“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.