PH880 Topics in Physics

Transcription

1 PH880 Topics in Physics Modern Optical Imaging (Fall 2010)

2 Overview of week 12 Monday: FRET Wednesday: NSOM

3 Förster resonance energy transfer (FRET) Fluorescence emission i FRET Donor Acceptor wikipedia

4 FRET: spectroscopic ruler Determines distances between biomolecules labeled with an appropriate donor and acceptor fluorochrome when they are within 10 nanometers of each other. (* normal diffraction limited it d fluorescence microscope resolution is insufficient i to determine whether an interaction between biomolecules actually takes place.) Teakjip Ha group

5 508 VOL.5 NO.6 JUNE 2008 NATURE METHODS

6 Overview of week 12 Monday: FRET Wednesday: NSOM

7 Things Natural The Scale of Things Nanometers and More 10-2 m 1 cm 10 mm Things Manmade Head of a pin 1-2 mm The Challenge Ant ~ m mm Dust mite 200 mm 10-4 m 0.1 mm 100 mm 1,000,000 nanometers = 1 millimeter (mm) e Microwave MicroElectroMechanic al (MEMS) devices mm wide Human hair ~ mm wide Red blood cells (~7-8 mm) ~10 nm diameter Fly ash ~ mm ATP synthase Microworld Nan noworld 10-5 m 10-6 m 10-7 m 10-8 m Infrared Visible Ultraviolet 0.01 mm 10 mm mm 100 nm Pollen grain Red blood cells 1,000 nanometers = Zone plate x-ray lens 1 micrometer Outer ring spacing ~35 nm (mm) 0.01 mm 10 nm Self-assembled, Nature-inspired structure Many 10s of nm Nanotube electrode O O S O O O O O O O O O O O S O S O S Fabricate and combine nanoscale building blocks to make useful devices, e.g., a photosynthetic reaction center with integral semiconductor storage. O S P O O O O S O O S O S DNA ~2-1/2 nm diameter Atoms of silicon spacing nm 10-9 m m Soft x-r ray 1 nanometer (nm) 0.1 nm Quantum corral of 48 iron atoms on copper surface positioned one at a time with an STM tip Corral diameter 14 nm Carbon buckyball ~1 nm Carbon nanotube diameter ~1.3 nm diameter Office of Basic Energy Science Office of Science, U.S. DOE Version , pmd

8 Scanning probe microscopes Scanning Tunneling Microscope STM Atomic Force Microscope AFM Nearfield Scanning Optical Microscope NSOM

9 Scanning Tunneling Microscope: STM Binnig and Rohrer won Nobel Prize in 1986 for the development of STM S. Woedtke, Ph.D. thesis, Inst.f.Exp.u.Ang.Phys. der CAU Kiel, When STM tip is close to the specimen (~ 1nm), a tunneling current, I T is established I T is exponentially proportional to the distance A feedback loop maintaining I T can change z position topographical p information

10 STM images "quantum corral" Atom Carbon Monoxide Man Iron on Copper Iron on Copper Carbon Monoxide on Platinum Don Eigler, IBM Lutz & Eigler, IBM Lutz & Eigler, IBM

11 Atomic Force Microscope AFM STM is a precursor of AFM Feedback Loop V Laser PZT Mirror Photodiode ~ deflection Tip ThermoMicroscopes Explorer AFM Substrate AFM relies on contact rather than current nonconductive materials can be imaged

12 AFM images the compaction of DNA in yeast caused by a protein ti called AbF2 nuclear pore complex LR Brewer, et al, Biophysical journal, 2003 D Stoffler et al, Current opinion in cell biology, 1999

13 AFM + Fluorescence imaging techniques A. Gaiduk et al, Chem. Phys. Chem. 6, no. 5, pp , 2005

14 Near field

15 sub wavelength aperture (a) ( nm) ~10 nm Image can be reconstructed point by point spatial resolution is limited by a (rather than λ)

16 the propagation of waves :the loss of spatialinformationinformation Hartschuh et al., Angewandte Chemie,2008

17 History of NSOM 1. Theoretically proposed in 1928, EH Synge, Philos. Mag. 6, 356 (1928) 2. Demonstration at microwave frequencies with a resolution of λ/60. EA Ash ad G. Nicholls, Nature (London) 237, 510 (1972) 3. At visible wavelengths ( optical stethoscopy ) was demonstrated. D. Pohl, W. Denk, and M. Lanz, Appl. Phys. Lett. 44, 651 (1984) 4. Betzig et al used fiber probes to image a variety of samples with a number of different contrast mechanisms. Betzig, E. & Trautman, JK Science 257

18

19

20

21 NSOM tip fabrication chemical etching (meniscus or tube etching) Micro fabrication Fast Large cone angle Fast, convenient process mass production. (Low cost, reproducible) Toxic (HF) vapor Difficult to control surface quality Smooth surface Low cone angle (low throughput) Fragile Complex fabrication process

22 NSOM tip: metal coating

23 NSOM tip: illumination Waveguide tip (Takashi et al.1999) SiO2 cantilevered tip (Mitsuoka et al. 2000) Fiber tip by Nanonics Inc.

24 NSOM tip: intensity distribution Probe to Probe configuration (Ohtsu et al. 2000) Minh et al Lu et al. 2001

25 NSOM tip: geometry and light throughput Ultramicroscopy 57 (1995)

26 Common NSOM illumination

27 Other focusing concepts using near field optics Hartschuh et al., Angewandte Chemie,2008 a) Far field focusing using a lens. The angular frequency range of propagating p gwaves kx,max, and thus the focus diameter, is limited by the aperture angle of the lens kx,max=nsin(q)2p/l, with n being the refractive index and l the wavelength of light. b) Aperture type scanning near field optical microscope (aperture SNOM). c) Tip enhanced near field optical microscopy (TENOM). d) Tip on aperture (TOA) approach, which combines the advantages of (b) and (c).

28 Oscillatory Feedback Methods Oscillating ~ 1 nm at resonance freq (~ 30 khz) Increases SNR for feedback methods Q factor ~ 500 (the oscillator's resonance frequency divided by its resonance width) 1. Shear force detection utilizes lateral oscillation shear forces generated between the tip andspecimen (parallel to the surface) to control the tipspecimen gap during imaging 2. Tapping mode detection relies on atomic forces occurring during oscillation of the tip perpendicular to the specimen surface (as in AFM) to generate the feedback signal for tip control.

29 Reading List BetzigE, Lewis A, Harootunian A, Isaacson M, & Kratschmer E (1986) Near Field ildscanning Optical Microscopy (NSOM): Development and Biophysical Applications. Biophys J 49(1):