MCB3020 Lecture 2: Observing the Microbial Cell PDF

LECTURE 2 Observing the Microbial Cell MCB3020 General Microbiology Instructor: Maksym Bobrovskyy Copyright © 2024 by W. W. Norton & Company, In...

LECTURE 2 Observing the Microbial Cell MCB3020 General Microbiology Instructor: Maksym Bobrovskyy Copyright © 2024 by W. W. Norton & Company, Inc.  OVERVIEW 2.1 OBSERVING MICROBES 2.2 OPTICS AND PROPERTIES OF LIGHT 2.3 BRIGHT-FIELD MICROSCOPY AND PHASE-CONTRAST MICROSCOPY 2.4 FIXATION AND STAINING FOR BRIGHT-FIELD MICROSCOPY 2.5 FLUORESCENCE MICROSCOPY 2.6 ELECTRON MICROSCOPY, SCANNING PROBE MICROSCOPY, AND X-RAY CRYSTALLOGRAPHY Observing Microbes ▪ Since Leeuwenhoek’s time, powerful microscopes have been devised to search for microbes in unexpected habitats e.g. human stomach, deep sea, peat bog Methane-oxidizing bacteria shown by microscopy. Resolution of Objects by Our Eyes ▪ The size at which objects become visible depends on the resolution of the observer’s eye ▪ Resolution is the smallest distance by which two objects can be separated and still be distinguished ▪ We define what is visible and what is microscopic in terms of the human eye Human Retina Defines Resolution ▪ The resolution of the human retina is about 150 µm (1/7 mm) Limited by the distance between foveal cones and neuron clusters Detection vs. Resolution ▪ Detection is the ability to determine the presence of an object ▪ Magnification means an increase in the apparent size of an image to resolve smaller separations between objects A. Bacterial culture of Rhodospirillum rubrum. The presence of bacteria is detected, though individual cells are not resolved. B. Individual cells of Oenococcus oeni are resolved when magnified using light microscopy Microbial Size ▪ Microbes differ in size, over a range of a few orders of magnitude: Eukaryotic microbes (protozoa, algae, fungi) ▪ 2-2000 µm Prokaryotic microbes (bacteria, archaea) ▪ 0.2-10µm Viruses ▪ 5-1000 nm Microbial Shape ▪ Prokaryotic cell structures are generally simpler than those of eukaryotes Microbial Shape ▪ Eukaryotic microbes have many shapes Eukaryotic microbes are large enough that details of internal and external organelles can be seen under a light microscope. A. Amoeba proteus. B. Trypanosoma brucei (cause of sleeping sickness) among blood cells. Microscopy at Different Size Scales * *Cryo-electron microscopy Participation Quiz 2 Code: Microscopy A fictional creature with eyes spaced out 2 meters apart, with each eye containing only one photoreceptor cell, would perceive humans as: 1) Giants 2) Normal size 3) Microbes 4) None of the above  OVERVIEW 2.1 OBSERVING MICROBES 2.2 OPTICS AND PROPERTIES OF LIGHT 2.3 BRIGHT-FIELD MICROSCOPY AND PHASE-CONTRAST MICROSCOPY 2.4 FIXATION AND STAINING FOR BRIGHT-FIELD MICROSCOPY 2.5 FLUORESCENCE MICROSCOPY 2.6 ELECTRON MICROSCOPY, SCANNING PROBE MICROSCOPY, AND X-RAY CRYSTALLOGRAPHY Light Carries Information ▪ Electromagnetic radiation is composed of electrical and magnetic waves perpendicular to each other ▪ Waves exist in a range of wavelengths (spectrum) Visible Light is a Tiny Portion of the Spectrum ▪ Visible light is part of electromagnetic radiation spectrum Wavelength of visible light is 400– 750 nm ▪ For electromagnetic radiation to resolve an object, certain conditions must exist: Contrast between object and its medium Wavelength smaller than the object Magnification Light Interacts with an Object ▪ Absorption means that the photon’s energy is acquired by the absorbing object ▪ Reflection means that the wavefront bounces off the surface of an object ▪ Refraction is the bending of light as it enters a substance that slows its speed ▪ Scattering occurs when the wavefront interacts with an object smaller than the wavelength of light  Magnification by the Lens ▪ Magnification requires the bending of light rays, as in refraction Wavefronts of light shift direction as they enter a substance of higher refractive index ▪ Parabolic lens refracts parallel rays such that all of the rays meet at a certain point, called the focal point Magnification by the Lens Limitations of Light Microscopy ▪ Resolution of detail in microscopy is limited by the wave nature of light Light rays form wavefronts, which undergo interference Two wavefronts approaching at an angle generate an interference pattern in which intensity alternately increases and decreases away from peak intensity in the center Limitations of Light Microscopy ▪ Wavelength of light limits the sharpness of the peak intensity of the point of detail Shorter wavelength=sharper peak intensity ▪ Interference widens peak intensity causing decrease in resolution ▪ Light microscopy can resolve objects at half the wavelength of visible light, so roughly 200 nm (0.2 µm)  OVERVIEW 2.1 OBSERVING MICROBES 2.2 OPTICS AND PROPERTIES OF LIGHT 2.3 BRIGHT-FIELD MICROSCOPY AND PHASE-CONTRAST MICROSCOPY 2.4 FIXATION AND STAINING FOR BRIGHT-FIELD MICROSCOPY 2.5 FLUORESCENCE MICROSCOPY 2.6 ELECTRON MICROSCOPY, SCANNING PROBE MICROSCOPY, AND X-RAY CRYSTALLOGRAPHY Bright-Field Microscopy ▪ Generates a dark image of an object over a light background ▪ Can magnify a specimen by as much as 1,000x ▪ Bright-field microscopes use an objective lens for magnification ▪ All lenses contain inherent aberrations that detract from perfect curvature The Achromat The Mitutoyo $30.00 $23,000.00 Bright-Field Microscopy ▪ Numerical aperture (NA) determines resolving power of the lens: NA= n sin θ Angle of aperture (θ) is half of the angle of the cone of light that enters the objective lens Refractive index (n) of the lens ▪ Minimum resolution distance (R) is defined by half the wavelength of light (λ/2) and NA λ 𝑹= 𝟐(𝐍𝐀) Bright-Field Microscopy ▪ As the lens strength increases the light cone widens ▪ Immersion oil is used to collect and focus more light entering a high power objective Immersion oil increases the refractive index in the space between the lens and the object Compound Microscope ▪ Compound microscope is a system of multiple lenses designed to correct or compensate for aberration Ocular lens Objective lens Need to be parfocal ▪ Total magnification is magnification of ocular multiplied by that of the objective Dark-field Microscopy ▪ Dark-field optics enables microbes to be visualized as halos of bright light against darkness ▪ Light shines at oblique angle Only light scattered by sample reaches objective Makes visible objects below resolution limit Phase-Contrast Microscopy ▪ Superimposes refracted light and specimen light and transmitted light out of phase transmitted light shifted out of phase ¼ wavelength Reveals differences in refractive index as patterns of light and dark Can be used to view live cells and ¼ wavelength cellular organelles  OVERVIEW 2.1 OBSERVING MICROBES 2.2 OPTICS AND PROPERTIES OF LIGHT 2.3 BRIGHT-FIELD MICROSCOPY AND PHASE-CONTRAST MICROSCOPY 2.4 FIXATION AND STAINING FOR BRIGHT-FIELD MICROSCOPY 2.5 FLUORESCENCE MICROSCOPY 2.6 ELECTRON MICROSCOPY, SCANNING PROBE MICROSCOPY, AND X-RAY CRYSTALLOGRAPHY Preparing Specimens for Microscopy ▪ Wet mount preparation is simply placing microbes in a drop of water on a slide with a cover slip ▪ Advantages: Simple Cells remain in their natural state ▪ Disadvantages: Low contrast of specimen vs background May dry out Fixation and Staining ▪ Fixation - cells are made to adhere to a slide in a fixed position ▪ Staining - cells are given a distinct color Stains are organic molecules that chemically interact with various target structures (e.g. phospholipid membranes, peptidoglycan) Generally cationic, targeting anionic surface structures Process of staining often fixes the cells Examples: Different Types of Stains ▪ A simple stain adds Yeasts Yeasts + M-blue dark color specifically to cells, but not to the external medium or surrounding tissue Most commonly used stain is methylene blue Gram-positive Gram-negative ▪ A differential stain Streptococcus pneumoniae Proteus mirabilis stains one kind of cell but not another The most famous differential stain is the Gram stain Gram Stain Other Differential Stains ▪ Acid-fast stain: carbolfuchsin used to stain Mycobacterium species (A) ▪ Spore stain: malachite green used to detect spores of Bacillus and Clostridium (B) ▪ Negative stain: congo red colors the background, which makes capsules more visible (C) Microscopy Animation (click to view)  OVERVIEW 2.1 OBSERVING MICROBES 2.2 OPTICS AND PROPERTIES OF LIGHT 2.3 BRIGHT-FIELD MICROSCOPY AND PHASE-CONTRAST MICROSCOPY 2.4 FIXATION AND STAINING FOR BRIGHT-FIELD MICROSCOPY 2.5 FLUORESCENCE MICROSCOPY 2.6 ELECTRON MICROSCOPY, SCANNING PROBE MICROSCOPY, AND X-RAY CRYSTALLOGRAPHY Fluorescence Microscopy ▪ In fluorescence microscopy, the specimen absorbs light of a defined wavelength, and then emits light of lower energy (longer wavelength) which is fluorescence Excitation wavelength - wavelength at which specimen absorbs light Emission wavelength - wavelength at which specimen emits light ▪ Utilizes physical properties of fluorophores – molecules that fluoresce when excited Fluorescence Microscope ▪ The optical system for fluorescence microscopy uses color filters ▪ Color filters: Limit incident light to the wavelength of excitation Limit emitted light to the wavelength of emission Fluorophores ▪ Fluorophore specificity can be determined by: Chemical affinity Labeled antibody DNA hybridization ▪ A variety of fluorophores: Dyes (Alexa, Cy3/Cy5) DNA labels (DAPI, ethidium bromide) Protein labels (Allophycocyanin) Fluorescent proteins (Green fluorescent protein (GFP) Fluorescent Proteins ▪ Fluorescent protein fusions are used in various applications: monitor gene expression determine protein location and movement in the cell Fluorescent Proteins Localization The replisome and the DNA origin of replication. Fluorescence microscopy reveals the DNA origin, labeled blue by a protein fused to cyan fluorescent protein, binding at a sequence near the origin (Ori-CFP). Replisomes are labeled yellow by fusion of a DNA polymerase subunit to yellow fluorescent protein (Pol-YFP) in dividing cells of Bacillus subtilis. Fluorescent Protein Tracking ▪ Single-molecule localization by computation: a form of super-resolution imaging Fluorescence In Situ Hybridization (FISH) ▪ FISH utilizes the specificity of DNA/RNA labeled with fluorophores (probes) ▪ Allows for simultaneous labeling with several probes Fluorescence In Situ Hybridization (FISH) Fluorescence in situ hybridization (FISH) of bacteria and archaea. Syntrophy between anaerobic methane-oxidizing archaea (red FISH) and sulfate-reducing bacteria (green FISH) from a deep-sea cold seep at Guaymas Basin in the Gulf of California.  OVERVIEW 2.1 OBSERVING MICROBES 2.2 OPTICS AND PROPERTIES OF LIGHT 2.3 BRIGHT-FIELD MICROSCOPY AND PHASE-CONTRAST MICROSCOPY 2.4 FIXATION AND STAINING FOR BRIGHT-FIELD MICROSCOPY 2.5 FLUORESCENCE MICROSCOPY 2.6 ELECTRON MICROSCOPY, SCANNING PROBE MICROSCOPY, AND X-RAY CRYSTALLOGRAPHY Electron Microscopy (EM) ▪ Electrons behave like light waves Very high frequency Allow great resolution (0.2 nanometers) ▪ Sample must absorb electrons Coated with heavy metal ▪ Electron beam and sample are in a vacuum Tungsten gun generates high-voltage electron beam Magnetic lenses generate magnetic fields that focus electron-beam Electron Microscopy (EM) Two major types: ▪ Transmission electron microscopy (TEM) Electrons pass through the specimen Reveals internal structures ▪ Scanning electron microscopy (SEM) Electrons scan the specimen surface Reveals external features in 3D Transmission Electron Microscopy (TEM) Transmission electron microscopy (TEM) ▪ Electron source- high-voltage current applied to a tungsten filament which gives off electrons when heated ▪ Magnetic lenses- correct the path of the electrons to focus the e- beam ▪ Fluorescent screen- used to visualize the image Transmission Electron Microscopy (TEM) A. Bacillus anthracis thin section, showing envelope and cytoplasm (uranyl acetate stain). B. “Injectisome” toxin injection devices from Salmonella enterica (phosphotungstate negative stain). Scanning Electron Microscopy (SEM) ▪ SEM microscope is arranged differently compared to TEM Scanning Electron Microscopy (SEM) A. Archaea of a wetland biofilm, with numerous hami. The archaea encase bacterial filaments. B. Helicobacter pylori adheres to the villi (small bulges) of the gastric epithelium. Bacteria are colorized green. EM Sample Preparation ▪ The specimens for electron microscopy can be prepared in several ways Embedded in a polymer for thin sectioning ▪ Microtome is used to cut very thin slices Sprayed onto a copper grid ▪ The specimen is then treated with a heavy-metal salt such as uranyl acetate ▪ For SEM, specimen is coated with heavy metal and it is not sliced Cryo-Electron Microscopy and Tomography ▪ In cryo-EM, or electron cryomicroscopy Specimen is frozen (vitrified) - suspended in water and frozen rapidly in a refrigerant Does not require staining Low contrast ▪ Cryo-electron tomography, or electron cryotomography Avoids the need to physically slice the sample for thin-section TEM The images are combined digitally to visualize the entire object in 3D Generates high-resolution models of virus particles and proteins Cryo-Electron Microscopy and Tomography Cryo-Electron Microscopy and Tomography Flagellar Motors from two bacteria Cryo-Electron Microscopy and Tomography Magnetotactic cell visualized by cryo- electron tomography. A. A single cryo-EM scan lengthwise through Magnetospirillum magneticum. B. 3D model based on multiple scans. C. Expanded view of the cell interior. Scanning Probe Microscopy ▪ Scanning probe microscopy (SPM) enables nanoscale observation of cell surfaces ▪ The atomic force microscope (AFM) is an example of an SPM It measures the van der Waals forces between electron shells of adjacent atoms of the cell surface and the sharp tip It can be used to observe live bacteria in water or exposed to air (unlike electron microscopy) Atomic Force Microscopy X-ray Crystallography X-ray diffraction analysis ▪ Sample is crystallized to fix position of specimen atoms in a symmetrical crystal structure ▪ X-rays beam is shot at a crystallized sample ▪ Diffraction pattern is generated when X-rays diffract according to position of atoms ▪ Compute position of atoms from pattern of scattered X- rays X-ray Crystallography ▪ Today, X-ray data undergo digital analysis to generate sophisticated molecular models ▪ e.g. anthrax lethal factor A toxin produced by Bacillus anthracis Note: the model was encoded in a protein data bank (PDB) text file (PDB code: 1J7N) Chapter Summary ▪ When observing microbes, resolution and magnification are paramount ▪ Different kinds of microscopes are required to resolve cells and subcellular structures: - Bright-field: employs various stains - Dark-field: detects unresolved objects - Phase-contrast: exploits differences in refractive indices - Fluorescence: employs fluorophores for labeling Chapter Summary ▪ Electron microscopes use beam of electrons instead of light rays - TEM: provides internal details in 2D - SEM: provides external details in 3D ▪ Scanning probe microscopes (SPMs) include the atomic force microscope (AFM) - Allow observation of living cells in water or in air ▪ Molecules can be visualized by X-ray crystallography Suggested Reading ▪ Microbiology: An Evolving Science, 6th Edition – CHAPTER 2 ▪ Resource to learn about optics: florida.pbslearningmedia.org/resource/arct15-sci-lensmirrorlab/lens- and-mirror-lab/?student=true ▪ Young’s double slit experiment to visualize wave interference https://www.khanacademy.org/science/in-in-class-12th-physics-india/in- in-wave-optics/x51bd77206da864f3:young-s-double-slit- experiment/v/youngs-double-split-part- 1#:~:text=We%20can%20see%20interference%20in,of%20constructive %20and%20destructive%20interfere  Lecture Slides This concludes the Slide Set for Chapter 2 Microbiology: An Evolving Science, Sixth Edition Copyright © 2024 by W. W. Norton & Company, Inc.