BIO 224
Intro to Molecular and Cell Biology
 Microscopes are tools frequently used in cell biology
 Type of microscope used depends on the specimen
being viewed and magnification desired
 Many types of microscopes are available
 Light microscopes remain the basic tools of cell
biologists
 Early microscopes used by Hooke and others were
simple light microscopes
 Earliest microscopes were capable of up to 300 X
magnification
 Early scientists viewed and documented an impressive
array of specimens
 Bacteria
 Human cells
 Paramecia
 Modern microscopes are capable of 1000X
magnification (mag)
 Most cells range from 10um to 100um diameter,
easily seen with light scope
 Some large organelles can be visualized as well
 Fine structural details can’t be viewed
 Requires higher resolution
 Ability of a microscope to distinguish items separated by small
distances
 More important than magnification
 Limit of light microscopes about 0.2 um based on equation
 Determined by two factors
 Wavelength of visible light: λ
 Numerical aperture of the lens: NA
 Calculated by an equation: (0.61 λ) ÷ NA
 Wavelength fixed at 0.5 for light microscope
 NA is found by equation NA=η sinα
 η is refractive index of the medium which light travels through between
the specimen and the lens (1.0 for air, 1.4 with immersion oil)
 Angle α is half the width of cone of light collected by the lens
(maximum value is 90)
 Highest possible value of NA is 1.4
 Maximum theoretical resolution (also called resolving
power) had been achieved by late 1800s, no increases
expected
 Several types used routinely
 Bright-field microscopy is simplest
 Light passes directly through the cell
 Ability to see parts depends on absorption of visible
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light by cell components
Usually requires use of dyes
Tissues usually fixed (preserved) prior to staining
Requires cells be killed
Histology labs routinely examine fixed and stained
tissues
 Phase-contrast and differential interference-
contrast microscopy commonly used for living
cells
 Achieve contrast due to variations in thickness in
cell parts
 Speed of light drops as it passes through
intracellular structures, altering its phase
compared to light in cytoplasm
 Changes in phase converted to differences in
contrast
 Allows for improved images of live, unstained cells
 Video and cameras can be added to enhance features
of microscopes
 Has allowed visualization of movement of organelles
along microtubules
 Location of certain molecules can be seen using labels
and dyes
 Fluorescence microscopy is used for studying
intracellular distribution of molecules
 Molecules of interest labeled with fluorescent dye
 Used in living or fixed cells
 Fluorescent dye absorbs light at one wavelength and
emits it at a second
 Filters detect the wavelength of light the dye emits
 Fluorescent labeled antibodies often used to detect
specific proteins
 Green fluorescent protein (GFP) of jellyfish used to see
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proteins inside living cells
Protein can be expressed in cells and viewed with
microscope
Other related proteins with blue, yellow, or red emissions
also available
Other methods developed to follow interactions of GFPlabeled proteins within living cells
Fluorescence recovery after photo-bleaching (FRAP)
Region of interest bleached by exposure to high-intensity
light
Unbleached molecules move into bleached region
Allows detection of rate of movement of protein within cell
 Fluorescence resonance energy transfer
(FRET)
 Two proteins coupled to fluorescent dyes, like two
GFP variants that emit different wavelengths of
light
 Light emitted by one GFP variant excites the
second
 If proteins labeled by the two GFP variants interact
within cells, the fluorescent molecules will be near
each other and light emission will occur
 Conventional microscopy produces blurred and out-of-
focus images
 Can be improved by deconvolution: a computer analyzes
images obtained by different depths of focus and generates
a sharper image
 Confocal microscopy can be used to allow images of
increased contrast and detail
 Obtained from fluorescence from a single point in specimen
 Light produced by laser focused on specimen at a certain depth
 Fluorescence emitted passes through pinhole aperture before
hitting detector
 Allows only light emitted from plane of focus to reach detector
 Results in sharper image from scanning across image
 Series of images may be used for 3D image of sample
 Multi-photon excitation microscopy
 Alternative to confocal microscopy
 Can be applied to living cells
 Specimen illuminated with wavelength needing
absorption of two or more photons to excite fluorescent
dye
 Photons only excite fluorescent dye at point in the
specimen where input beam is focused
 Fluorescence only emitted from plane of focus
 Allows for 3D resolution without need of pinhole
aperture
 Minimal damage, allowing 3D image of living cells
 More powerful than light microscopy
 Developed in 1930s, first used on biological specimens
in 1940s and 1950s
 Higher resolution due to wavelength of electrons
 Practical limit of resolution for biological specimens is
1 to 2nm
 Over 100X improvement on light scopes
 Transmission electron microscopy (TEM)
 Similar to observation of stained cells with bright field
scope
 Specimens fixed and stained with heavy metal salts
 Scatter electrons to provide contrast
 Electron beam passed through specimen, focused to
form image on fluorescent screen
 Electrons encountering heavy metal are deflected,
causing stained areas to be dark
 Can use positive or negative staining
 3D views can be obtained using electron tomography
 Uses computer to generate 3D image from 2D scans over range of
directions
 Metal shadowing is technique used to see surface of
structures or molecules
 Evaporated metal sprayed on specimen from angle so surfaces
facing source are more heavily coated
 Creates shadow effect, allowing 3D look
 Freeze fracture used in studies of membrane structure,
usually interior faces
 Specimens frozen in liquid N2 and fractured by knife blade
 Followed by shadowing with Pt and dissolving biological
material with acid
 Produces metal replica of sample surface
 Freeze etching is variation the allowing visualization of
external surface of membranes along with interior
 Scanning electron microscopy
 Used to provide 3D image of cells
 Surface of specimen coated with heavy metal
 Beam of electrons used to scan across specimen
 Electrons scattered or emitted from specimen surface
collected by detector
 Resolution only about 10nm, restricted to whole cells
rather than macromolecules or organelles
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