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PH880 Topics in Physics
Modern Optical Imaging (Fall 2010)Modern Optical Imaging (Fall 2010)
KAIST PH880 11/17/2010
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Overview of week 12
• Monday:• Monday:
‐ FRET
• Wednesday:
‐ NSOM
KAIST PH880 11/17/2010
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Förster resonance energy transfer (FRET)Fl i iFluorescence emission
FRET
Donor Acceptor
KAIST PH880 11/17/2010wikipedia
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FRET: spectroscopic rulerDetermines distances between biomolecules labeled with an appropriate donor andDetermines distances between biomolecules labeled with an appropriate donor and acceptor fluorochrome when they are within 10 nanometers of each other.
(* l diff ti li it d fl i l ti i i ffi i t t d t i(* normal diffraction‐limited fluorescence microscope resolution is insufficient to determine whether an interaction between biomolecules actually takes place.)
KAIST PH880 11/17/2010 http://www.olympusmicro.comTeakjip Ha group
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KAIST PH880 11/17/2010508 | VOL.5 NO.6 | JUNE 2008 | NATURE METHODS
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Overview of week 12
• Monday:• Monday:
‐ FRET
• Wednesday:
‐ NSOM
KAIST PH880 11/17/2010
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Things NaturalThings Natural Things ManmadeThings Manmade1 cm10-2 m
The Scale of Things The Scale of Things –– Nanometers and MoreNanometers and More
Head of a pin1-2 mm
Ant~ 5 mm
The Challenge
1 millimeter (mm)
10 mm10 m
10-3 m
e
1,000,000 nanometers =
mm
Dust mite
200 mm 0.1 mm100 mm
(mm)
10-4 m
Mic
row
ave
MicroElectroMechanical (MEMS) devices10 -100 mm wide
Red blood cells
Fly ash~ 10-20 mmHuman hair
~ 60-120 mm wideO O
O
OO
O OO O OO OO
O
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
PO
O
Mic
row
orld
0.01 mm10 mm
10-5 m
Infra
red
Red blood cells
Pollen grain
(~7-8 mm)
Fabricate and combine nanoscale building blocks to make useful0 1
1 micrometer (mm)
10-6 m
Visi
ble
1,000 nanometers = Zone plate x-ray “lens”Outer ring spacing ~35 nm
blocks to make useful devices, e.g., a photosynthetic reaction center with integral semiconductor storage.
0.01 mm10
0.1 mm100 nm
10-7 m
10-8 mnow
orld Ultra
viol
et
Self-assembled,Nature-inspired structureMany 10s of nm
ATP synthase
~10 nm diameter Nanotube electrode
1 nanometer (nm)
10 nm
10-9 m
Nan
ray
Carbon buckyball
Many 10s of nm
Quantum corral of 48 iron atoms on copper surfacepositioned one at a time with an STM tip
Corral diameter 14 nm
Carbon nanotube~1.3 nm diameter
0.1 nm10-10 m
Soft
x-r
Office of Basic Energy ScienceOffice of Science, U.S. DOE
Version 05-26-06, pmd
~1 nm diameter
Atoms of silicon
spacing 0.078 nm
DNA~2-1/2 nm diameter
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Scanning probe microscopes
Scanning Tunneling Microscope STM
Atomic Force Microscope AFM
Nearfield Scanning Optical Microscope NSOM
KAIST PH880 11/17/2010
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Scanning Tunneling Microscope: STMBinnig 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, 2002.
When STM tip is close to the specimen (~ 1nm), a tunneling current, IT is established
, , p g y ,
IT is exponentially proportional to the distance
A feedback loop maintaining IT can change z‐position topographical information
KAIST PH880 11/17/2010
p g T g p p g p
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STM images
“Carbon Monoxide Man”“Atom”"quantum corral"
C Iron on Copper Carbon Monoxide on Platinum Iron on Copper
Don Eigler, IBM
Lutz & Eigler, IBM Lutz & Eigler, IBM
KAIST PH880 11/17/2010
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Atomic Force Microscope AFMSTM is a precursor of AFM
Feedback Loop
V
Laser
Photodiode
MirrorPZT
~ deflection
ThermoMicroscopes Explorer AFM
Tip
SubstrateThermoMicroscopes Explorer AFM
AFM relies on contact rather than current nonconductive materials can be imaged
KAIST PH880 11/17/2010
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AFM images
the compaction of DNA in yeast d b t i ll d AbF2
nuclear pore complex
caused by a protein called AbF2
LR Brewer, et al, Biophysical journal, 2003
D Stoffler et al, Current opinion in cell biology, 1999
KAIST PH880 11/17/2010
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AFM + Fluorescence imaging techniques
A. Gaiduk et al, Chem. Phys. Chem. 6, no. 5, pp. 976‐983, 2005
KAIST PH880 11/17/2010
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Near field
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sub‐wavelength aperture (a)(20 ‐ 200 nm)(20 200 nm)
~10 nm
b d i b iImage can be reconstructed point by point spatial resolution is limited by a (rather than λ)
KAIST PH880 11/17/2010
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the propagation of waves :the loss of spatial information:the loss of spatial information
Hartschuh et al., Angewandte Chemie,2008
KAIST PH880 11/17/2010
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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
KAIST PH880 11/17/2010
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KAIST PH880 11/17/2010
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KAIST PH880 11/17/2010
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KAIST PH880 11/17/2010
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NSOM tip fabrication
chemical etching(meniscus or tube etching)
Micro‐fabrication(meniscus or tube etching)
Fast, convenient processFastLarge cone angle (Low cost, reproducible)
mass production.
Smooth surface
Low cone angle (low throughput)
Large cone angle
Toxic (HF) vapor
( p )
Complex fabrication processLow cone angle (low throughput)Fragile
Toxic (HF) vaporDifficult to control surface quality
p p
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NSOM tip: metal coating
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NSOM tip: illumination
Waveguide tip (Takashi et al.1999)
SiO2 cantilevered tip (Mitsuoka et al. 2000)
Fiber tip by Nanonics Inc.
KAIST PH880 11/17/2010
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NSOM tip: intensity distribution
Probe‐to‐Probe configuration (Ohtsu et al. 2000)
Minh et al 2000
KAIST PH880 11/17/2010
Minh et al. 2000
Lu et al. 2001
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NSOM tip: geometry and light throughput
KAIST PH880 11/17/2010
Ultramicroscopy 57 (1995) 204‐207
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Common NSOM illumination
KAIST PH880 11/17/2010
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Other focusing concepts using near field optics
a) Far‐field focusing using a lens. The angular frequency range of propagating waves
Hartschuh et al., Angewandte Chemie,2008
) g g g q y g p p g gkx,max, and thus the focus diameter, is limited by the aperture angle of the lenskx,max=nsin(q)2p/l, with n being the refractive index and l thewavelength 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).
KAIST PH880 11/17/2010
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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 and specimen
2. Tapping mode detection
relies on atomic forces occurring during oscillation of the tip perpendicular to thegenerated between the tip and specimen
(parallel to the surface) to control the tip‐specimen gap during imaging
oscillation of the tip perpendicular to the specimen surface (as in AFM) to generate the feedback signal for tip control.
KAIST PH880 11/17/2010http://www.olympusmicro.com/primer/techniques/nearfield/nearfieldintro.html
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Reading List
i i i & h ( ) i ld iBetzig E, Lewis A, Harootunian A, Isaacson M, & Kratschmer E (1986) Near Field Scanning Optical Microscopy (NSOM): Development and Biophysical Applications. Biophys J 49(1):269‐279.
KAIST PH880 11/17/2010