molecular cell biology light microscopy in cell biology cooper modified from a 2010 lecture by...
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Molecular Cell Biology
Light Microscopy in Cell BiologyCooper
Modified from a 2010 lecture byRichard McIntosh, University of Colorado
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Images from a light microscope can be strikingly informative about cells
How are these images made? What questions can they answer?What are their limitations? Can you make and use them?
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Scales of absolute size: powers of 10
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Wavelength sets limitson what one can see
Light behaves as a Wave
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Resolution = 0.61 x wavelength of light NA (numerical aperture)
The effect of NAon the image ofa point.
The need forseparation to allow resolution
θθ
θ
Lower limits on spatial resolution aredefined by the Rayleigh Criterion
NA = nsinθn = refractive index of the mediumθ = semi-angle of an objective lens
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Contrast in the Image is Necessary:Types of Optical Microscopy Generate Contrast
in Different Ways
•Bright field - a conventional light microscope•DIC (Differential Interference Contrast -
Nomarski)•Phase contrast•Fluorescence•Polarization•Dark field
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Bright-field Optics: Light Passing Straight Through the Sample
•Most living cells are optically clear, so stains are essential to get bright field contrast
•Preserving cell structure during staining and subsequent observation is essential, so cells must be treated with “fixatives” that make them stable
•Fixing and staining is an art
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Classic drawings and modern images made from Giemsa-stained blood smears
Plasmodium falciparum Histidine-rich Protein-2
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Generating Contrast
• Staining• Coefficients of absorption among different materials
differ by >10,000, so contrast can be big• Without staining
• Everything is bright• Most biological macromolecules do not absorb visible
light• Contrast depends on small differences between big
numbers• Need an optical trick
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Mammalian Cell: Bright-field and Phase-contrast Optics
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Principles of bright fieldand phase contrast optics
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Differential Interference Contrast (DIC)
•Optical trick to visualize the interference between two parts of a light beam that pass through adjacent regions of the specimen
•Small amounts of contrast can be expanded electronically
•Lots of light: Video camera with low brightness & high gain
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Brightfield vs DIC
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DIC has shallow depth-of-field:Image a single plane in a large object
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Worm embryo
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DIC: Good contrast. Detection vs Resolution.Microtubules: 25 nm diameter (1/10 res.lim.) but visible in DIC
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Fluorescent staining:High signal-to-noise ratio (white on black)
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Principle of Fluorescence
• Absorption of high-energy (low wavelength) photon
• Loss of electronic energy (vibration)
• Emission of lower-energy (higher wavelength) photon
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Design of a Fluorescence Microscope
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Fluorescent tubulin injected into aDrosophila embryo, plus a DNA stain
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Green Fluorescent Protein - Considerations
• Color - Not just green• Brightness • Time for folding• Time to bleaching
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Live-cell Imaging of Microtubule Ends:EB1-GFP chimera
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GFP-Cadherin in cultured epithelial cells
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Immunofluorescence
•Primary Abs recognize the antigen (Ag)•Secondary Abs recognize the primary Ab•Secondary Abs are labeled
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Immunofluorescence Example
•Ab to tubulin•Ab to kinetochore
proteins•DNA stain (DAPI)
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Biological microscopy problem: Cells are 3D objects, and pictures are 2D images.
•Single cells are thicker than the wavelength of visible light, so they must be visualized with many “optical sections”
•In an image of one section, one must remove light from other sections
•Achieving a narrow “depth-of-field”•A “confocal light microscope”
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Laser-Scanning Confocal Light
Microscopy
• Laser thru pinhole• Illuminates sample with
tiny spot of light• Scan the spot over the
sample• Pinhole in front of
detector: Receive only light emitted from the spot
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Light from points that are in focus versus out of focus
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Spinning-disk confocal microscopy:Higher speed and sensitivity
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Example: Confocal imaging lessensblur from out-of-focus light
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Optically Sectioning a Thick Sample: Pollen Grain
Multiple optical sections assembled to form a 3D image
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3D Image Reconstructed From Serial Optical Sections Obtained with a Confocal Microscope
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Fluorescence can Measure Concentration of Ca2+ Ions in Cells:
Sea Urchin egg fertilization
Phase Contrast Fluorescence
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Summary
•Light microscopy provides sufficient resolution to observe events that occur inside cells
•Since light passes though water, it can be used to look at live as well as fixed material
•Phase contrast and DIC optics: Good contrast•Fluorescence optics: Defined molecules can be
localized within cells•“Vital” fluorescent stains: Watch particular
molecular species in live cells
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