Bio 301 - Ch. 2 Book Notes

Chapter 2 - Observing the Microbial Cell

  • fluorescence microscopy captures single molecules within a living cell

    • the fluorophores can label specific kinds of DNA or proteins

  • electron microscopy explores the cell’s interior

  • cryo-electron microscopy reveals the cells the 3D & models the shape of viruses

  • Example of how the technology has advanced since Leeuwenhoek’s first invention

    • spiral-shaped methane oxidizing bacterium were isolated in a peat bog in northern Russia

    • these bacteria are significant for curbing the release of methane from the archaea that produce it

    • these bacteria were visualized as a whole by phase-contrast microscopy

      • phase contrast utilizes the wave property of light to bend “out of sync” at the edge between a microbe and its surrounding medium to generate high contrast

  • electron transmission microscopy

    • the sample is sliced into very thin sections, stained with electron-dense metal atoms and bombarded with a beam of electrons

    • upon viewing the specimen, the electron beam reveals layers of membranes stacked like pancakes

      • these intracellular membranes are packed with proteins that transfer electrons from methane into oxygen

      • much of the methane is trapped in the cell’s biomass which prevents an escape of a greenhouse gas into the atmosphere

  • resolution of objects by our eyes

    • animals’ eyes observe an object by focusing its image on a retina packed with light-absorbing photoreceptor cells

      • the image appears sharp if the eye’s lens and cornea bend all the light rays from each point of the object to converge at one point on the retina

    • in the human eye, the finest resolution of two separate points is perceived by the fovea, the portion of the retina where the photoreceptors are packed at the highest density

      • the foveal photoreceptors are cone cells which detect primary colors and finely resolved detail

      • the distance between two foveal cones limits our resolution to 100-200 um

  • our eyes can detect a large population of microbes

    • examples include: a spot of mold or a cloudy tube of bacteria

    • observing these examples means the eye can detect the presence of mold or bacteria but it cannot resolve distinct cells

    • magnification is needed to resolve microbial cells individually to their natural shapes

      • as the distance increases between points of detail, our eyes can now resolve the object’s shape as a magnified image

  • microbial size & shape

    • eukaryotic microbes have a range of cell sizes to which a light microscope can resolve intracellular compartments such as the nucleus and vacuoles

    • protists show complex shapes and appendages

      • example includes an ameba from a freshwater ecosystem that has a large nucleus and pseudopods to engulf prey

      • another example includes Trypanosoma brucei which is an insect-borne blood parasite that causes African sleeping sickness; a nucleus and flagellum can be observed under a light microscope

    • certain shapes of bacteria are common to many taxonomic groups

      • both bacteria & archaea form similarly shaped rods, bacilli and cocci

      • it can be noted rods and spherical shapes evolved independently within different taxa

    • another shape distinction is a spirochete in which species of this shape cause diseases such as Lyme borreliosis and syphilis; the spiral form is maintained by internal axial filaments, flagella, and an outer sheath

    • an unrelated spiral form is known as spirillum which is a wide, rigid spiral cell that is similar to a rod-shaped bacillus

  • microscopy at different size scales

    • light microscopy (LM) - resolves images of individual bacteria by their absorption of light

      • bright-field microscopy - the specimen is commonly viewed as a dark object against a light-filled field

      • advanced techniques based on special properties of light include phase-contrast and fluorescence microscopy

    • electron microscopy (EM) - uses beams of electrons to resolve details several orders of magnitude smaller than those seen under LM

    • scanning electron microscopy (SEM) - uses a beam of electrons that is scattered from a metal-coated surface of an object which can generate an appearance of 3D depth

    • transmission electron microscopy (TEM) - a type of EM microscope where the electron beam travels through the object and the electrons are absorbed by an electron-dense metal stain

    • chemical imaging microscopy - uses spectrometry to map the chemical contents of a specimen like distribution of nitrogen and carbon compounds

    • X-ray crystallography - detects the interference pattern of X-rays entering the crystal lattice of a molecule; from this pattern a computational model of the structure can be built of the individual molecule

  • optics and properties of light

    • regions of the electromagnetic spectrum are defined by wavelength

      • visible spectrum ranges from 400-750 nm

      • radiation of longer wavelengths includes infrared and radio waves

      • radiation of shorter wavelengths includes ultraviolet rays and x-rays

    • information carried by radiation can be used to detect objects

      • all electromagnetic radiation travels through a vacuum at 3 Ă— 10^8 m/s (speed of light)

      • c is constant; the longer the wavelength, the lower the frequency of v (Hz)

      • wavelength limits the size of objects that can be resolved as separate from neighboring objects

  • requirements for resolution

    • contrast between the object and its surroundings

      • if an object and its surroundings absorb or reflect radiation equally, then the object will be undetectable

    • wavelength smaller than the object

      • the radiation must be equal to or smaller than the size of the object

      • if the wavelength of the radiation is larger than the object, then most of the wave’s energy will simply pass through the object

    • magnification

      • the smallest distance our retina can resolve is 150 um which means we are virtually unable to access all the information contained in the light than enters our eyes

      • we must adapt and spread the light rays far enough apart for our retina to perceive the resolved image

  • light interacts with an object

    • the physical behavior of light resembles a beam of particles, photons, and a waveform

      • each photon has an associated wavelength that determines how the photon will interact with a given object

      • the combined properties of particle and wave enable light to interact with an object in several different ways

    • absorption: the absorbing object gains the photon’s energy which is converted to a different form of energy; usually heat

      • when a microbial specimen absorbs light, it can be observed as a dark spot against a bright field (bright-field microscopy)

      • some molecules exhibit fluorescence which is when the absorbed light of a specific wavelength reemits energy as light with a longer wavelength

    • reflection: the wavefront redirects from the surface of an object at an angle equal to its incident angle

    • refraction: the light bends as it enters a substance / medium that slows its speed

      • a substance that exhibits this type of property is said to be refractive and has a higher refractive index than air

    • scattering: a portion of the wavefront is converted to a spherical wave originating from the object

      • if a large # of particles simultaneously scatter light, we observe a haze

  • magnification by a lens

    • refraction magnifies an image when light passes through a refractive material shaped so as to spread its rays

      • a parabolic curve is a type of shape that would spread light rays

    • when light rays enter a lens of refractive material with a parabolic surface, parallel rays each bend at an angle so that all the rays meet at a focal point

      • from this focal point, the light rays continue to spread out with an expanding wavefront

      • this expansion magnifies the image carried by the wave

      • focal distance is determined by the degree of curvature of the lens and by the refractive index of its material

  • resolution of detail

    • empty magnification occurs when there is magnification without increasing detail

    • the resolution of detail in microscopy in limited by the wave nature of light

      • interference occurs because the width of the lens is infinite but light rays form wavefront of infinite extent so only part of the wavefront enters

      • the converging edges of the waves interfere with each other to form alternation regions of light and dark

      • when viewing a specimen with LM, each point source generates a central peak of intensity to which the width of this central peak will define the resolution between any two points of the object

      • this resolution determines the degree of detail that can be observed

  • factors that limit the resolution of an image

    • the wavelength of light limits the sharpness of the peak of intensity of a point of detail

    • the finite width of the wavefront captured by the lens leads to interference and widens the peak intensity

      • bright-field microscopy resolves only details that are greater than half the wavelength of light (about 200 nm)

    • super-resolution imaging can allow for the tracking of cellular molecules at a precision of 20-40 nm

  • magnification

    • wavelength & resolution

      • the greatest magnification that can improve our perception of what is usually 100-200 um is about 1,000x

      • any greater magnification expands the image size but the peaks expand without resolution between them (empty magnification)

    • light and contrast

      • balanced amount of light yields the highest contrast between the dark specimen and the light background

      • high contrast is needed to perceive the full resolution at a given magnification

    • lens quality

      • all lens possess aberrations that detract from perfect curvature

      • optical properties limit the perfection of a single lens but manufactures can construct microscopes with a series of lens to multiply each other’s magnification as a correction

    • an object at the focal point of a lens sits at the tip of a cone of light formed by rays from the lens converging at the object

      • the angle of the light cone is determined by the curvature and refractive index of the lens

      • as theta increases, the horizontal width of the light cone increase giving way to a wider cone of light to pass through the specimen

      • the wider the cone of light rays, the less the interference between the wavefronts and the narrower the peak intensities in the image

      • a wider light cone enables us to resolve smaller details

      • the greater the angle of aperture of the lens will give better resolution

  • the compound microscope

    • a series of lower-power lens is used to multiply their magnification with minimal aberration (condenser & ocular lens)

    • lower-power lens require lower light levels because the excess light makes it impossible to observe the darkening effect of specimen absorbance

    • for higher-power lenses, they spread the light rays further and require an open diaphragm to collect sufficient light for contrast between the dark specimen and the bright field

    • condenser lenses increase light available for contrast but do not participate in magnification

    • in a parfocal system, when a specimen is viewed from one lens and the lens are rotated, it remains in focus

  • steps for observing a specimen under a compound microscope

    • position the specimen centrally in the optical column

    • optimize the amount of light to produce the optimal amount of contrast

    • focus the objective lens - typically start with a low-power objective which generates a greater depth of field

  • preparing a specimen for microscopy

    • wet mount; to observe microbes by placing a drop of water on a slide with a coverslip

      • advantages of wet mounting include: the organism being viewed is in it’s most natural state without artifacts resulting from chemical treatment

      • disadvantages of wet mounting includes: there is little contrast with the external medium and the specimen of interest (detection and resolution are minimal) & the sample can get easily dried out

    • the microbe observed must adhere to a specially coated slide within the temperature-controlled flow cell to avoid overheating

  • focusing the objects

    • the shape of the dark object is defined by points of light surrounding its edge when in focus

      • at higher power, these points of light are only partially resolved (limit of resolution reached)

      • partially resolution generates interference effects like extra rings of light

    • to observe motile bacteria, you must adjust the settings to have a higher magnification but a narrower depth of field

  • phase contrast microscopy

    • exploits differences in refractive index between the cytoplasm and the surrounding medium or between different organelles

    • depends on the principle of interference where two wavefronts interact with each other by addition or subtraction which results in alternating zones of constructive and destructive interference

      • addition - amplitudes in phase | subtraction - amplitudes out of phase

    • due to the fact that living cells have relatively high contrast because of their concentration of solutes, after light passes through the cell, about one-quarter of a wavelength behind the phase of light is transmitted directly through the medium

      • optical system is designed so that by the light refracted through the cell is slowed by a total of half a wavelength and when out of phase, it produces destructive interference (a region of darkness in the image of the specimen)

    • the light transmitted through the medium needs to be separated from the light interacting with the object

      • the transmitted light is separated by a ring-shaped slit (annular ring) which generates a hollow cone of light

  • fixation & staining for bright-field microscopy

    • fixation - a process which cells are adhered to a slide in a fixed position

      • cells can be fixed by methanol or heat treatments in order to denature the cell’s proteins so the side chains are exposed and bind to the glass

    • staining - the use of molecule that absorbs the incident light, usually over a wavelength that results in a distinctive color

    • how stains work:

      • stain molecules contain conjugated double bonds or aromatic rings that absorb visible light & have positively charged groups that bind cell-surface components with negative charge

      • different stains will depend on the strength of their binding and the degree of binding to different parts of a cell

  • simple stains

    • adds a dark color to the specimen but not to the external medium or immediate surrounding tissue

      • most common simple stain used is methylene blue

  • differential stains

    • this is a type of stain that differentiates among objects by staining only particular types of cells or specific subcellular structures

    • Gram stain - distinguishes cells that have a thick cell wall & retain a positively charged stain from cells that have a thin cell wall and outer membrane & fail to retain the stain

      • gram-negative cells usually stain red | gram-positive cells usually stain purple

    • Procedure for gram-staining

      • stain process: crystal violet dye, mordant (binding agent), decolorizer like ethanol, and counterstain (safranin)

      • during the stage of using a decolorizer, gram-positive cells appear dark purple and gram-negative cells appear colorless

    • the timing is important for each step because it can release the retained stain

      • in the decolorizer stage if over time then the gram-positive cells will release their crystal violet stain

      • in the counterstain stage if over time then both the gram-positive and gram-negative cells appear pink because of the safranin

  • acid-fast stain

    • a stain for mycobacteria that retain the dye fuchsin because of the presence of mycolic acids in the cell wall

  • giemsa stain for blood film

    • a mixture of methylene blue, eosin, and Azure B is used to stain blood cells & associated parasites

  • negative stain

    • a stain that colors the background and leaves the specimen unstained

    • a suspension of opaque particles such as India ink that is added to darken the surrounding medium and reveal transparent components of pathogens & microbes

  • antibody stains

    • stains linked to antibodies which can help identify precise strains of bacteria or molecular components of cells

      • the antibody may be conjugated to a reactive enzyme for detection

  • fluorescence microscopy

    • helpful to identify specific kinds of microbes and reveals specific cell parts function

    • for this type of microscopy, the specimen absorbs light of a defined wavelength and then emits light of lower energy

      • some microorganisms can fluoresce on their own due to the presence of chlorophyll

      • specific parts of the cell are labeled with a fluorophore

    • two different colors of fluorescence distinguish between the cyanobacterial cells performing photosynthesis and the heterocysts performing nitrogen fixation

  • excitation and emission

    • fluorescence occurs when a molecule absorbs light of a specific wavelength to raise an electron to a higher-energy orbital (excitation wavelength)

      • because this higher-energy electron state is unstable the electron will decay to an orbital of slightly lower energy, some E lost as heat

      • it will then fall back it’s original level by emitting a photon of less energy and longer wavelength (emission wavelength)

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    • the optical system for fluorescence microscopy uses filters to limit the source light to the wavelength range of excitation and the specimen’s emitted light to the wavelength range of emission

      • the wavelengths of excitation and emission are determined by the choice of fluorophore

  • fluorophores for labeling

    • a fluorophore’s properties is determined by the molecular structure of each fluorophore determines its peak wavelength of excitation and emission & its binding properties

    • the specificity of a fluorophore can be determined by:

      • chemical affinity - especially for biological molecules

      • labeled antibodies - antibodies that specifically bind to a cell component are covalently attached to a fluorophore which forms a “conjugated antibody” (immunofluorescence)

      • DNA hybridization - a short sequence of DNA attached to a fluorophore will hybridize to a specific sequence in the genome

      • gene fusion reporter - cells can engineered with a gene fusion which expresses a bacterial protein combined with either GFP or one of many GFP variants

  • super-resolution imaging

    • enables scientists to pinpoint the location of a DNA-binding protein with a precision tenfold greater than the resolution of an ordinary optical microscope

    • when tasked with tracking a single molecule, we must consider the shape of the magnified image of a point source of light

      • the uncertainty of the central peak position is about a tenth wide on the intensity profile and computation based on this profile can reveal the peak positions with high precision

  • confocal laser scanning microscopy

    • a microscopic laser light source scans across the specimen in such a way that the excitation light from at laser and the emitted light from the specimen are focused together to produce a high-resolution image

    • this type of imaging can be used for 3D imaging of pathogens embedded within host cell or tissue

  • Fluorescence In Situ Hybridization (FISH)

    • FISH can map specific taxa of microbes within an environment & can show the spatial location of microbial taxa

    • the FISH technique makes use of a fluorophore-labeled oligonucleotide probe that hybridizes to a microbe’s DNA or rRNA

      • doing so increases sensitivity because rRNA is present in approx 100-fold to 10,000-fold excess over DNA

  • chemical imaging microscopy

    • the use of mass spectrometry to visualize the distribution of chemicals within living cells

    • a high res method for chemical imaging is referred to as nanoscale secondary ion mass spectrometry (NanoSIMS)

      • has an ionizing probe which provides a source of energy that breaks up the large organic molecules of a sample

      • this instrument measures the fragment masses of the secondary ions that fly off the source which generates a mass spectrum

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