1. Presented by: Ziah Dean Date: November 30, 2010 Course: EE 230

2. Overview  Intro: Single Molecule Fluorescence Microscopy  Single Molecule Localization Microscopy  The principle of STORM  Photoswitching labels  Resulting images  Conclusion

3. Single Molecule Fluorescence Microscopy  Single molecule detection offers new possibilities in achieving sub-diffraction-limited resolution.  Position of a single-molecule can be determined to high accuracy if sufficient number of photons are collected  One of the initial techniques to demonstrate this, is known as FIONA: Fluorescence Imaging One- Nanometer Accuracy.

4. FIONA Collects photons of light from a single fluorescent molecule. Obtains center of image using the point spread function of image. Curve fitted onto a Gaussian function

5. Single Molecule Localization Microscopy (SMLM)  Closely spaced fluorescent molecules are diffraction limited when imaged with conventional fluorescent microscopes.  This is primarily due to simultaneous fluorescence of molecule  When applying SMLM each individual fluorophore is imaged one at a time  This allowed high precision positioning of each molecule

6. SMLM Cont.

7. Definitions  Photoactivation: the process of activating a substance by means of radiant energy and especially light.  Photobleaching: The destruction of a photochemical fluorescence by high-intensity light in a particular wavelength.  Photoswitching: The modification of the structure of a compound by light, especially when accompanied by a change in function.

8. Principle of STORM Utilizes a photoactivation and photobleaching technique to allow stochastic photoswitching of fluorescent dyes. Photoswitching allows localization of precisely positioned fluorophores Through multiple cycles of photoswitching the position of many fluorophores are determined resulting in a super-resolution image.

9. Principle of STORM Cont.

10. Photoswitching Labels

11. Kinesin II labeled with Cy3-Cy5 Photoswitching of Labeled Cells

12. Resulting Images

13. Conclusion  STORM was able to go below the current diffraction-limit by having the capability to produce images at 20nm resolution.  New techniques have shown STORM as having the capability to produce multi-color and 3D images.  Some of the disadvantages to STORM include lack of imaging speed, sample specific, and limited to in situ and immunofluorescence imaging.

14. References  Ann L McEvoy, Derek Greenfield, Mark Bates and Jan Liphardt. BMC Biology 2010, 1741-7007- 8-106  Yildiz, A. et al.. Science 300, 2061–2065 (2003).  Rust MJ, Bates M, Zhuang X: Sub-diffraction- limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 2006, 3:793-796

15. Questions? Thanks For Your Attention!