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Visualizing and Enumerating Micro- to FemtoPlankton OCN 626 Lecture 5 (Steward) Light Microscopy (Transmitted Light) • Advantages • Can see internal structure • inexpensive • Disadvantages • Limited resolving power (ca. 0.25 µm) Specimen on • Performs poorly with material on filters Dinophysis spp. from Ross Sea, Antarctica (Photo by Dawn Moran, Mark Dennett, and Julie Rose, Woods Hole Oceanographic Institution) glass slide Tungsten Lamp Light Microscopy (Traditional Epifluorescence) • Advantages • Can image cells on a filter • Can also count viruses even though they are not resolved • Reveals chlorophyl containing organisms • Can detect presence and number of any cells that can be specifically labeled (fluorescence-based probing) • Disadvantages • For most field samples, need to concentrate Mercury Arc Lamp Specimen on glass slide • Only stained or naturally fluorescent structures are visualized • Adds expense to the cost of a light microscope Tungsten Lamp Photo: Barry and Evelyn Sherr Traditional Membrane Filters were Unsuited for Epifluorescence Microscopy Traditional Membrane Filters Glass fiber (GF) Polyethersulfone (PES) Cellulose acetate (CA) Diatom, bacteria, viruses (SYBR Green I stain (Photo: J. Fuhrman) Newer Very Flat Filters polycarbonate track-etched aluminum oxide New: polystyrene (not yet on market) Green = Eubacteria Probe! Red = Archaea Probe! Archaea and Bacteria Stained by polynucleotide Probe (Photo: E. DeLong) Light Microscopy (Photoactivated Localization Microscopy or PALM) http://www.sflorg.com/sciencenews/av/vdscn081006_01_01.mov Eric Betzig - Janelia Farm Research Campus Jan Liphardt- Lawrence Berkeley National Lab • Advantages • Overcomes the resolution limit of traditional light imaging • Disadvantages • Expensive, great for studies of cellular structure; not a tool for routine looking and counting Light Scattering (Flow Cytometry) • Advantages Intensity of forward scatter varies as a function of size • Limited sample preparation • Small sample volume • Fast, automated counting • Multidimensional discrimination among different cell types • Possibility for sorting • Disadvantages • Does not provide an image • Viruses near detection limit Side scatter varies as a function of internal structure Flow Cytometry - fluoresence/multidimensional discrimination Can also measure fluorescence properties of each particle using autofluoresence (photosynthetic pigments) or fluorescent probes Marie et al. Current Protocols in Cytometry 2001 Flow Cytometry - sorting Zehr et al. Science 2008 Genomics Light Scattering (NanoSight) • Advantages • Abundance by light scatter and sizing based on particle diffusivities • Can distinguish DNA- and nonDNA-containing • Good for quick counts of homgeneous or limited mixtures of particles • Disadvantages • Limited sensitivity - cannot resolve biological particles < 40 nm • Not suitable for larger cells • Fluorescence sensitivity low FlowCAM - combined flow cytometry and imaging • Advantages • Automated counting AND image capture on the fly • Disadvantages • Limited to larger cells Electron Microscopy (Transmission and Scanning) • Advantages • Much higher resolution than light microscope (1 nm) • Disadvantages • Very expensive to buy, operate, and maintain Table Top! (Hitachi) Scanning (Hitachi) (JEOL) Transmission (JEOL) Electron Microscopy (Transmission) • Advantages • Very high magnification and resolution (1 nm) • Can reveal detailed internal structure using thin sectioning • Disadvantages • Expensive to buy, maintain, and operate • Considerable sample preparation required, esp. for sectioned material Electron Microscopy (Scanning) • Advantages • Very high magnification 3D detailed views • Excellent tool for taxonomy • Disadvantages • Resolution of very fine structure (nm scale) can be obscured by the coating process • Only see external structure • Expensive to buy, maintain, and operate Atomic Force Microscopy • Advantages • Highest Possible Resolution (individual atoms) • Disadvantages • External structure only • Great for structure, not for routine counts Impedance Pulse-based Instruments • Advantages • Simple, quick, direct sizing and counting • Disadvantages • Cannot distinguish cells or viruses from detritus or organic colloids Bead Standards • Advantages • 0.4 to 1200 µm • Easy, and accurate, precise http://www.microparticles.de/certified_particles.html Does not distinguish cells from detritus Tunable nanopore • Works down to 50 nm • Requires frequent calibration • Many viruses < 50 nm • Distinguishes based only on size Cell count Salinas et al. Ann. Rheum. Dis. Distinguishes only based on size Coulter • • • Disadvantages • Disadvantages • • Advantages Manual
