Download 0.61 x wavelength of light

Molecular Cell Biology
Light Microscopy in Cell Biology
Cooper
Modified from a 2010 lecture by
Richard 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
Light behaves as a Wave
Wavelength sets limits
on what one can see
Lower limits on spatial resolution are
defined by the Rayleigh Criterion
Resolution = 0.61 x wavelength of light
NA (numerical aperture)
NA = nsinθ
n = refractive index of the medium
θ = semi-angle of an objective lens
θ
θ
The effect of NA
on the image of
a point.
θ
The need for
separation to
allow resolution
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 field
and 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
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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
Fluorescent tubulin injected into a
Drosophila embryo, plus a DNA stain
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Green Fluorescent Protein - Considerations
• Color - Not just green
• Brightness
• Time for folding
• Time to bleaching
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
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 lessens
blur from out-of-focus light
Optically Sectioning a Thick Sample: Pollen Grain
Multiple optical sections assembled to
form a 3D image
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
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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