Lecture 4 Microscopy.pdf

Stage 1 Fundamentals of Microbiology BIO1314 Coordinator : Dr Linda Stewart LECTURE 4 – MICROSCOPY Microscopy Principles of Light Microscopy Bright field microscopy Dark field Microscopy Phase contrast Differential interface contrast Fluorescence microscopy Confocal scann...

Stage 1 Fundamentals of Microbiology BIO1314 Coordinator : Dr Linda Stewart LECTURE 4 – MICROSCOPY Microscopy Principles of Light Microscopy Bright field microscopy Dark field Microscopy Phase contrast Differential interface contrast Fluorescence microscopy Confocal scanning laser microscopy Preparation and Staining of specimens Probing cell structure Electron microscopy Cryo-Electron Microscopy Microscopy Microorganisms range in size from the smallest viruses which are measured in nanometers (nm), to the largest protists and bacteria, which can be about 200 micrometers (μm). Microscope Microbiologists oldest and most basic tool for studying microbial structure Light microscopes were first invented and still widely used today Enlarge the image of an object Many types now used and some are extremely powerful All light microscopes use lenses that magnify the image Resolution limit is 0.2µm Lenses Create images by bending light Light is refracted (bent) when passing from one medium to another Refractive index ◦ a measure of how greatly a substance slows the velocity of light Direction and magnitude of bending is determined by the refractive indices of the two media forming the interface Lenses Focus light rays at a specific place called the focal point (F) Distance between center of lens and focal point is the focal length (f) Strength of lens related to focal length short focal length more magnification The Light Microscope Many varieties ◦ Bright-field microscope ◦ Dark-field microscope ◦ Phase-contrast microscope ◦ Fluorescence microscope ◦ Confocal microscope Compound microscopes ◦ Image formed by action of 2 lenses 7 The Bright-Field Microscope Used to examine both stained and unstained specimens. Produces a dark image against a brighter background. Has several objective lenses producing different magnification. Total magnification: Product of ocular lenses and objective lenses 8 Some Principles of Light Microscopy Magnification: the ability to make an object larger – not the limiting factor in the ability to see small objects (magnification can be increased without limit) Resolution: the ability to distinguish two adjacent objects as separate and distinct – governs the ability to see the very small, as resolution is a function of the physical properties of light ◦ Resolution is determined by the wavelength of light used and numerical aperture of lens – ◦ shorter wavelength  greater resolution ◦ Limit of resolution for light microscope is about 0.2 μm ie two objects that are closer together than 0.2 μm cannot be resolved as distinct and separate  Working distance — distance between the front surface of lens and surface of cover glass or specimen when it is in sharp focus 10 11  THE OIL IMMERSION OBJECTIVE (100X) If air is replaced with immersion oil, many light rays that did not enter the objective due to reflection and refraction at the surfaces of the objective lens and slide will now do so. This results in an increase in resolution and numerical aperture. 12  Many microbes are not colored. Unpigmented microbes are not visible because Visualizing there is not enough contrast between the cells, subcellular structures, and water. Living, Three types of light microscopes can be used: Unstained ◦ Dark-field microscope Microbes ◦ Phase-contrast microscope ◦ Differential interference contract (DIC) microscope 13  Visualizing living unstained microbes Dark-field microscopy ◦ Image is formed by light reflected or refracted by specimen ◦ Light reaches the specimen from the sides only ◦ The only light reaching the lens has been scattered by specimen ◦ Produces a bright image of the object against a dark background ◦ Used to observe living, unstained preparations used to observe internal structures in eukaryotic microorganisms used to identify bacteria such as Treponema pallidum, the causative agent of syphilis Cells visualized by different types of light microscopy Visualizing living unstained microbes Phase-contrast microscopy ◦ Invented in 1936 by Frits Zernike ◦ Based on the principle that cells differ in refractive index from their surroundings ◦ Light passing through the cells differs in phase from light passing through the surrounding liquid ◦ Deviated and undeviated light are combined in a condenser to generate an image ◦ Improves the contrast of a sample without the use of a stain ◦ Resulting image is dark cells on a light background ◦ Excellent way to observe microbial movement and detecting bacterial structures The Differential Interference Contrast Microscope (DIC) Creates image by detecting differences in refractive indices and thickness of different parts of specimen Uses a polarizer to create two distinct beams of polarized light that pass through the specimen into the objective where they combine as one. Combined beams not in phase as refractive indices are different enhancing subtle differences in cell structures Structures not visible by bright-field microscopy are sometimes visible by DIC Excellent way to observe living cells ◦ live, unstained cells appear brightly colored and three-dimensional ◦ cell walls, endospores, granules, vacuoles, and nuclei are clearly visible 16 The Fluorescence Microscope Produces image by exciting a specimen with a wavelength of light that triggers the object to emit fluorescence light Specimens stained with fluorochromes that absorb light and emit visible fluorescent light Shows a bright image of the object resulting from the fluorescent light emitted by the specimen Has applications in medical microbiology and microbial ecology studies 17  Fluorochrome-labeled probes, such as antibodies, or fluorochrome dyes tag specific cell constituents for identification of unknown pathogens Localization of specific proteins in cells Mycobacterium bovis specific binders Live cells green Dead cells red Cytoskeleton protein of Bacillus subtilis 18 Improving Contrast in Light Microscopy Confocal scanning laser microscopy (CSLM) ◦ Uses a computerized microscope coupled with a laser source to generate a sharp, composite 3D image of specimens ◦ Computer can focus the laser on single layers of the specimen ◦ Different layers can then be compiled for a three-dimensional image ◦ Resolution is 0.1 μm for CSLM ◦ Numerous applications including study of biofilms Preparation and Staining of Specimens Staining Increases visibility of specimen helps to Accentuates specific morphological features visualize and identify Preserves specimens microbes Fixation Preserves internal and external structures by inactivating enzymes that can disturb cell morphology and toughens cell structures, and fixes them in position Organisms usually killed and firmly attached to microscope slide Two Types of Fixation Heat fixation – routinely used with bacteria and archaea preserves overall morphology but not internal structures Chemical fixation – used with larger, more delicate organisms eg protists protects fine cellular substructure and morphology Dyes Staining improves contrast, dyes make internal and external structures of cell more visible by increasing contrast with background resulting in a better final image ◦ Dyes are organic compounds - each dye has an affinity for specific cellular materials ◦ Many dyes are positively charged and are therefore called basic dyes Simple Staining Simple stains ◦ a single stain is used ◦ use can determine size, shape, and arrangement of bacteria  Differential Staining Can be used to: i. Divide organisms into groups based on their staining properties. Gram stain Acid-fast stain ii. Detect presence or absence of structures. Capsules Flagella 24 Gram Staining Most widely used differential staining procedure Divides bacteria into two groups: Gram-positive (purple) and Gram-negative (pink), based on differences in cell wall structure Acid-Fast Staining Particularly useful for staining members of the genus Mycobacterium e.g., Mycobacterium tuberculosis – causes tuberculosis e.g., Mycobacterium leprae – causes leprosy High lipid content in cell walls (mycolic acid) prevents dyes from binding to cells is responsible for their staining characteristics Ziehl-Neelson method of staining used concentrated phenol and carbol fuchsin to drive the stain into the cell Staining Specific Structures Endospore staining ◦ heated, double-staining technique ◦ bacterial endospore is one color and vegetative cell is a different color Capsule stain used to visualize polysaccharide capsules surrounding bacteria ◦ negative stain - capsules may be colorless against a stained background Flagella staining to provide information on the presences and distribution pattern of flagella ◦ mordant applied to increase thickness of flagella Limits of microscopic resolution 28 Probing Cell Structure: Electron Microscopy Electrons replace light as the ‘illuminating’ beam that is focused. Wavelength of electron beam is 100,000x shorter than visible light, resulting in a higher resolution image. Allows for microbial morphology to be studied in detail. Rhodospirillum rubrum (a) phase contrast 29 (b) TEM Probing Cell Structure: Electron Microscopy Use electrons rather instead of visible light to image cell and cell structures Electromagnets function as lenses and the whole system operates in a vacuum Transmission electron microscopy (TEM) The electron microscope. ◦ High magnification and resolution (0.2 nm) as wavelengths of electrons are much shorter than wavelengths of light ◦ Electrons scatter when they pass through thin sections of a specimen ◦ Transmitted electrons are under vacuum which reduces scatter and produce clear image ◦ Denser regions in specimen scatter more electrons and appear darker ◦ Enables visualization of structures at the molecular level ◦ Specimen must be very thin (20–60 nm) (electrons are poor at penetrating) and be stained (using osmic acid, permanganate, uranium to scatter the electrons and improve contrast) Transmission Electron micrographs. 31 Other Preparation Methods Negative staining ◦ Specimen spread out in a thin film with chemicals. ◦ Produces images bright against a dark background. ◦ Used for study of viruses and cellular microbes. Shadowing ◦ Specimen is coated with a heavy metal on one side. ◦ Useful for virus particle morphology, flagella, and D N A. 32 Preparation Methods Without Metals Freeze-etching ◦ Rapidly freeze cells in liquid nitrogen. ◦ Frozen cells are brittle and fracture along lines of greatest weakness (that is, internal membranes). ◦ Allows for observation of shapes of intracellular structures. 33  Probing Cell Structure: Scanning Electron Microscopy (SEM) An electron beam scans the object and uses electrons excited from the surface of a specimen to create detailed image Specimen is coated with a thin film of heavy metal (e.g., gold) Scattered electrons are collected by a detector, and an image is produced Magnification range of 15✕–100,000✕ but only surface is visualized Produces a realistic 3D image of specimen’s surface features Can determine actual in situ location of microorganisms in ecological niches Scanning Electron micrographs. Cryo-Electron Microscopy Earned Nobel Prize in chemistry in 2017. Used to visualize biomolecules (that is, proteins) and generate high resolution structures. Samples rapidly frozen, then multiple TEM images are captured at different angles. These images combine to generate a 3-D image. 35 Take Home Message There are many different varieties of microscopy. Understanding the basics of each, and how they work, will allow you to better grasp which one would be best for a research question. Specimen preparation and different staining methods are just as crucial to microscopy. ◦ Understanding staining is just as critical as understanding microscope methods. 36 Intended learning outcomes ◦ Evaluate the parts of a light microscope in terms of their contribution to image production and use of the microscope ◦ Compare and contrast the various types of light microscopes in terms of their uses, how images are created, and the quality of images produced. ◦ Recommend a fixation process to use when the microbe is a bacterium or archaeon and when the microbe is a protist ◦ Plan a series of appropriate staining procedures to describe an unknown bacterium as fully as possible ◦ Compare what happens to Gram-positive and Gram-negative bacterial cells at each step of the Gram-staining procedure. ◦ Evaluate light microscopy and electron microscopy in terms of their uses, resolution, and the quality of images created