Upload
victor-stevenson
View
220
Download
5
Embed Size (px)
Citation preview
Molecular Cell Biology
Light Microscopy in Cell BiologyCooper
Modified from a 2010 lecture byRichard McIntosh, University of Colorado
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?
Scales of absolute size: powers of 10
Wavelength sets limitson what one can see
Light behaves as a Wave
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
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
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
Classic drawings and modern images made from Giemsa-stained blood smears
Plasmodium falciparum Histidine-rich Protein-2
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
Mammalian Cell: Bright-field and Phase-contrast Optics
Principles of bright fieldand phase contrast optics
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
Brightfield vs DIC
DIC has shallow depth-of-field:Image a single plane in a large object
QuickTime™ and aCinepak decompressor
are needed to see this picture.
Worm embryo
DIC: Good contrast. Detection vs Resolution.Microtubules: 25 nm diameter (1/10 res.lim.) but visible in DIC
Fluorescent staining:High signal-to-noise ratio (white on black)
Principle of Fluorescence
• Absorption of high-energy (low wavelength) photon
• Loss of electronic energy (vibration)
• Emission of lower-energy (higher wavelength) photon
Design of a Fluorescence Microscope
QuickTime™ and aCinepak decompressor
are needed to see this picture.
Fluorescent tubulin injected into aDrosophila embryo, plus a DNA stain
Green Fluorescent Protein - Considerations
• Color - Not just green• Brightness • Time for folding• Time to bleaching
QuickTime™ and aCinepak decompressor
are needed to see this picture.
Live-cell Imaging of Microtubule Ends:EB1-GFP chimera
QuickTime™ and aCinepak decompressor
are needed to see this picture.
GFP-Cadherin in cultured epithelial cells
Immunofluorescence
•Primary Abs recognize the antigen (Ag)•Secondary Abs recognize the primary Ab•Secondary Abs are labeled
Immunofluorescence Example
•Ab to tubulin•Ab to kinetochore
proteins•DNA stain (DAPI)
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”
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
Light from points that are in focus versus out of focus
Spinning-disk confocal microscopy:Higher speed and sensitivity
Example: Confocal imaging lessensblur from out-of-focus light
Optically Sectioning a Thick Sample: Pollen Grain
Multiple optical sections assembled to form a 3D image
QuickTime™ and aCinepak decompressor
are needed to see this picture.
3D Image Reconstructed From Serial Optical Sections Obtained with a Confocal Microscope
QuickTime™ and aCinepak decompressor
are needed to see this picture.
Fluorescence can Measure Concentration of Ca2+ Ions in Cells:
Sea Urchin egg fertilization
Phase Contrast Fluorescence
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
End