Microscopy Lec (1) PDF

THE MICROSCOPE Basics and Beyond Prepared by: Catherine Denise M. Palabyab, RMT, MD LEARNING OBJECTIVES Be able to define terms used in histology Know the significance of histology in your profession Be able to know the different types of microscope Properly identify the part...

THE MICROSCOPE Basics and Beyond Prepared by: Catherine Denise M. Palabyab, RMT, MD LEARNING OBJECTIVES Be able to define terms used in histology Know the significance of histology in your profession Be able to know the different types of microscope Properly identify the parts and functions of the microscope Be able to manipulate the microscope properly DEFINITION HISTOLOGY “Histos” - tissue “logia” - knowledge Branch of anatomy that deals with the tissues of the body and how these are arranged to make up an organ Deals with how cell’s structure and arrangement optimize functions specific to each organ Study of healthy tissues. DEFINITION HISTOPATHOLOGY the clinical application of histological methods to examine diseased cells and tissues for diagnosis medical conditions like cancer. MICROSCOPY The science of investigating small objects using an instrument called a microscope The Advent of Microscopy Anton van Leeuwenhoek (1632-1723): Used magnifying lens of 400 microscopes First to observe live microorganisms in teeth scrapings, and rain water, to which he called “animalcules” Father of Microscopy HISTORY OF MICROBIOLOGY The Advent of Microscopy Anton van Leeuwenhoek (1632-1723): The Advent of Microscopy Robert Hooke (1635-1703): Used a crude type of microscope Published "Micrographia" reported that living things were composed of little boxes called “cork” Coined the term “cell" to describe the basic unit of life as it reminds him of the cells in monasteries The Advent of Microscopy MICROSCOPY IMPORTANT PARAMETERS IN MICROSCOPY 1. MAGNIFICATION: Ratio between the size of an image produced by the mircoscope and its actual size 2. RESOLUTION: ability of a microscope to distinguish between two distinct points in a specimen 3. CONTRAST: extent to which features in a specimen can be distinguished from their surroundings. MICROSCOPY MAGNIFICATION MICROSCOPY RESOLUTION MICROSCOPY CONTRAST MICROSCOPY MAJOR KINDS OF MICROSCOPE: 1. Light Microscope - based on the interaction of light with tissue components a. Bright field microscopy b. Dark field microscopy c. Fluorescence microscopy d. Phase contrast microscopy e. Confocal Microscopy f. Polarizing microscopy 2. Electron Microscope- uses an electron beam BRIGHT FIELD MICROSCOPY Commonly used type of microscope Also known as the compound light microscope Microscopic field in brightly lightened and the specimen appears dark Greater contrast and color differentiation It is mainly used with stained preparations. BRIGHT FIELD MICROSCOPY BRIGHT FIELD MICROSCOPY BRIGHT FIELD MICROSCOPY BRIGHT FIELD MICROSCOPY BRIGHT FIELD MICROSCOPY OPTICAL COMPONENTS: (3 lenses) 1. Condenser: collects and focuses a cone of light that illuminates the object 2. Objective lens: -enlarges and projects the image of the object in the direction of the eyepiece - Interchangeable objectives with different magnifications X4: scanner observing a large field X10: LPO: medium magnification of a smaller field X40: HPO : higher magnification X1000: OIO 3. Eyepiece or Ocular lens: further magnifies the object by x 10 and projects it into the viewer BRIGHT FIELD MICROSCOPY RESOLVING POWER/ RESOLUTION Smallest distance between two particles at which they can be seen as separate objects Light microscope max RP: 0.2 um Any structure smaller than this (ribosomes, membranes. filaments) cannot be distinguished by a LM. TOTAL MAGNIFICATION Magnifying power of the objective lens x the ocular lens. Ex: HPO total magnification: 40 x 10 = 4000x magnification BRIGHT FIELD MICROSCOPY The stage ○ found just below the objectives ○ where the specimen is placed ○ allowing movement of the specimen around for better viewing with the flexible knobs and it is where the light is focused on. Fine and Coarse Adjustment knobs ○ Found on the microscope’s arm. ○ Ensure production of a sharp image with clarity. BRIGHT FIELD MICROSCOPY Coarse Adjustment Knob ○ Causes the stage to move upward or downward at a fast rate and is used to focus on a specimen Fine Adjustment Knob ○ Causes the stage to move upward or downward at a slow rate and is used to focus on a specimen Microscope tube ○ The microscope tube is attached on top of the arm. It can be of the monocular or binocular type. It supports the eye-piece on the upper end. BRIGHT FIELD MICROSCOPY Arm ○ backbone of the microscope, ○ used to carry and move the microscope Illuminator Revolving Nosepiece ○ Carries the objective lenses ○ can move round to any position Aperture diaphragm- controls the diameter of the beam of light that passes through the condense ○ Closed condenser: creates a high contrast image ○ Open condenser: the image is very bright with very low contrast. BRIGHT FIELD MICROSCOPY DARK FIELD MICROSCOPY enhances the contrast of light-absorbing or refractive specimens against a dark background. useful for observing transparent or low-contrast specimens, such as live cells, bacteria, or thin tissue sections Aids in visualization of flagella, cilia and cell nucleus Spirochetes, Spirilla, Campylobacter, Vibrio FLUORESCENCE MICROSCOPY specimens are stained with fluorochromes/ fluorochrome complexes Light of high energy or short wavelengths (from halogen lamps or mercury vapour lamps) is then used to excite molecules within the specimen These excited molecules emit light of different wavelengths, often of brilliant colours Ex: Auramine dye for acid-fast bacilli Appear brilliant on a dark background PHASE- CONTRAST MICROSCOPY based on the principle that light changes its speed when passing through cellular and extracellular structures with different refractive indices causes the structures to appear lighter or darker in relation to each other. allow the examination of cells without fixation or staining Advantage: allows you to visualize live organisms CONFOCAL MICROSCOPY achieves high resolution and sharp focus by using: ○ a small point of high-intensity ○ a plate with a pinhole aperture in front of the image detector The key feature of confocal microscopy is its ability to selectively illuminate and collect light from a specific focal plane within the specimen. POLARIZING MICROSCOPY designed to observe specimens that exhibit optical anisotropy, meaning that their optical properties vary with the direction of light Used to measure birefringence Birefringence: ○ ability to rotate the direction of vibration of polarized light ○ a feature of crystalline substances or substances containing highly oriented molecules, such as cellulose, collagen, microtubules, and actin filaments. ELECTRON MICROSCOPY uses a beam of electrons instead of visible light to achieve much higher resolution in imaging. Resolving power is 100,000x more than the light microscope ELECTRON MICROSCOPY 2 types of EM: 1. Transmission EM: a. a beam of electrons passes through the tissue section to produce an image with black, white, other shades of gray b. allows isolated particles magnified as much as 400,000 times to be viewed in detail. c. studying internal structures of biological specimens, at the subcellular and atomic levels 2. Scanning EM: a. SEM is widely used for surface imaging (surfaces of cells, tissues, and organs) b. unlike the TEM, this instrument the beam does not pass through the specimen THANK YOU QUIZ LABORATORY ACTIVITY Draw and label the parts of a microscope