Super-Resolution Fluorescence
Microscopy
Clif Thivierge
08/12/10
Prof. Kevin Burgess
Texas A&M University
The Bane of Imaging: Diffraction
Limit
practical limit obtained when imaging very small
objects by magnification
diffraction causes blurring of objects when
imaging smaller than ~200-500 nm (diffraction
limit)
“broadening” of a point caused by diffraction is
known as the “point spread function” ()
x-y = (0.61 )/( sin())
 = refractive index medium
 = half-cone angle of focused light
Examples of Diffraction Limit
Ways to Circumvent Limit
• Near Field Microscopy (NSOM)
• Far Field Microscopy
– Confocal, 4pi and I5M, SIM
• Super-Resolution
– Spatially Patterned Excitation
• STED
• RESOLFT
• SSIM
– Localization Methods
• STORM
• PALM
• FPALM
Near Field Imaging (NSOM)
-place microscope distance less than 1
wavelength from sample
-20-50 nm resolution
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problem: cannot image into sample because of
wavelength restriction
Far Field: Confocal Microscopy
• Non-linear 2-photon excitation and pinhole
detection decrease SPF beyond classical
limits
• 21/2 improvement in resolution
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problem: 2-photon excitation uses high
wavelengths which increase SPF:
x-y = (0.61 )/( sin())
Structured-Illumination
Micropscopy (SIM)
100 nm resolution
possible
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Conclusions
• methods use common dyes (good)
• confocal is easiest, most widely used
• best resolution obtainable only 100
nm (SIM)
• single molecule is problematic
Super-Resolution Microscopy
Goal: obtain sub-100 nm resolution
pioneered by Stefan Hell in mid-1990s
Max Plank Institute (Germany)
two methods:
(i) Spatially Patterned Excitation
STED, RESOLFT, SSIM
(ii) Localization Methods
STORM, PALM, FPALM
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Stimulated Emission Depletion
Microscopy (STED)
Spontaneous VS Stimulated Emission
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when an excited molecule encounters a photon matching it’s
emission energy, another “clone” photon is created and ground
state results
STED
(i) sample is excited with laser and blur is obtained due to
diffraction (exc)
(ii) another “doughnut” shaped laser excites at emission
wavelength (STED) of dye and switches outside dyes to “dark
state”
(iii) observing in between exc and STED, very resoved image is
produced
observe
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exc
STED
STED Microscopy
resolutions 20 - 30 nm common, best 6 nm
STED Dyes
-dyes need to be very photostable
excitation laser: 107 W/cm2
STED laser: 109 W/cm2
-dye needs large stimulated emission cross-section
-most common: Atto 532 and Atto 647N
Rhodamine derivatives?
Dyes Used for STED
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Reversible Saturable Optically
Linear Fluorescence Transitions
(RESOLFTs)
same concept as STED but dyes are made to “dark
state” by other mechanisms:
-switch to triplet state
-switch to ground state
-use reversibly photoswitchable dyes
advantages:
-less powerful lasers need to be used (100
W/cm2)
-this leads to many more dyes and even fluorescent
proteins being used
Single Molecule Imaging
the exact location of single dyes can be determined
by doing multiple excitation/emission cycles
Things Become More Complicated
with Multiple Dyes
Considerable overlap. Hard to identify
individual fluors in real live.
Super-Resolution Single Molecule
Localization Methods
• Stochastic Optical Reconstruction Microscopy
(STORM)
• Photoactivated Localization Microscopy
(PALM)
• Fluorescence Photoactivated Localization
Microscopy (FPALM)
all work by same principle: image only some
dyes at one time
Consider Previous Example
Considerable overlap. Hard to identify
individual fluors in real live.
Most Dyes in “Off-state”
localization of “on-state” dyes possible
Switch the State of the Dyes
localization of other dyes possible
Combine the Images
position of each dye is known
Dyes for Localization Microscopy
• have on and off state
• easily able to switch from on/off state
• on/off can be non-fluorescent or have a change
in either excitation or emission wavelengths
• best if reversible but not necessary
Common Dyes for Localization Mic.
Examples of Photoswitchable Dyes
Conclusions on Localization
Microscopy
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using this technique 3D localization of
labels can be achieved with 20 nm
resolution
Multicolor Imaging
methods discussed so far are valuable in the elucidation
of structures
to study interactions, multicolor imaging can be used
multicolor imaging has been done with both STED and
Localization Microscopy
Multicolor STED
2 methods:
(i) use set of dyes with non-overlapping
excitation, emission, and STED wavelengths
(hard to find)
(ii) find dyes with same STED excitation but
non-overlapping absorbance
Multicolor STED: Synaptic Proteins
Synaptophysin (red)
Syntaxin 1 (green)
Conclusions
still very young technology and best dyes have
yet to be discovered
impact is big since imaging is such a popular
tool
a lot of opportunity for innovation/development
The End