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 × 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