1. More on smFRET and high-resolution microscopy

2. You give a (brief) talk? (4 per class: 10 minute.) Apr 21 Apr 28 May 5

3. Last time: smFRET Have problems with getting 100% labeling Have problems synchronizing. G-quadruplex: at least 3G, repeated 4 times

4. Another reason to do smFRET Labeling is stochastic Imagine protein is made up of 5 identical subunits, want 1D, 1A D: Alexa 488 A: Alexa 568 Require: ONE Donor ONE Acceptor on ONE Channel Reality: Labeling is stochastic. (even with optimization) Mechanosensitive Channel

5. Virtually Sort Out Channels with 1D & 1A by Photobleaching Use photobleaching to count the number of attached fluorophores. Wang et al. eLife, 2014 How many distances expect to see? E? RfRf RnRn Donor trace

6. Labeling Stoichiometry Note: Donor : Donor & Acceptor-Acceptor, thrown away D:nA ; nD:A ; nD:nA ; also gotten rid of by photobleaching 2 donor’s 1 donor, 1 acceptor There is direct excitation; subtracted out.

7. Eukaryotic MSCs Lichtman et.al, Hematology Zarychanski et al, Blood, 2012 Coste et al, Nature, 2012 Garcia et al, J. Neurosci, 1998 Corey et al, Nature, 2004 Hearing Cell growth (tumor metastasis) Kirber et al, Pflugers Arch, 1988 Simon et al, J. Physio, 2009 Touch

8. Mechanosensitive Channels of Large Conductance (MscL) Last “safety valves” Large nonselective pore High conductivity ~ 3 nS Crystalized in the closed state (Mycobacterium tuberculosis) Homo-pentamer Each subunit: TM1, TM2 and CP TM1 & TM2 for gating; CP as a pre- filter BUT: NO crystal structure for the open state! Chang et al, Science, 1998

9. Distribution of FRET efficiency (E) Closed (~23% open) Open closed E = 0.23 M42C After adding LPC, the high-E peak decreases, clearly showing the opening of the channels. Why are there THREE peaks? Wang et al. eLife, 2014 Expect 2 E, for closed; 2 E’s for open some open some closed open Closed (not all opened) Primarly opened Closed (not all opened)

10. How does MscL open? Barrel-Stave Model Helix-Tilt Model TM1 TM2 Membrane Perozo et al, Nat Rev Mol Cell Biol, 2006

11. Other techniques for high-resolution (besides STORM/PALM) STED, 2-Photon microscopy

12. STORM & PALM Most Super-Resolution Microscopy Inherently a single-molecule technique Huang, Annu. Rev. Biochem, 2009 Zhuang, 2007 Science STochastic Optical Reconstruction Microscopy PhotoActivation Localization Microscopy Betzig, 2006 Science 2-color secondary antibodies Cy2-Alexa 647 Cy3-Alexa 647 1. Activation (405 nm) (widefield) 2. Fluorescence ~ 500-650nm (widefield)

13. Correlative PALM-EM imaging 1  m TIRFPALMEM Mitochondrial targeting sequence tagged with mEOS Patterson et al., Science 2002 PhotoActivation Localization Microscopy (F)PALM (Photoactivatable GFP)

14. STORM/PALM: Get fantastic resolution (1-30 nanometers) by repeatedly localizing point-spread- function very accurately (based on FIONA) center width Collect from ~ 1-10k photons STimulated Emission Depletion: STED Get fantastic resolution (6-50 nanometers) by narrowing point-spread-function. Downside:

15. STimulated Emission Depletion (STED) http://www.mpibpc.gwdg.de/groups/hell/ Net result is a smaller Point Spread Function Sharpen the fluorescence focal spot is to selectively inhibit the fluorescence at its outer part. Make the molecules lase by using high- power STED beam! Huang, Annu. Rev. Biochem, 2009 S. Hell 200nm Recent development in super-resolution microscopy

16. Biological Example of STED Hell, PNAS, 2007 Analysis of spot size for Confocal (A) and STED (B) images of TRPM5 immunofluorescence layer of the olfactory epithelium. (A, C Inset) Confocal image at a lower (higher; box) magnification taken with a confocal microscope. (B) STED image. Effective point-spread function in the confocal (189 nm) and STED (35 nm) imaging modes. The transient receptor potential channel M5

17. You get Z-axis for free! Automatic confocal detection with 2-photon microscopy …plus other advantages Two-Photon Microscopy (Watt Webb, Science, 2003)

18. emission 1p two-photon One-photon wavelength Intensity 2p Simultaneous absorption of two photons Reasonable power if use pulsed laser Two-Photon Microscopy Inherently confocal, long wavelength (less scattering)

19. One photon Inherent spatial (z-) resolution Low light scattering (scattering like  4 ) Single-color excitation with multiple emission colors Disadvantage: Huge Instantaneous Excitation Powers: must use photostable dyes (e.g. quantum dots) objective two photon (Dis-)Advantages of 2-Photon Excitation

20. 2-Photon Widefield Excitation of Single Quantum Dot Blinking and emission intensity – laser power plot prove that it is single Qdots and 2-photon excitation Qdot585, 655 in PBS buffer, no reductants (no deoxygenation) = ~150 W/cm 2, 30 msec/frame, scale bar 1 um. 160 nm effective pixel size 449 mW 46 mW 50x lower power with Single Quantum Dot than with single fluorophores

21. 2P- 3D FIONA Super-Accuracy Imaging