bio 224 intro to molecular and cell biology. microscopes are tools frequently used in cell biology...
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BIO 224
Intro to Molecular and Cell Biology
Microscopy
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 availableLight microscopes remain the basic tools of
cell biologists
Microscopy
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 specimensBacteriaHuman cellsParamecia
Light Microscopy
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 viewedRequires higher resolution
Ability of a microscope to distinguish items separated by small distances
More important than magnification
Light Microscopy
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
Resolution
Several types used routinelyBright-field microscopy is simplest
Light passes directly through the cellAbility to see parts depends on absorption of
visible light by cell componentsUsually requires use of dyesTissues usually fixed (preserved) prior to
stainingRequires cells be killedHistology labs routinely examine fixed and
stained tissues
Light Microscopy
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
Light Microscopy
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
Light Microscopy
Molecules of interest labeled with fluorescent dye
Used in living or fixed cellsFluorescent dye absorbs light at one
wavelength and emits it at a secondFilters detect the wavelength of light the dye
emitsFluorescent labeled antibodies often used to
detect specific proteins
Fluorescence Microscopy
Green fluorescent protein (GFP) of jellyfish used to see 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 GFP-labeled 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 Microscopy
1.28 Fluorescence recovery after photobleaching (FRAP)
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
Fluorescence Microscopy
1.29 Fluorescence resonance energy transfer (FRET)
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
Fluorescence Microscopy
1.30 Confocal microscopy
1.31 Confocal micrograph of human cells
Multi-photon excitation microscopyAlternative to confocal microscopyCan be applied to living cellsSpecimen 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 focusAllows for 3D resolution without need of
pinhole apertureMinimal damage, allowing 3D image of living
cells
Fluorescence Microscopy
1.32 Two-photon excitation microscopy
More powerful than light microscopyDeveloped in 1930s, first used on biological
specimens in 1940s and 1950s Higher resolution due to wavelength of
electronsPractical limit of resolution for biological
specimens is 1 to 2nmOver 100X improvement on light scopes
Electron Microscopy
Transmission electron microscopy (TEM)Similar to observation of stained cells with bright
field scopeSpecimens fixed and stained with heavy metal salts
Scatter electrons to provide contrastElectron beam passed through specimen, focused to
form image on fluorescent screenElectrons encountering heavy metal are deflected,
causing stained areas to be darkCan use positive or negative staining3D views can be obtained using electron
tomographyUses computer to generate 3D image from 2D scans over
range of directions
Electron Microscopy
1.33 Positive staining
1.34 Negative staining
Metal shadowing is technique used to see surface of structures or moleculesEvaporated metal sprayed on specimen from angle so
surfaces facing source are more heavily coatedCreates shadow effect, allowing 3D look
Freeze fracture used in studies of membrane structure, usually interior facesSpecimens frozen in liquid N2 and fractured by knife
bladeFollowed by shadowing with Pt and dissolving
biological material with acidProduces metal replica of sample surface
Freeze etching is variation the allowing visualization of external surface of membranes along with interior
Transmission Electron Microscopy
1.35 Metal shadowing
1.36 Freeze fracture
Scanning electron microscopyUsed to provide 3D image of cellsSurface of specimen coated with heavy metalBeam of electrons used to scan across
specimenElectrons scattered or emitted from specimen
surface collected by detectorResolution only about 10nm, restricted to
whole cells rather than macromolecules or organelles
Electron Microscopy
1.37 Scanning electron microscopy
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