 1000x. Compound refers to the fact that in order to enlarge an image - a single light path passes through a series of lenses in a line where each lens magnifies the image over the previous one. In the standard form – 2 lenses an objective lens (closest to the object or specimen) an eyepiece lens (closest to the observers’ eye) Uses light to illuminate the specimen The objective lens usually consists of three or four lenses. The most used light method is trans-illumination. At 400x much detail can be seen at the cellular level of biological specimens. Applications: Learn about cells and microorganisms in both medical and science field. 24 25 Parts of a Compound Microscope The parts of a compound microscope are of two categories Mechanical Parts Optical Parts 1. Base or Metal Stand: 1. Light Source: 2. Pillars: 2. Diaphragm: 3. Inclination joint: 3. Condenser: 4. Curved Arm: 4. Objective: 5. Body Tube: 5. Eyepiece: 6. Draw Tube: 7. Coarse Adjustment: 8. Fine Adjustment: 9. Stage: 10. Mechanical Stage (Slide Mover): 11. Revolving Nosepiece 26 Objective lenses One of the most important parts of a compound microscope, as they are the lenses closest to the specimen. A standard microscope has three, four, or five objective lenses that range in power from 4X to 100X. Objectives vary in power from 1x to 160x in compound microscopes but the most common power range is from 4x to 100x. Most compound microscopes have three or four (occasionally five) objectives usually of 4x, 10x, 40x, and 100x (oil immersion) which revolve on a nosepiece (turret) to give different magnifying powers. 27 Numerical Aperture NA of a microscope objective is a measure of its ability to gather light The more light (higher NA) the better the resolving power of the lens Better resolution The N.A. will be marked on the objective and the typical N.A. for the following are; 4x=0.10, 10x=0.25 40x=0.65 100x=1.25. 28 The objective 29 Ocular Lens or Eye piece The eyepiece consists of a series of lenses mounted in a tube (barrel) at the upper end of the microscope. Its basic function is to look at the focused, magnified image projected by the objective lens and magnify that image a second time before your eye looks at the image of the specimen. The eyepieces are usually 10x but also come in 5x, 12.5x, 15x, and 20x. The “x” refers to the amount of magnification (power) that this lens adds as a multiplier to the magnification of the objective. For special applications, eyepieces can have scales, pointers, crosshairs, markers, etc. on them. The eyepoint is the location (or position) of the eye from the eyepiece which allows for the best possible viewing of the image. 30 Eyepiece Also known as the ocular Contains the first lens you look through - usually a magnification of 10x Located on the top of the body tube 31 Objective Lenses Used in combination with the eyepiece to provide a range of magnification Magnification ranges from 40x to 400x Located on the nose-piece at the bottom of the body tube 32 Nosepiece Holds the objective lenses Rotates to enable magnification Located at the bottom of the body tube 33 Arm Supports the upper parts of the microscope Used to carry the microscope When carrying a microscope, always have one hand on the arm and one hand on the base. Use two hands!! 34 Base Supports the whole microscope Used to carry the microscope When carrying a microscope, always have one hand on the arm and one hand on the base. Use two hands!! 35 Stage Supports the slide The slide contains the specimen or object that you are viewing with the microscope. 36 Stage Clip Helps to hold the slide in place Usually one on each side of the hole (stage opening) = 2 stage clips The stage opening allows light to pass from the light source to the lenses. 37 Light Source Provides light necessary for viewing the specimen Usually either a mirror or illuminator Sends light through the stage opening to the diaphragm 38 Diaphragm Wheel or lever located below the stage opening Regulates the amount of light that can enter the lenses May need to be adjusted based on the thickness of the specimen being studied 39 Coarse Adjustment Knob Raises and lowers the stage or objective lenses Used only when focusing the low power (4x) objective lens 40 Fine Adjustment Knob Raises and lowers the stage or objective lenses a small distance for exact focusing Used when focusing the medium power (10x) and high power (40x) objective lenses 41  Phase contrast Principle: Incident light [Io] is out of phase with transmitted light [I] as it was slowed down while passing through different parts of the sample and when the phases of the light are synchronized by an interference lens, a new image with greater contrast is seen. Phase ring I0 not aligned aligned Phase stops I https://www.youtube.com/watch?v=fC Zw4X7V5Pw 42 Phase contrast Application: Phase contrast is the most commonly used contrasting technique All tissue culture microscopes and the time-lapse microscopes are set up for phase. brightfield wrong phase stop right phase stop 43 Applications Determine morphologies of living cells such as plant and animal cells Studying microbial motility and structures of locomotion To detect certain microbial elements such as the bacterial endospores 44 Fluorescence https://www.youtube.com/watch?v=SfzmW7EMdLE 45 Applications Used in the visualization of bacterial agents such as Mycobacterium tuberculosis. Used to identify specific antibodies produced against bacterial antigens/pathogens in immunofluorescence techniques by labeling the antibodies with fluorochromes. Used in ecological studies to identify and observe microorganisms labeled by the fluorochromes It can also be used to differentiate between dead and live bacteria by the color they emit when treated with special stains 46  Darkfield Principle: The illuminating rays of light are directed through the sample from the side by putting a dark disk into the condenser that hinders the main light beam to enter the objective. Only light that is scattered by structures in the sample enters the objective. Application: People use it a lot to look at Diatoms and other unstained/colourless specimens Darkfield Symbiotic Diatom colony (www1.tip.nl/~t936927/making.html) Brightfield 47 Applications It is used to visualize the internal organs of larger cells such as the eukaryotic cells Identification of bacterial cells with distinctive shapes such as Treponema pallidum, a causative agent of syphilis. https://www.youtube.com/watch?v=W9Mhk myfMLQ 48  Electron Microscopy ▪ Electron microscopes use electrons instead of photons(visible light) to image cells and structures. ▪ Electromagnets function as lenses in EM , whole system operates in a vacuum. ▪ EM are fitted with cameras to allow a photograph to be taken 49 Electron microscopes are scientific instruments that use highly energetic electrons to examine objects on a very fine scale which yields the following information 1. Topography: The surface features of an object (hardness, reflectivity etc) 2. Morphology: The shape and size of the particles 3. Composition: The elements and compounds that the object is composed of and the relative amount of them. 4. Crystallographic information: How the atoms are arranged in the objects. An Electron Microscope can magnify structure from 10-250000 times than a light microscope 50 Electron Microscopy Two types of electron microscopes: Transmission electron microscopes (TEM) : Allows one, the study of inner surfaces. (need thin section),negative stain Scanning electron microscopes (SEM): Used to visualize the surface of objects Coat with heavy metal 51  Electron Microscopy ▪ Transmission electron microscopy is used to examine cells and cell structure at very high magnification and resolution , even enabling one to view structures at the molecular level. ▪ This is because the wavelength of electrons is much shorter than the wavelength of visible light and wavelength affects resolution. ▪ Unlike visible light , electron beams can not penetrate very well. So, special techniques of thin sectioning are needed to prepare specimens before observing them. ▪ To obtain sufficient contrast, the preparation are treated with stains such as osmic acid , permanganate , uranium , lanthanum ; because these substances are composed of atoms of high atomic weight , they scatter electrons well and thus improve contrast. 52 Figure 2.9 Electron source Evacuated chamber Sample port Viewing screen 53 © 2012 Pearson Education, Inc. Electron Microscopy ▪ Scanning electron microscopy used to observed external features of an organisms or cell. ▪ No need for thin sections ▪ The specimen is coated with a thin film of a heavy metal such as gold. ▪ An electron beam then scans back and forth across the specimens. Electrons scattered from the metal coating are collected and activate a viewing screen to produce an image. 54  Cytoplasmic DNA Septum Cell wall (nucleoid) membrane 55 © 2012 Pearson Education, Inc. Electron Microscopy ▪ Electron micrographs taken either TEM or SEM are black and white images. ▪ Some false color is added to these images to boost their artistic appearance. 56 THANK YOU 57