1. Stochastic Optical Reconstruction Microscopy (STORM)
美国哈佛大学年轻的中国学者庄小威教授将荧光光谱和显微分析技术应用于单分子测量，发明了三维随机光重构显微术（3D-STORM）。

3. Example from Science Science, February 8, 2008
Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy Bo Huang,1,2 Wenqin Wang,3 Mark Bates,4 Xiaowei Zhuang1,2,3*
Science, February 8, 2008
1 Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA. 2 Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA. 3 Department of Physics, Harvard University, Cambridge, MA 02138, USA. 4 School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA. 3

4. Members of team
Xiaowei Zhuang, Prof of Chemistry & Chemical Biology, Prof of Physics, HHMI, Harvard
Bo Huang, post-doctoral fellow in Zhuang lab.
Wenqin Wang, graduate student, Dept of Physics, Harvard; member Zhuang lab
Mark Bates, graduate student, Division of Engineering and Applied Sciences, Harvard; member of Zhuang lab 4

5. Finding Out the Position of a Molecule
FIONA
σ  σPSF / N1/2
Thompson at al., Biophys. J., 2002
Yildiz et al., Science, 2003
A few molecules
Localization by sequential photobleaching
or QD blinking
Gordon et al., PNAS, 2004
Qu et al., PNAS, 2004
Lagerholm et al., Biophys. J., 2006
Lydke et al., Optics Exp., 2005 5

6. Demonstration of the difference between single- and two-photon excitation
The cuvette is filled with a solution of a dye, safranin O, which normally requires green light for excitation. Green light (543 nm) from a continuous-wave helium-neon laser is focused into the cuvette by the lens at upper right. It shows the expected pattern of a continuous cone, brightest near the focus and attenuated to the left. The lens at the lower left focuses an invisible 1046-nm infrared beam from a mode-locked Nd-doped yttrium lanthanum fluoride laser into the cuvette. Because of the two-photon absorption, excitation is confined to a tiny bright spot in the middle of the cuvette.
2·hν excitation
Image source: Current Protocols in Cytometry Online Copyright © 1999 John Wiley & Sons, Inc. All rights reserved.
Slide credit: Brad Amos, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom 6 6

7. Wide-field vs. confocal vs. 2-photon
Drawing by P. D. Andrews, I. S. Harper and J. R. Swedlow 7 7

8. Many Molecules

9. The principle of STORM

10. The principle of STORM

11. The principle of STORM

12. The principle of STORM

13. The principle of STORM

14. The principle of STORM

15. The principle of STORM

16. The principle of STORM

17. The principle of STORM

18. The principle of STORM

19. The principle of STORM

20. The principle of STORM

21. A Photo-switchable Probe
Imaging laser (657 nm) 2 1 5
Activation laser pulses
Cy5 fluorescence
Time (s)
Activator
Reporter
Cy3
Cy5
Deactivation
6000 photons
Activation
Cy3
Cy5
Cy3
Cy5
Activation laser (532 nm) 21

22. More Colors Activator absorption (nm) Reporter emission (nm) 550 490
Activation pulses 2 1 5 t i m e ( s )
Fluorescence
Cy3
550
532
457
405 nm
Activation pulses
Activator absorption (nm)
Cy2
490 3 2 1 t i m e ( s )
Fluorescence
Alexa 405
400
665
690
775
Cy5
Cy5.5
Cy7
Activation pulses
Alexa 647
Fluorescence
Reporter emission (nm)
Bates et al, Science 317, 1749 – 1753 (2007) 22

23. 5 μm
B-SC-1 cell,
Microtubules stained with anti-β tubulin
Cy3 / Alexa 647 secondary antibody

24. 5 μm
Bates et al, Science 317, 1749 – 1753 (2007) 24

25. 500 nm
5 μm

26. █ Cy2 / Alexa 647: Microtubule
█ Cy3 / Alexa 647: Clathrin
█ Cy2 / Alexa 647: Microtubule
5 μm
Bates et al, Science 317, 1749 – 1753 (2007) 26

27. 1 μm

28. Avg = 172 nm
200 nm

30. Class evaluation
What was the most interesting thing you learned in class today?
2. What are you confused about?
3. Related to today’s subject, what would you like to know more about?
4. Any helpful comments.
通过长时间的积累和图像重合迭代，在计算机上重构出分子团个各分子的空间位置，从而重构出整个分子团的三维图景，其图像分辨率可以达到横向20～30纳米，轴向50～60纳米。

31. Slot Waveguide (WG) for On-chip Functions
Multi-slot WG: vertical, UCSD, 2008
Slot WG with polymer for thermal stability, ETRI, 2008
Slot WG w/ polymer for low-power E/O modulation, Univ. Washington, 2008
Biomolecules manipulation, Cornell, 2009
Multi-slot WG: horizontal, MIT, 2008
Photonic crystal slot WG for slow light, St. Andrews Univ., 2008
Si nano-crystal slot WG for nonlinearity, Univ. Trento, 2008
Gas sensor, Cornell, 2008

32. Biomolecules manipulation,
Cornell, 2009
Cornell大学的研究人员在2009年初在Nature期刊上发表了他们突破传统衍射极限的工作：在一个芯径为100纳米宽（亚波长）充满液体的缝隙波导中，成功地将光能量限制在60纳米的尺度内，可以对直径为75纳米的电解质微粒和DNA分子进行捕获和输运。

33. Building chips from collapsing nanopillars
Although Berggren and his colleagues didn’t know it when they began their own experiments, for several years Aizenberg’s group has been using the controlled collapse of structures on the micrometer scale to produce materials with novel optical properties. But “particularly interesting applications would come from this sub-100-nanometer scale,” Aizenberg says. “It’s a really amazing level of control of the nanostructure assembly that Karl’s group has achieved.”
Ironically, the work was an offshoot of research attempting to prevent the collapse of nanopillars. “Collapse of structures is one of the major problems that lithography down at the 10-nanometer level will face,” says Karl Berggren, the Emanuel E. Landsman (1958) Associate Professor of Electrical Engineering and Computer Science, who led the new work.

34. Building chips from collapsing nanowalls
--By turning a common problem in chip manufacture into an advantage, MIT researchers produce structures only 30 atoms wide.