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“Nanostructure characterization techniques”
UT-Austin
PHYS 392 T, unique # 59770 ME 397 unique # 19079
CHE 384, unique # 15100
Instructor:Professor C.K. Shih
Subject: Nanoscale optical microscopy
Lecture Notes - Oct 29 and Nov 3, 2009
*Some slides are courtesy of Professor David A. Vanden BoutDept of Chemistry, University of Texas
Resolution limit of an optical microscope
Diffraction limit:
..64.1 ANR
* Such diffraction limit does not apply to “Negative Index” materials (superlens)
Confocal Microscopy
N.A.0.37
trans
d
2z (N.A.)0.89
d
EmissionFilter
Out-of-focus Light Rays
In-focus Light Rays
Epifluorescence or Reflectance Mode
Sample
Objective
Dichroic Mirror
Excitation Filter
Light Source
CCD Detector
Resolution
Focus (z)
Optical Characterization
• Chemical Information• Molecular Orientation• Electronic Properties• Wide Array of Techniques
• Problem: Limited resolution due to diffraction
3-D Optical Sectioning
Images of four dye labeled 3 micron latex spheres suspended in immersion oil
Scan Mirror in conjugate image planeBeam pivots in back focal plane of objectiveScan lens allows use of full N.A. of the objective
Beam Scanning Confocal
Difficulties with single photon confocal
Scan box
objective
dichroic
pinhole
sample
Signal is localized in detection. Therefore fluorescence path must follow excitation back through scan box.
Losses due to scattering (limiting depth of sample)
Losses in scan box from passing through many optics
Losses at pinhole in limiting detection region
Images generated electronically by scanning the focal excitationin the sample
Fluorescence most commonTransmission & Polarization variants are possible
Beam ScanningFastScanning Galvo Mirror Mirrors placed so beam pivots at back of the objective
Sample ScanningSlow, slow, sloweasy to implement as optics are fixed
Multiphoton Excited Fluorescence Microscopy
Nonlinear optical processSignal nonlinear with the excitation intensity
Extremely low cross sectionRequired high intensity (short pulses, tight focus, …)
Intensity requirements limit excitation volumeFluorescence excited only at the focus
Low scattering of red lightLess worry about fluorescence scattering
Deep depth profiling
• Japanese physicists have created a model bull just 10 micrometres long - about the size of a red blood cell. Satoshi Kawata and co-workers of Osaka University (S Kawata et al 2001 Nature 412 697).
White bar = 2 m.
Functional micro oscillator system
• The spring, anchor cub, and the beads are all made using two-photon absorption• The diameter of the coil is only 300 nm.• The oscillator was kept in ethanol so that the buoyancy would balance gravity• The bead is manipulated using laser trapping (trapping force ~ 3 pN)• The force constant of the spring is determined by damping curve k = 8.2 nN/m
Harmonic Microscopy
Nonlinear Optical Technique
Small volume excitation3-D sectioning
Coherent MicroscopyPhase matching requirements provide new contrast
Third harmonic generation image of a spiral algae formation. The image is rendered using a series of cross sectional images produced by third harmonic generation at interfaces within the specimen. The excitation wavelength was 1.2 microns, and the detected wavelength was 400 nm. the laser pulse duration was 100 fs. Each cross sectional image was acquired in 1.6 seconds. The specimen in the image measures approximately 100 microns long by 50 microns wide.
Reported by: Jeff Squier at the CLEO/QELS '99 meeting.
More Microscopies
RamanVibrational Information
CARSCoherent Anti-Stokes Raman ScatteringNon-linear3-D with Raman
NIH-3T3 cell in interphase. Lasers tuned to CH2 stretching vibration. The nuclear envelope and the mitochondrian network can be discerned
X. Sunney Xie, Harvard
E
Near Field Optics
aNear Field
Far Field
Resolution is only a function of aperture size!
a/2
1928: Proposal of concept (E. Synge, Phil. Mag. 6, 356, 1928)
1944: Calculation of sub wavelength aperture coupling (H. Bethe, Phys. Rev. 66, 163, 1944)Correct by Bouwkamp
1972: demonstration using microwaves (Ash et al., Nature 237, 510, 1972)
1980’s Work by Pohl and Lewis
How do you break the diffraction limit?
Resolution determined by size of aperture
<< ( 5 nm)
Optical Fiber
Far Field
Al Coating
Near Field
one wavelength
Sample
Near-Field Optics
500 nm
NSOM ConventionalFar Field
C12 Polyfluorene Annealed
Near-Field vs. Far-Field Imaging
NSOM modes of operation
From L to R : Tranmission mode, Reflection Mode, Collection Mode and Illumination/Collection mode.
http://www.nanonics.co.il/main/twolevels_item1.php?ln=en&item_id=34&main_id=14
D.W. Pohl, APL_44_651 (1984)
Science_251_1468
A. SEM
B. Optical microscopy(NA = 0.9)
C. NSOM
D. NSOM after deconvolution
Science_257_189 (1992)
Science_262_1422
Ex2 Ez
2
Ey2
ElectricFields at SubwavelengthAperture
Bethe &Bouwkamp
Feedback Methods
In all cases force is measured as a function of distance from the sample surface. Tip is held at a constant distance to ensure sample is in the near field.Topographic map is recorded by monitoring z-piezo motion.
Distance (nm)
0 10 20
sign
al
SEM Images of pulled and Silver coated NSOM probes
Final tip diameter ~250 nmAperture ~ 50 nm
Images from www.chem.ucsb.edu/ ~buratto_group/nsom.htm
Optical Spectroscopy and Laser Desorption on aNanometer ScaleDieter Zeisel, Bertrand Dutoit, Volker Deckert, Thomas Roth, and Renato Zenobi, “Optical Spectroscopy and Laser Desorption on a Nanometer Scale”, Anal. Chem., 69,749-754 (1997).
Organic overlayer
50% HF
Chad E. Talley, Gregory A. Cooksey, and Robert C. Dunn, “ High resolution fluorescence imaging with cantilevered near-field fiberoptic probes”, Appl. Phys. Lett. 69, 3809 (1996).
Cantilevered probes
Lewis design
Nanofabricated ProbeWitec commercial probeshttp://www.witec.de/pdf/alphaSnom/snomcantilever.pdf
Tip DesignsStraight Pulled Fibers
“standard” design. Can be fabricated in house with some effortreproducible tips. Aperture no always perfect. Low throughput
Bent Pulled FibersAllow for normal force feedback. Throughput can be low. Slightly harder to make
Etched FibersCheap and easy to make. Poor reproducibility. Significantly higher throughput compared to pulled
Nanofabricated CantileversNew designs. High throughput. Normal force feedbackRequires Si processing. Can be mass produced
Focus Ion Beam Milled FibersIdeal pulled or etched tip. Near perfect aperturesHard to make. Requires FIB
NSOM Spectroscopy
Transmission
Fluorescence
Polarization
Time-resolved
Raman
IR
Low Temperature
ABCD
550 700 850W avelength ( nm )
550 700 850
Fluo
resc
ence
(arb
. uni
ts)
Localized Spectra
Parking the tip in a fixed position allows fluorescence spectra to be acquired from a small region of the the sample
Spectra taken at points noted in image of PIC dye crystal
David A.Vanden Bout, Josef Kerimo, Daniel A. Higgins, and Paul F. Barbara, “Spatially Resolved Spectral Inhomogeneities in Small Molecular Crystals Studied by Near-Field Scanning Optical Microscopy”, J. Phys. Chem. 100, 11843-11849 (1996).
Fluorescence imaging of PIC dye crystalsWavelength images acquired using
bandpass filters
1 m A
Topography
B
580 nm Fluorescence (10x)
DC
620 nm Fluorescence 700 nm Fluorescence
David A.Vanden Bout, Josef Kerimo, Daniel A. Higgins, and Paul F. Barbara, “Spatially Resolved Spectral Inhomogeneities in Small Molecular Crystals Studied by Near-Field Scanning Optical Microscopy”, J. Phys. Chem. 100, 11843-11849 (1996).
Topography Phase
Amplitude Phase w/directions
PM-NSOM images of Rhodamine 110 crystals
Daniel A. Higgins, David A. Vanden Bout, Josef Kerimo, and Paul F. Barbara,“Polarization-Modulation Near-Field Scanning Optical Microscopy of MesostructuredMaterials”, J. Phys. Chem. 100, 13794-13803 (1996).
Polarization Modulation of Defects in Inorganic CrystalSrTiO3 Bicrystals
topo transmission PM-NSOM
• Clearly images defects at crystal interface
• Strain is seen in birefringence
Eric McDaniel, Anthony Louis Campillo, Julia W.P. HsuUniversity of Virginia, Physics
NSOM Spectroscopy
Transmission
Routine. Difficult to interpret contrast
Fluorescence
Routine. Work horse
Polarization
Advanced. Excellent results in transmission and fluorescence
Can avoid difficulties in fluctuations of total intensity
Probe molecular orientations
Time-resolved
Time resolved fluorescence. Demonstrated. Good results.
As fluorescence lifetime is perturbed by tip best results look at contrast
Pump-Probe experiments. Extremely difficult. A few demonstrations with fs
time resolution
Raman
Difficult. Some demonstrations. Only challenge is signal size.
Excellent results for point spectra or line scans. Imaging very slow
IR
Extremely difficult. Few demonstrations. Probes limiting factor as well as
transmission problems. Small signals.
Low Temperature
Extremely difficult. Some excellent work with specialized microscopes
Sharp metal tip will focus the electric field
Tot al Int ernal Reflect ion Excit at ion Scat t ering Collect ion
Convent ional Excit at ion Transmission Collect ion
• Less ex cit at ion light c ollect ed
• Scat t er ing measurement po ssible
• Sample on TIR cell
• Lower collect io n ef f iciency
• More background f rom excit at ion
• Higher collec t ion ef f iciency
• Or excit at ion t hrough object ive
Apertureless NSOM
AFM ANOM
APL_65_1623 (1994)
AFMANOM
Glass slide
Contrast due to perturbation of refractive index beneath surface
Science_269_1083 (1995)
Attractive force AFM SIAM image
Attractive force AFM SIAM image
Mica
Oil droplets on mica
Ion beam milled tip designed to enhance in-plane electric field
Calculated field enhancement at tip is 1000:1
Eric. J. Sanchez, Lukas Novotny and X. Sunney Xie, “Near-Field Fluorescence Microscopy Basedon Two-Photon Excitation with Metal Tips”, Phys. Rev. Lett. 82, 4014-4017 (1999).
photosynthetic membrane fragments
Two photon fluorescence of PVS/dye J-aggregates
Confocal Raman Image of Carbon NanotubeDetecting at G’ band – Raman shift = 2615 cm-1
Excitation source: 633 nm.
TopagraphyScanning Raman Image
• Variations in the Raman spectrum reflect changes in the molecular structure which can have several origins such as external stress, due to the catalyst particles, or local defects in the tube structure. The observed spectral variations between 1 and 3 can also be explained by a change of the tube structure, which modifies the electronic state energies
• G band remains at 1596 cm-1; G’ band movers from 2619 cm-1 to 2610 cm-1, and possibly a splitting of G’ band.
To establish a strong field enhancement at the tip, the electric field of the exciting laser beam needs to be polarized along the tip axis.
I-z curve dependence confirms the near-field effect.
Thick vs. Thin
Topographic coupling
Topography vs NSOM resolution
Difficulties with NSOM imaging
Problems and Limitations of NSOM
• Variations in tips make quantitative work difficult
mostly qualitative work, comparative results, multiple measurement simultaneously
• Very small light levels
High sensitivity detectors (SAPD, cooled PMT, LN CCD)
• Mixing of Near-field and Far-field signals
Take care in interpretation of thick samples
• Interaction of tip with sample
Understand CPS theory
• Slow scanning
Don't plan on too many scans
• Tips very fragile
Don't be a klutz
• Nonlinearity in piezo scanners
Linearized scanners, can't average scans
Selected General NSOM references
Michael A. Paesler, Patrick J. Moyer, Near-Field Optics: Theory, Instrumentation, and Application. John Wiley and Sons, New York, 1996.
E. Betzig, J. K. Trautman, T. D. Harris, J. S. Weiner, and R. L. Kostelak, “Breaking the Diffraction Barrier: Optical Microscopy on a Nanometric Scale,” Science 251, 1468-1470 (1991).
E. Betzig and J. K. Trautman, “Near-Field Optics: Microscopy, Spectroscopy, and Surface Modificaion Beyond the Diffraction Limit,” Science 257 (5067), 189-195 (1992).
D. W. Pohl, “Scanning Near-Field Optical Microscopy (SNOM),” Advances in Optical and Electron Microscopy 12, 243 (1991).
Aaron Lewis, Klony Lieberman, Nily Kuck Ben-Ami, Galina Fish, Edward Khachatryan, Alina Strinkovski, Shmuel Shalom et al., “Near-field optical microscopy in Jerusalem,” Isr. J. Chem. 36 (1), 89-96 (1996).
Application to Inorganic Materials
S.K. Buratto, “Near-Field Scanning Optical Microscopy (NSOM),” Current Opinions in Solid State and Materials Science 1 (4), 485-492 (1996).
Hsu, J. W. P “Near-field scanning optical microscopy studies of electronic and photonic materials and devices”, Mater. Sci. Eng., R33(1), 1-50 (2001).
Application to Organic Materials
D. A. Vanden Bout, J. Kerimo, D. A. Higgins, and P. F. Barbara, “NSOM of Organic Thin Film Materials,” Acc. Chem. Res. 30 (5), 204-212 (1996).
P. F. Barbara, D. M. Adams, and D. B. O’Connor "Characterization of organic thin film materials with near-field scanning optical microscopy (NSOM)” Annu. Rev. Mater. Sci. 29, 433–69 (1999)
Application to Biological Materials
R.C. Dunn, “Near-field Scanning Optical Microscopy,” Chemical Reviews 99, 2891-2927 (1999).
S1
S0
k23
k31
Light State "ON"
Dark State "OFF"
T1 or cation/e-...hex hem
How can we detect a single molecule?
Repeatedly excite the molecule to its electronic excited state.If it fluoresces thousands of times, then we can detect hundreds of photons. Background must be essentially zero!
How do you see the molecule?
One molecule give fluorescence of ~10 kHz.
What about everything else?
GET RID OF EVERYTHING ELSE
Spread the molecules out
No fluorescence from matrix
Turn off the lights
Very good detection system
10x10 microns 5x5 microns
NSOMConfocal
By delimiting the excitation and dispersing fluorescence dye molecules the fluorescence from individual molecules can be detected.
Because of fluorescence and raman from the NSOM probe the background is actually lower in the confocal experiment despite the larger excitation volume
How do you know they are single molecules?
Photon anti-bunching hard
No fluorescence from blank
Number density same as expected
Number density varies linearly with concentration
Linearly polarized excitation and emission (Dipole)
Intermittent fluorescence (blinking)
Single step photobleaching
Now you’ve seen them. So what?
Follow translational motion (imaging)Follow rotational motion (polarization)Proximity to other probes (FRET)Spectroscopic probe of environment (spectrum)
Almost any fluorescence experiment
Biggest limitation is photochemistry of probe
Single step photobleaching
Time
FluorescenceIntensity (A. U.)
Molecules rotate slowly, on the timescale of the scan (11 s per line).(OTP at Tg+10 K)
By “parking” atop a molecule, its polarization resolved fluorescence can becollected for a long time. By measuring two orthogonal polarization the projection of the transition dipole in the plane of the sample can be measured. From this a trajectory of the angle change per unit time can be created.
1 m
Example. Characterizing Local “Viscosity” following rotational motions
0 2500500 1000 1500 2000
|d(
)/d(t)
| de
g/se
c
Time (s)
200-20-40 40200-20-40 40 200-20-40 400
10
20
30
Occ
urre
nce
d()/d(t) deg/sec
0 2500500 1000 1500 2000
|d(
)/d(t)
| de
g/se
c
Time (s)
d/d(t) deg/s0 20 40-40 -20
0
20
40
60
Occ
urre
nce
Confocal Raman spectra of crystalline met-Hb A, dense layer of HbAgaggregates B, and two time series (C1 C6 and D1 D6) of “hot sites”obtained at the single molecule detection limit. The vertical lines indicate the Hb marker modes discussed in the text. All spectra were measured with the same collection time 30 s and collection efficiency. The incident laser power was 1 mW in A and 1 W in B-D.
Calculated electromagnetic enhancement factor for the midpoint between two Ag spheres separated by d =5.5 nm and for a point d/2 outside a single sphere. The solid and open circles indicate the position of the Ag spheres and the Hb molecule, respectively, in relation to the incident polarization vector (double arrows). The calculations have been performed for spheres of diameters D = 60 (dashed curves), 90 (solid curves) and 120 nm (dotted curves). Inset shows the enhancement versus D for = 514.5 nm and a Stokes shift of 1500 cm-1
for configuration a.
Molecule
Metal tip
Metal nanoparticles
Hamamatsu Commerical
NA enhancement using Solid Immersion Lens
Freshman E&M Textbook
P = ∞ q = R(n/(n-1))
n1 = 1, n2 = n
Rt = R/(n-1)
Within the “paraxial approximation”
By proper choice of t focal point right at the surface of the lens
Diffraction limit is improved by a factor of n
Requirement: surface to be imaged needs to be place in intimate contact with the lens surface
Note that effective NA is improved by a factor of n; the field of view is decreased by the same factor
Depending on the thickness of the wafer, one can adjust the extra thickness “t”to compensate
APL_57_2615
Schematic diagram of the near-field scanning solid immersion microscope with the SIL probe mounted to a cantilever in an AFM.
APL_74_501 (1999)
(a) AFM scan of lines in photoresist exposed with the SIL at a scan rate of 1 cm/s, 15 m x15 m scan size (b) 5 m x 5 m AFM surface plot (c) Cross-section of the lines in photoresist showing 190 nm FWHM and, 50 nm depth.