1.HW due today. (HW not turned back today because a person is sick and still hasn’t turned it in; will return Next Monday) 2. Another HW assigned today, due next Wednesday. Today’s Announcements

1.Outline of rest of course a.April 3: Lec 18: More Fluorescence b.April 8: Lec 19: FRET c.April 10: Lec 20: Diffusion d.April 15: Lec 21: Diffusion II e.April 17: Student presentation f.April 19: Special day! Student presentation g.April 22: Lec 22: Ion Channels, Nerves h.April 24: Lec 23: Tom Kuhlman—Single Cells i.April 29: Lec 24: Ion Channels II j.May 1: Lec 25: Vision k.May 3rd (Friday) Papers due by Midnight l.May 6, 7-10 pm: Final Exam (136 LLP) Today’s Announcements

SHRImP Super High Resolution IMaging with Photobleaching In vitro Super-Resolution: Nanometer Distances between two (or more) dyes ± 0.93 nm 72.1 ± 3.5 nm 8.7 ± 1.4 nm Distance can be found much more accurately than width (250 nm) Resolution now: Between 2-5 molecules: <10 nm (Gordon et al.; Qu et al, PNAS, 2004) Next slides gSHRIMP: > 5-40 molecules ~ nm Via 2-photon: ~ 35 nm (next time)

Rhodamine-labeled microtubules, TIR Actual 24 nm; Measured 300 nm Regular Microtubules (In vitro) Image Take regular Image. Then one fluorophore photobleaches. Subtract off, get high resolution, repeat. Imaging resolution 300 nm 1000 nm 60

Microtubules in a COS-7 cell 2 um Regular- resolution image 500 nm Spots localized versus frame Standard FWHM: 560 ± 20 nm 500 nm Super- gSHRImP FWHM: 96.5 ± 1 nm

Why do you get ≈ 60 nm, rather than ≈1 nm resolution? It’s like taking an absorption spectra, instead of fluorescence spectra. Can you just subtract off background? Background is large. You can subtract off average of background, but not fluctuation—sqrt N!

3-D sectioning with Confocal Three-dimensional reconstruction of a series of 2D images of PMMA spheres

Basic Set-up of Fluorescence Microscope Semwogerere & Weeks, Encyclopedia of Biomaterials and Biomedical Engineering, 2005 (Lasers, Arc Lamps) (Electronic Detectors: CCD, EMCCDs, PMTs, APDs) Nikon, Zeiss, Olympus, Leica—Microscope Manufacturer Andor, Hamamatsu, Princeton Instruments, other…make EMCCDs

Sample is 3-D. Detectors are 2-D. How do you get z-axis sectioning with Microscopy? A pinhole allows only in-focus light through 3-D sample Detector (Intensity) (Regular microscopy) Confocal Detection Focused Light creates fluorescence which gets to detector Light mostly gets rejected Smaller the pinhole, better out-of-focus discrimination but lose more signal. Scan sample in x, y, z and reconstruct entire image

3-D sectioning with Confocal Three-dimensional reconstruction of a series of 2D images of PMMA spheres

STimulated Emission Depletion (STED) Net result is a smaller Point Spread Function Sharpen the fluorescence focal spot is to selectively inhibit the fluorescence at its outer part. Huang, Annu. Rev. Biochem, 2009 S. Hell 200nm Recent development in super-resolution microscopy

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

You get automatic confocal detection with 2-photon microscopy …plus other advantages Two-Photon Microscopy (Watt Webb, Science, 2003)

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)

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

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

2P- 3D FIONA Super-Accuracy Imaging

Class evaluation 1. 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. Answer, and turn in at the end of class.