1. Super-Resolution Fluorescence Microscopy
Clif Thivierge
08/12/10
Prof. Kevin Burgess
Texas A&M University

2. 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 ~ 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

3. Examples of Diffraction Limit

4. 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

5. Near Field Imaging (NSOM)
-place microscope distance less than 1 wavelength from sample
nm resolution
problem: cannot image into sample because of wavelength restriction

6. Far Field: Confocal Microscopy
Non-linear 2-photon excitation and pinhole detection decrease SPF beyond classical limits
21/2 improvement in resolution
problem: 2-photon excitation uses high wavelengths which increase SPF:
x-y= (0.61 /( sin())

7. Structured-Illumination Micropscopy (SIM)
100 nm resolution
possible

8. Conclusions methods use common dyes (good)
confocal is easiest, most widely used
best resolution obtainable only 100 nm (SIM)
single molecule is problematic

9. Super-Resolution Microscopy
Goal: obtain sub-100 nm resolution
pioneered by Stefan Hell in mid-1990s
Max Plank Institute (Germany)
two methods:
Spatially Patterned Excitation
STED, RESOLFT, SSIM
(ii) Localization Methods
STORM, PALM, FPALM

10. Stimulated Emission Depletion Microscopy (STED)
Spontaneous VS Stimulated Emission
when an excited molecule encounters a photon matching it’s emission energy, another “clone” photon is created and ground state results

11. STED
sample is excited with laser and blur is obtained due to diffraction (exc)
another “doughnut” shaped laser excites at emission wavelength (STED) of dye and switches outside dyes to “dark state”
observing in between exc and STED, very resoved image is produced
observe
exc
STED

12. STED Microscopy
resolutions nm common, best 6 nm

13. 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?

14. Dyes Used for STED

15. 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

16. Single Molecule Imaging
the exact location of single dyes can be determined by doing multiple excitation/emission cycles

17. Things Become More Complicated with Multiple Dyes
Considerable overlap. Hard to identify individual fluors in real live.

18. 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

19. Consider Previous Example
Considerable overlap. Hard to identify individual fluors in real live.

20. Most Dyes in “Off-state”
localization of “on-state” dyes possible

21. Switch the State of the Dyes
localization of other dyes possible

22. Combine the Images
position of each dye is known

23. 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

24. Common Dyes for Localization Mic.

25. Examples of Photoswitchable Dyes

26. Conclusions on Localization Microscopy
using this technique 3D localization of labels can be achieved with 20 nm resolution

27. 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

28. 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

29. Multicolor STED: Synaptic Proteins
Synaptophysin (red)
Syntaxin 1 (green)

30. 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

31. The End