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 coursea. April 3: Lec 18: More Fluorescenceb. April 8: Lec 19: FRETc. April 10: Lec 20: Diffusiond. April 15: Lec 21: Diffusion IIe. April 17: Student presentationf. April 19: Special day! Student presentationg. April 22: Lec 22: Ion Channels, Nervesh. April 24: Lec 23: Tom Kuhlman—Single Cellsi. April 29: Lec 24: Ion Channels II j. May 1: Lec 25: Visionk. May 3rd (Friday) Papers due by Midnightl. 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

-100

0

100

200

300

400

500

600

0

200

400

600

800

1000

0200

400600

8001000

132.9 ± 0.93 nm

-100

0

100

200

300

400

500

600

200

400

600

800

1000

1200

200400

600800

1000

72.1 ± 3.5 nm

-100

0

100

200

300

400

500

600

700

0

200

400

600

800

1000

0200

400600

8001000

8.7 ± 1.4 nmDistance 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

~ 20-100 nmVia 2-photon: ~ 35 nm (next time)

Rhodamine-labeled microtubules, TIRActual 24 nm; Measured 300 nm

Regular Microtubules (In vitro) Image

•Take regular Image. •Then one fluorophore photobleaches.•Subtract off, get high resolution, repeat.

Imaging resolution300 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 ManufacturerAndor, 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)

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.

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-photonOne-photon

wavelength

Inte

nsity

2p

Simultaneous absorption of two photonsReasonable power if use pulsed laser

Two-Photon MicroscopyInherently confocal, long wavelength (less scattering)

One photon

Inherent spatial (z-) resolutionLow light scattering (scattering like 4)

Single-color excitation with multiple emission colorsDisadvantage: 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) <P> = ~150 W/cm2 , 30 msec/frame, scale bar 1 um. 160 nm effective pixel size

449 mW46 mW

50x lower power with Single Quantum Dot than with single fluorophores

2P- 3D FIONA Super-Accuracy Imaging

Class evaluation1. 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.