Structure of Cells in the Microscope

 Typical animal cell is 10-20um in diameter

 Until early part of 19th century when light microscopes available that plant and animal tissues discovered to be
made of individual cells
o This discovery marks formal birth of cell biology (proposed as the cell doctrine by Schleiden and
Schwann)
 Animal cells are colourless and translucent
o Discovery of main internal features depended on development of stains that gave contrast to make
features visible
o Also introduction of electron microscope needed development of new techniques for staining and
preserving cells
o Today, microscopy depends on techniques as much on the performance of the microscope itself
 Resolving power—ability of microscope to distinguish small or closely adjacent images as separate
The Light Microscope can resolve Details 0.2um apart
 In general, given type of radian cannot be used to probe structural details smaller than its own wavelength
o Limitation of all microscopes
o Limit to resolution of light microscopes set by wavelength of visible lights (ranges from 0.4um to 0.7um)
o Bacteria, mitochondria generally smallest that can be clearly discerned by light microscope
 Limit of resolution—the limiting separation at which two objects can still be seen as distinct
Living Cells are Seen Clearly in a Phase Contrast or Differential Interference Contrast Microscope
 Possibility some component of cell may be lost or distorted during specimen preparation challenge for
microscopists
o Only way to avoid this issue is to examine cells while alive without fixing or freezing
o For this purpose, light microscopes with special optical systems useful
 Phase contrast microscope (differential-interference-contrast microscope)
o When light pass through cell, phase of light wave change according to cell’s refractive index: if through
relatively thick area, light becomes retarded—its phase is shifted relative to light that has passed through
thinner adjacent region
o Exploit interference effects produced when two sets of waves recombine, creating an image of the cell’s
structure
 Dark field microscope
o Illuminated rays of light directed from the side so only scattered light enters microscope lenses
o Cell appears as a bright object against a dark background
Tissues Usually Fixed and Sectioned for Microscopy
 To make permanent preparation that can be stained and viewed, one must treat cells in a fixative to immobilize,
kill and preserve them
o Fixation makes cells permeable to staining reagents and cross links their macromolecules so they are
stabilized and locked in position
Different Components of the Cell can be Selectively Stained
 There is little in the contents of most cells (70% water by weight) to impede the passage of light rays
o Most cells even if fixed and sectioned, almost invisible in ordinary light microscope—use stains
 Some dyes found to show a preference for parts of the cell making these internal structures visible
o Ex. hematoxylin as affinity for negatively charged molecules so reveals distribution of DNA, RNA and
acidic proteins
 Chemical basis of specificity of many dyes are unknown
Specific Molecules Can be Located in Cells by Fluorescence Microscopy
 Fluorescent molecules absorb light at one wavelength and emit it at another longer wavelength
o If such a compound illuminated at absorbing wavelength then viewed through a filter that allows only light
of the emitted wavelength to pass, it is seen to glow against a dark background
o As background is dark, even minute amount of glowing fluorescent dye can be detected
o Same number of molecules of an ordinary stain viewed conventionally practically invisible as they would
give only faint tinge of colour
 Most often used to detect specific proteins or other molecules in cells and tissues
Imaging of Complex 3D Objects is Possible with the Optical Microscope
 In the process of sectioning for light microscope the information about 3D is lost (thinner the better for light)
 Optical microscope focused on a particular focal plane within complex 3D specimens but also all the other parts of
the specimen above and below the plane of focus are illuminated also
o Light originating from these regions contributes to the image as out of focus
o Make it hard to interpret the image in detail and can lead to fine image structure being obscured
 Two solutions: computations and optical
 o Optical—from a series of optical sections taken at different depths and stored in a computer, easy to
reconstruct a 3D image
o Computational—often called image deconvolution
Confocal Microscope Produces Optical Sections by Excluding Out of Focus light
 Achieves a result similar to that of deconvolution but does so by manipulation of the light before it is measured
 It is an analog technique rather than digital
 Confocal fluorescence microscope
o Generally used with fluorescence optics but instead of illuminated the whole specimen at once, the optical
system at any instant focuses a spot of light onto a single point at a specific depth in the specimen
o Bright source of pinpoint illumination is required
 Usually supplied by a laser whose light passed through a pinhole
Electron Microscope Resolves the Fine Structure of the Cell
 Relationship between limit of resolution and wavelength of illuminating radiation holds true for light and electrons
as well
 With electrons, limit of resolution can be made very small
 Wavelength of electrons decrease as its velocity increases
 100x better than the resolution of light microscope
 Overall design, Transmission electron microscope (TEM) is similar to light microscope
Biological Specimens Require Special Preparation for the Electron Microscope
 In early days of application, electron microscope revealed previously unimagined structures
 Since specimen exposed to high vacuum in electron microscope, no possibility of viewing it in the living wet state
o Tissues usually preserved by fixation
 Because electrons have limited penetrating power, fixed tissues normally have to be cut in very thin sections
o Achieved by dehydrating specimen and permeating it with a resin to form block of plastic which is cut with
fine glass or diamond knife
 Thin sections, free of water and other volatile solvents placed on circular metal grid for viewing
 Issue: how can we be sure that the fixed specimen is the same as when alive
o Rapid freeing—water supercooled into rigid but noncrystalline state—vitreous ice
 Contrast in electron microscope depends on atomic number of atoms in specimen; higher AN#, the more
electrons are scattered and greater contrast
Images of Surfaces Can be obtained by Scanning Electron Microscopy (SEM)
 Directly produces an image of the 3D structural off the surface of a specimen
 Smaller, simple and cheater than TEM
 SEM uses electrons that are scattered or emitted from surface of specimen rather than TEM which uses electrons
that have passed through the specimen
Freeze Fracture and Freeze Etch Electron Microscopy Provide Views of Surfaces Inside the Cell
 Freeze fracture EM provides way of visualizing the interior of cell membranes
o Cells are frozen and then the frozen block cracked with a knife blade
o Fracture plane often passes through hydrophobic middle of lipid bilayers and expose the interior
o Fracture faces shadowed with platinum
 Freeze Etch EM can be used to examine either the exterior or interior of cells
o Frozen block is cracked with knife blade
o Ice level is lowered around the cells by the sublimation of ice in a vacuum as the temperature is raises—
called freeze drying
o Parts of the cells exposed by this etching process are shadowed to make platinum replica
o Exposes structure in the interior of the cell and can reveal their 3D organization with clarity
Negative Staining and Cryoelectron Microscopy Allow Macromolecules to be Viewed at High Resolution
 Negative staining
o Molecules supported on a thin film of carbon are washed with a aconcentrated solution of a heavy metal
salt
o After sample dried, thin film of metal salt covers carbon film everywhere except wehre it has been
excluded by presence of adsorbed macromolecule
o As macromolecule allows electorns to pass more readily than the surrounding heavy metal stain,
reversed or negative image of molecule created
o Useful for viewing large macromolecular aggregates such as viruses or ribosomes and for seeing the
subunit structure of protein filaments
Summary
 Cells that have been fixed and stained can be studied in a conventional light microscope
o Antibodies coupled to fluorescent dyes can be used to locate specific molecules in cells in a fluorescence
microscope
 Living cells can be seen with phase contrast, differential interference contrast, dark field, or bright field
microscopes
 All forms of light microscopy are facilitated by electronic image processing techniques which enhance sensitivity
and refine the image
 Confocal microscopy and image deconvolution provide thin optical sections and can be used to reconstruct 3D
images
 Determining detailed structure of membranes and organelles require higher resolution in TEM
 Specific macromolecules can be localized with colloidal gold linked to antibodies
 3D views of the surfaces of cells and tissues obtained by SEM
 Shapes of isolated macromolecules that have been shadowed with heavy metal or outlined by negative staining
can be readily determined by EM
using computational methods, multiple images, and views from different direction are combined to produce
detailed reconstructions of macromolecules and molecular complexes through a technique known as electron
microscope tomography