The Microscope PDF

The Microscope  LEARNING OBJECTIVES 1. Identify the parts of the microscope. 2. Describe and give the function/s of each part of the microscope. 3. Explain the correct use of the microscope. 4. Define terms related to microscopy. 5. Compare and contrast other types of microscope. 7...

The Microscope  LEARNING OBJECTIVES 1. Identify the parts of the microscope. 2. Describe and give the function/s of each part of the microscope. 3. Explain the correct use of the microscope. 4. Define terms related to microscopy. 5. Compare and contrast other types of microscope. 7/30/2024 2  WHAT IS MICROSCOPY? is a set of skills/techniques used to magnify images of samples, collecting details not perceptible to the human eye. 7/30/2024 3  A BRIEF HISTORY.. 7/30/2024 4  14th century : Invention of the first spectacles in Italy 1590 : Hansen and Zacharias Janssen created the 1st microscope (Compound microscope) by experimenting with different lenses 1667 : “Micrographia” by Robert Hooke was published 7/30/2024 5  17th century: Antonie Van Leeuwenhoek – Father of Microscopy, viewed different specimens under the microscope with a specially grinded lens that could exceed the capacity of a magnifying glass 18th – 19th century: The discovery of the Abbe’s condenser 19th - 20th century: Robert Koch discovered the Anthrax bacillus, Tuberculosis Bacillus and Vibrio cholerae 7/30/2024 6  Introduction to the First UV microscope Phase contrast was discovered by microscope from ZEISS Fritz Zernike. 1931 Present 1904 1941 Invention of the first Digital microscopes electron microscope that can be attached by Max Knoll and to a TV or computer Ernst Ruska screen which can emit live viewing of the slide. 7/30/2024 7  THE MICROSCOPE Used to magnify an object to a point where it can be seen with the human eye Total magnification --> the product of the magnifications of these two lenses The image seen in a microscope is upside down and reversed Resolution --> indicates how small and how close individual objects (dots) can be and still be recognizable Numerical aperture (NA) --> the quantitative expression of the light gathering ability of a microscope 7/30/2024 8 7/30/2024 9  PARTS OF THE MICROSCOPE 7/30/2024 10 7/30/2024 11  FRAMEWORK 1 2 3 BASE - firm, ARM - structure that STAGE - the horizontal horseshoe-shaped supports the platform, or shelf, on foot on which the magnifying and which the object being microscope rests adjusting systems observed is placed 7/30/2024 12  ILLUMINATION SYSTEM LIGHT SOURCE (BULB) CONDENSER - directs and focuses the beam of light from the bulb onto the material under examination APERTURE IRIS DIAPHRAGM - controls the amount of light passing through the material under observation 7/30/2024 13  MAGNIFICATION SYSTEM OCULAR (EYEPIECE) - lens that magnifies the image formed by the objective o What is the magnification of the ocular lens? o Interpupillary distance – the distance between the two oculars o Diopter adjustment - focus on one of the oculars 7/30/2024 14  OBJECTIVES The most important components of an optical microscope Responsible for primary image formation and play a central role in determining the quality of images the microscope is capable of producing FOCAL LENGTH - the distance from the object being examined to the center of the objective lens MAGNIFICATION SYSTEM WORKING DISTANCE - the distance from the bottom of the objective to the material being studied 7/30/2024 15  MAGNIFICATION SYSTEM THREE OBJECTIVES: A. Low-Power Objective - Usually a 10× lens with a 16-mm working distance - Used for the initial scanning and observation in most microscope work - Employed for the initial focusing and light adjustment of the microscope 7/30/2024 16  MAGNIFICATION SYSTEM THREE OBJECTIVES: B. High Power Objective - Is usually a 40× lens with a 4-mm working distance - Used for more detailed study - Used to study histologic sections and wet preparations (e.g., urine sediment) in more detail 7/30/2024 17  MAGNIFICATION SYSTEM THREE OBJECTIVES: C. Oil-Immersion Objective - Generally a 100× lens with a 1.8-mm working distance - Requires that a special grade of oil called immersion oil be placed between the objective and the slide or cover glass - Routinely used for morphologic examination of blood films and microbes 7/30/2024 18  FOCUSING SYSTEM BODY TUBE - part of the microscope through which the light passes to the ocular COARSE ADJUSTMENT - gives rapid movement over a wide range and is used to obtain an approximate focus FINE ADJUSTMENT - gives very slow movement over a limited range and is used to obtain exact focus after coarse adjustment 7/30/2024 19  TYPES OF MICROSCOPES (ILLUMINATION SYSTEMS) BRIGHTFIELD MICROSCOPE The type used in most clinical laboratories Consists of two magnifying lenses, the objective, and the eyepiece (ocular) 7/30/2024 20  TYPES OF MICROSCOPES (ILLUMINATION SYSTEMS) PHASE-CONTRAST MICROSCOPE Facilitates the study of unstained structures without prior dehydration and staining The structures observed with this system show added contrast compared with the brightfield microscope 7/30/2024 21 TYPES OF MICROSCOPES (ILLUMINATION SYSTEMS) The net effect of phase contrast is to slow the speed of light by one-fourth of a wavelength. This diminution of the speed of light makes the system very sensitive to differences in refractive index Objects with differences in refractive index, shape, and absorption characteristics show added differences in the intensity and shade of light passing through them. 7/30/2024 22  TYPES OF MICROSCOPES (ILLUMINATION SYSTEMS) INTERFERENCE-CONTRAST MICROSCOPE Gives the viewer a three-dimensional image of the object under study Especially useful for wet preparations such as urine sediment, showing finer details without the need for special staining techniques Modified by the addition of a special beam- splitting (Wollaston) prism to the condenser. 7/30/2024 23  TYPES OF MICROSCOPES (ILLUMINATION SYSTEMS) POLARIZING MICROSCOPE Polarizer - a sieve that takes ordinary light waves, which vibrate in all orientations and allows only light waves of one orientation (north-south or east-west) to pass through the filter Analyzer - a second polarizing filter that is placed above the specimen, between the objective and the eyepiece Birefringence - they rotate (or polarize) light; bend light so it can be visualized when viewed through crossed polarizers 7/30/2024 24 TYPES OF MICROSCOPES (ILLUMINATION SYSTEMS) POLARIZING MICROSCOPE An object that polarizes light (or is birefringent) will appear light against a dark background 7/30/2024 25  TYPES OF MICROSCOPES (ILLUMINATION SYSTEMS) DARKFIELD MICROSCOPE A special darkfield condenser is used that causes light waves to cross on the specimen rather than pass in parallel waves through the specimen The object under study appears light against a dark background Used to observe spirochetes in exudates from leptospiral or syphilitic infections 7/30/2024 26 TYPES OF MICROSCOPES (ILLUMINATION SYSTEMS) FLUORESCENCE MICROSCOPE Further refinement of the darkfield microscope A special barrier filter is placed on the tube or eyepiece that will pass only the desired wavelength of emitted light for the particular fluorescent system Objects in the specimen that do not fluoresce will not emit light of that wavelength and will not be seen 7/30/2024 27  TYPES OF MICROSCOPES (ILLUMINATION SYSTEMS) ELECTRON MICROSCOPE For magnification of up to about 50,000× A. Transmission Electron Microscope (TEM) o Electrons are accelerated by a high-voltage potential and pass through a condenser lens system o The electron beam is concentrated onto the specimen, and the objective lens provides the primary magnification o The final image is not visible and cannot be viewed directly; rather, it is projected onto a fluorescent screen or a photographic plate 7/30/2024 28 TYPES OF MICROSCOPES (ILLUMINATION SYSTEMS) ELECTRON MICROSCOPE B. Scanning Electron Microscope (SEM) o focuses on the surface of the specimen and produces a 3D image by striking the sample with a focused beam of electrons 7/30/2024 29  GENERAL ALIGNMENT PROCEDURE FOR MICROSCOPE USE 1. Eyepieces (preliminary step): The eyepieces have focus rings on them (turn the very top). Set the “0” to the white dot. 2. Objective: Bring the objective into focus for the sample by “focusing away.” Start by looking outside the microscope at the distance between the front of the objective and the sample. Rotate the coarse focus knob away from you to raise the stage as close as possible to the objective. Then, while looking in the microscope, rotate the knob gently toward you until the image comes into sharp focus. Touch up with the fine focus. 7/30/2024 30  GENERAL ALIGNMENT PROCEDURE FOR MICROSCOPE USE 3. Condenser: Look under the stage for the small focusing knob for the condenser carrier. Raise the condenser until it is almost touching the back of the slide. Note which direction you turned the knob to raise the condenser. a. Close the field iris diaphragm. While watching in the microscope, gently rotate the condenser focus in the other direction until the dark edges of the field iris image come into focus. b. Open the field iris just outside the field of view. (Note: If you have a highly scattering sample, try moving the feature of interest to the center of the field and leaving the field iris mostly closed.) c. c. Adjust the condenser aperture iris until you have clean, crisp edges and as clear a background as possible. 7/30/2024 31 7/30/2024 32