FULL ARTICLE

Signal generation and Raman-resonant imagingby non-degenerate four-wave mixingunder tight focusing conditions

Tyler Weeks1, Iwan W. Schie1, Sebastian Wachsmann-Hogiu1; 2, and Thomas Huser*; 1; 3

1 NSF Center for Biophotonics Science and Technology, University of California, Davis, Sacramento, CA 95817, USA2 Department of Pathology and Laboratory Medicine, University of California, Davis, Sacramento, CA 95817, USA3 Department of Internal Medicine, University of California, Davis, Sacramento, CA 95817, USA

Received 15 August 2009, revised 14 November 2009, accepted 14 November 2009Published online 1 December 2009

Key words: four-wave mixing, microscopy, Raman scattering, CARS, nonlinear optics

PACS: 87.63.lt, 87.64.kp, 87.64.mn

# 2010 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1. Introduction

Non-linear spectroscopies, such as coherent anti-Stokes Raman scattering (CARS), have demon-strated a number of key advantages over linearprobing methods including directional signal genera-tion and large signal intensities resulting in short ac-

quisition times [1–5]. For such processes a maximumconversion efficiency, e, can be achieved by reachingthe global maximum of:

e ¼ 4p2was

nasc2

� �j3cð3Þj2 I2

pL2 sinc ðDk l=2Þ2 ; ð1Þ

# 2010 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Journal of

BIOPHOTONICS

The authors demonstrate Raman-resonant imaging basedon the simultaneous generation of several nonlinear fre-quency mixing processes resulting from a 3-color coher-ent anti-Stokes Raman scattering (CARS) experiment.The interaction of three coincident short-pulsed laserbeams simultaneously generates both 2-color (degener-ate) CARS and 3-color (non-degenerate) CARS signals,which are collected and characterized spectroscopically –allowing for resonant, doubly-resonant, and non-reso-nant contrast mechanisms. Images obtained from both 2-color and 3-color CARS signals are compared and foundto provide complementary information. The 3-colorCARS microscopy scheme provides a versatile multi-plexed modality for biological imaging, which may ex-tend the capabilities of label-free non-linear microscopy,e.g. by probing multiple Raman resonances.

10 µm

CD

CH2

Doubly-resonant difference CARS image of a section ofa C. elegans nematode imaged at the CD resonance(upper half) and the CH2 resonance (lower half)

* Corresponding author: e-mail: trhuser@ucdavis.edu

J. Biophoton. 3, No. 3, 169–175 (2010) / DOI 10.1002/jbio.200900068

where was and nas are the frequency and index ofrefraction of the CARS signal, cð3Þ is the third ordersusceptibility, Ip is the pump laser intensity, l is theinteraction length, and Dk is the phase mismatch [2].The global maximum of Eq. (1) is reached as the ar-gument of sinc ðDk l=2Þ2 approaches zero. Tradi-tional CARS spectroscopic schemes rely on fixed an-gles between interacting beams to minimize thephase mismatch, thereby maximizing signal genera-tion [6–8]. This is typically implemented in theboxCARS geometry. In this case each distinctphoton combination typically requires slightly differ-ent beam geometries to satisfy those conditions,which results in limiting any given configuration tothe analysis of a relatively narrow range of signals.For spectroscopic purposes these geometric con-straints can be beneficial since they can minimizespectral overlap. For imaging applications, such as inCARS microscopy, however, the ability to multiplexsignals can be desirable since several signals of dif-ferent origin are generated simultaneously and allowfor their relatively straightforward comparison. Un-der tight focusing conditions a maximum conversion

efficiency can be achieved by minimizing the interac-tion length, l, which relaxes the phase matching cri-teria and produces strong signals across a broadspectral range [9–12].

CARS microscopy takes advantage of this tightfocusing condition and has received considerableattention for its potential for chemically specific, la-bel-free imaging [4, 5, 10, 11, 13–24]. CARS uti-lizes a pump pulse, wp, and a Stokes pulse, ws,where (wp � ws) match a vibrational mode and thesignal is produced at wCARS ¼ ðwp � wsÞ þ wp (seeFigure 1a). As a degenerate four-wave mixing(FWM) process, the interacting CARS photons, wp,ws, and wCARS, have only one available combina-tion with which a Raman mode can be probed. If,however, another discrete photon source is avail-able, this degeneracy can be avoided, and undertight focusing conditions up to 6 unique Raman-re-sonant photon combinations may be available. Theorigin of these signals is due to not only 2-colorCARS (see Figure 1a), but also 3-color CARS anddoubly-resonant FWM (or: doubly-resonant CARS– see Figure 1b) [8, 25–29]. Increasing the numberof discrete signals simultaneously generated withina focal volume without increasing the total laserpower delivered to the sample can increase theamount of retrievable information while minimizingsample damage induced by multiple scans. Here,we characterize several possible FWM combina-tions produced in 3-color CARS microscopy, weverify the nature of the signals by measuring theirdependence on laser input power, and we demon-strate the utility of imaging with all available non-linear optical signals.

2. Materials and methods

2.1 Optical setup

Our CARS system is described in detail elsewhere[30], but utilizes two important changes for the cur-rent work. First, the Nd : YVO4 laser (10 W, 7 ps,76 MHz, 1064 nm – PicoTrain, HighQ Laser) syn-chronously pumps two optical parametric oscillators(OPO’s, Levante, APE). 9 W of the original laserpower are split up to pump each OPO individuallyand the remaining 1 W is used as a Stokes pulse.The result of this configuration is a fixed 1064 nmStokes pulse and two pump pulses that can be tunedindependently between 770 nm and 960 nm. Thepower and polarization of all three beams are alsocontrolled independently. The three beams are over-lapped in time and space and then coupled into aninverted optical microscope (Olympus IX71). Whilethe epi-CARS signal is coupled into a spectrometer

CARSa.

~1~3~1 S1

R1

~2~3~2 S4

R2

~1~2~1 S5

R3

3-Color CARSb.

~1~3~2 S2

R1

~2~3~1 S3

R2

~1~2~3 S6

R3

Figure 1 Energy diagrams of 6 Raman-resonant four-wavemixing processes that can occur when three input beamsare used, where w1 > w2 > w3. (a) Potential 2-color CARSsignals, and (b) potential 3-color CARS signals producedin this process. R1, R2, and R3 refer to three different Ra-man resonances probed by the combination of three dis-tinct input beams. S1, S2, S3, S4, and S5 correspond to peaks1–5 listed in Figure 3b. Note that signals S2 and S3 havethe same photon energy leading to doubly-resonantCARS.

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(Spectra Pro 2300i, Princeton Instruments), the for-ward CARS signal is focused onto a single photoncounting avalanche photo diode (APD) (SPCM-AQR-141, Perkin-Elmer) using a 0.55 NA conden-ser lens (see Figure 2). By using different combina-tions of all 3 beams, we are able to tune theCARS system to any Raman line from �100 cm�1

to �3600 cm�1.

2.2 Power dependence

To verify the nature of each collected signal thepower dependence was analyzed. A microscopecover slip was coated with a thin layer of polystyr-ene beads, which exhibit a very strong CH vibrationat 3054 cm�1. OPO1 of our system was tuned to803 nm in order to match the 3054 cm�1 Raman re-sonance with respect to the 1064 nm Stokes laser.OPO2 was tuned to 877 nm in order to probe aband at �2000 cm�1 (see Figure 3b) with respect tothe Stokes laser, which is a purely non-resonant re-gion for most biological samples. OPO1, OPO2, andthe Stokes laser were set to an average power of40 mW, 11 mW, and 12 mW, respectively, at thesample. To measure the power dependence of thedifferent CARS and ND-FWM signals the averagepower of two of the beams was held fixed while thethird was incrementally decreased using a half-waveplate and a polarizing beam splitter cube. The anti-Stokes Raman signals were recorded on a spectro-meter with an integration time of 0.2 s within abroad spectral window that showed the signals fromthe degenerate and non-degenerate CARS pro-cesses simultaneously (see Figure 3a). For eachcombination of input powers the anti-Stokes Ramanspectrum was recorded. The set of spectra repre-senting the data were then exported to Matlab1

where the relative intensities of the four-wave mix-ing signals were extracted and fit by a polynomialfit function.

Figure 2 (online color at: www.biophotonics-journal.org)Diagram of the experimental setup. Nd : YVO4 laser(Van), Optical Parametric Oscillator (OPO), Delay Stage(DS), Half-Wave plate (HW), Polarizing Beams SplitterCube (PB), Pulse Picker (PP), Dichroic Mirror (DM), Ob-jective (O), Sample (S), Condenser (C), Lens (L), Band-pass filter (F), Mirror (M).

Process Combination Raman Line Signal Assignment

Peak 1 CARS 2ω1-ω3 3054 cm-1 644 nm C-H Stretch

Peak 2 (ω1-ω3)+ω2 3054 cm-1 687 nm C-H Stretch

Peak 3 (ω2-ω3)+ω1 2113 cm-1 687 nm Not-Resonant

Peak 4 CARS 2ω2-ω3 2113 cm-1 734 nm Not-Resonant

Peak 5 CARS 2ω1-ω2 1058 cm-1 746 nm Not-Resonant

Peak 6 3CARS (ω1-ω2)+ω3 1058 cm-1 967 nm Not-Resonant

Laser 1*Laser 3Laser 2

Laser 3Laser 2 Laser 1* Laser 2

Laser 1* Laser 3

0 0.5 10

0.5

1 ******** 0 0.5 10

0.5

1 ********* 0 0.5 10

0.5

1

0 0.5 10

0.5

1

** * * * * ** *

b.

c. d. e. f.

1

2,3

4

5

a.

640 660 680 700 720 7400

0.2

0.4

0.6

0.8

1

1.2

Wavelength (nm)

3CARS

3CARS

Figure 3 (online color at: www.biophotonics-journal.org) Raman resonances probed by each combination of three distinctshort laser pulses. The three input beams, w1, w2, and w3 were set to 803 nm, 877 nm, and 1064 nm respectively. (a) Thefour measured peaks as collected in a spectrometer. (b) Table identifying the origin and assignment of each observed peak.3CARS is used to indicate 3-color CARS processes. Note that peaks 2 and 3 coherently add to produce a doubly-resonantCARS signal. Also note that peak 6, as described in Figure 3b is not observable due to the filters required to generatea clean CARS signal in an inverted microscope. (c) Peak 1 intensity as a function of the input power of lasers 1 and 3.(d) Peak 2/3 intensity as a function of the input power of lasers 1, 2, and 3. (e) Peak 4 intensity as a function of the inputpower of lasers 2 and 3. (f) Peak 5 intensity as a function of the input power of lasers 1 and 2.

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2.3 Imaging

Imaging with each of the available FWM signalswas also performed. C. elegans worms fixed in 4%paraformaldehyde, then washed in phosphate buf-fered saline (PBS) solution were allowed to settleon a glass cover slip and covered with an aqueoussolution of 2.1 M deuterated glucose. The CD vi-brations associated with the deuterated glucoseprovide a well-isolated and unique Raman peak at�2121 cm�1. In this case OPO2 was tuned to868.3 nm to probe this CD stretch vibration relativeto the 1064 nm Nd: Vanadate laser. OPO1 was set to817 nm to probe the 2845 cm�1 CH2 symmetricstretching vibration. The CARS signal generated asa result of mixing between OPO1 and OPO2 at723 cm�1 was not recordable due to the short passfilters used to prevent the input lasers from saturat-ing the spectrometer. The worms and surroundingmedium were imaged using the 2 different CARSsignals as well as the doubly-resonant Raman signalresulting from the coherent addition of the CD andCH stretching vibrations [28]. Images were gener-ated by collecting both the forward and epi-directedsignals simultaneously on the same detector [30].The resulting signals were normalized to laser inputpowers so that the resonant response of each processcould be compared directly.

3. Results and discussion

3.1 Power dependence of the nonlinearsignals generated from polystyrene beads

The combination of three photons (w, w2, and w3),where w1 > w2 > w3, leads to the generation of6 FWM signals (see Figure 1), of which 5 can be de-tected separately. The laser wavelengths used were:Laser 1: 803 nm, i.e. w1 ¼ 12,453.3 cm�1, laser 2:877 nm, i.e. w2 ¼ 11,402.5 cm�1, and laser 3:1064 nm, i.e. w3 ¼ 9,398.5 cm�1. Based on the inter-action of these 3 beams at the sample, we collectedthe spectrum shown in Figure 3a on a grating spec-trometer. Although 6 peaks are listed in Figure 3b,Figure 3a only shows 4 distinct peaks. The reason forthis disparity is that, as illustrated in Figure 1b and3b, peaks 2 and 3 result in a combined doubly-reso-nant signal, and peak 6 occurs in the band betweenthe pump and Stokes lasers, which is not recordablewith our current setup since it is cut off by the bandpass filters that allow us to collect the other signals.It is also important to note that the relative intensi-ties of these peaks are not preserved due to detector

response and the performance of the filters used inthe scheme.

Power dependence can be used to distinguish the3-color from the 2-color CARS signals. Figure 3dclearly shows that the signal at 687 nm is a 3-colorCARS signal since it increases linearly as a functionof input power from all three beams. On the otherhand, Figures 3c, e, and f clearly demonstrate thequadratic dependence on the power of the pump la-ser and a linear dependence on the power of theStokes laser expected from 2-color CARS. As out-lined in Figure 3b all these signals can be accountedfor in terms of the participating photons, which, inthe case of the 3-color CARS process, can result inthe coherent addition of two Raman-resonant signals.

3.2 Image formation by multiple Ramansignals as demonstrated on C. elegansnematodes

An interesting advantage of FWM-based microscopyis the wide assortment of signals that are all gener-ated simultaneously. For instance, by adjusting thelaser wavelengths such that they simultaneouslyprobe the CD stretch vibration of deuterated glucose(2121 cm�1) and the characteristic lipid CH2 stretch-ing vibration (2845 cm�1) it is possible to performmultispectral imaging in a single scan [10]. Figure 4ashows the 2845 cm�1 CARS image of a C. elegansworm in an aqueous solution of deuterated glucosewhere the CARS signal is generated by the distribu-tions of lipids in the nematode’s body. Indeed, thesurrounding solution only contributes a non-reso-nant background signal in this case. Figure 4b showsthe doubly-resonant CARS image due to the coher-ent addition of the 2121 cm�1 d-glucose and the2845 cm�1 lipid resonances. Here, the resonant signalis produced in the image due to the presence ofeither lipids or glucose and their coherent additionwherever the molecules overlap within the focus vo-lume of the microscope. Figure 4c shows the2121 cm�1 CARS image in which the resonant signalis provided by the d-glucose-containing aqueous so-lution. A comparison of these images shows thatwhile regions outside of the worm body haveroughly the same intensity in both Figures 4b and c,due to the presence of just the CD vibrational reso-nance, regions within the worm are brighter in 4bthan in either 4a or 4c due to the presence of boththe CD and CH2 resonances (see Figure 4f). At theselocations the signals of both resonances mix coher-ently to provide a stronger, combined signal. To high-light this effect, Figure 4d was produced by simplysubtracting the upper halves of Figure 4a from b, re-sulting in an enhanced contrast for the CD vibration.

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In this image, areas outside of the worm body whichcontain just the D-glucose solution have a roughlyuniform intensity. However several bright featureswithin the organism indicate regions where the D-glucose and lipids can be found within the same focalvolume leading to the coherent addition of the CDresonance with the CH2 resonance. This observationmakes sense, because the worms were fixed by para-formaldehyde application and the deuterated glucosesolution is expected to penetrate the worm body.Likewise, by subtracting the lower halves of Figure 4cfrom b we obtain Figure 4e, which reveals only theenhanced CH2 resonance. As is expected, by extract-ing the lipid signal only noise remains outside of theworm’s body, while the CH2 resonance is enhancedby the presence of the CD resonance within the or-ganism. Only half of each image is shown to providefor a better, direct comparison between the resultingimages. Figure 4f represents averaged line-sectionstaken from each image as indicated by the dashedbox in Figure 4a. An average was used to reduce theamount of noise and enable comparison of the 5 linesections (note that the line sections were taken fromthe same position within each of the full images). In-terestingly, sharp features in line c (the CD CARSsignal), presumably resulting from localized regionsof increased CD density, result in a disproportionatelocalized increase in intensity in line b (the doubly-resonant CARS signal) over line a (the CH2 CARS

signal) as indicated by the vertical arrows. These ob-servations highlight the possible advantages of dou-bly-resonant FWM for imaging weak signals that areotherwise difficult to detect by CARS. By producingthese data simultaneously we can readily identifysuch effects as well as potentially perform similaranalyses in real-time.

4. Conclusions

We have demonstrated that the use of three colli-near, tightly focused laser beams provides an ex-perimentally simple way to not only probe multipleRaman resonances, but also collect signals frommultiple FWM processes simultaneously. We veri-fied the nature of the FWM signals, based onphoton mixing arithmetics and their power depen-dence on the input beams, and demonstrated thatall available FWM signals can be useful for effi-cient imaging and analysis by providing enhancedcontrast for specific Raman resonances. While pre-vious work has utilized the simultaneous genera-tion of multiple CARS signals either by usingbroadband sources, or, in a few cases using threeinput lasers, it is important to recognize that everypossible combination of the interacting lasers willproduce FWM signals that are often discarded.

Inte

nsity

[a.u

.]

Position [µm]

d.

Inte

nsity

[a.u

.]

abc

ed

2

3

4

1 0 2 0 3 0 4 00

5

10

15

e.

f.

a. b. c.

0

4

8

12

16

10 µ m

Figure 4 (online color at: www.biophotonics-journal.org) (a) CARS image of a C. elegans worm imaged at 2845 cm�1. (b)Doubly-resonant CARS image of the same worm imaged by combination of the 2845 cm�1 CH2 vibration and 2121 cm�1

CD vibration. (c) CARS image of the deuterated glucose solution imaged at 2121 cm�1. (d) Difference image of Figures4b and 4a highlighting the enhanced CD signal. (e) Difference image of Figures 4c and b highlighting the enhanced CDsignal. (f) Averaged cuts across the entire width of images 4a–e as indicated by the dashed box in Figure 4a. The legendindicates from which figure each cross section was taken. The arrows highlight regions that have been disproportionatelyenhanced by the mixing of two signals, indicating homodyning of the signals.

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These signals, however, can contain valuable infor-mation or in some cases interfere with measure-ments. For example, in a dual-pump CARS set-up[10, 31], if the two pump lasers differ in wave-length by only �10 nm the resulting 2-color and 3-color CARS signals will occur spectrally very closetogether and bandpass filters may not adequatelydiscriminate the signals. This could then potentiallylead to an inferior signal-to-background ratio be-cause the signal intensities simply add up and donot interfere as in the doubly-resonant case. Wehave also shown that the multiplicity of signalsgenerated and the inherent differences betweenthese signals could be important for improving theapplicability of Raman-based imaging techniques tosuch problems as label-free cytometry and ultra-sensitive chemical imaging. In particular, probingtwo resonances simultaneously leads to an amplifi-cation of the signal while the non-resonant back-ground remains the same [28]. This scheme enablesboth, higher sensitivity for weak resonances, andhigher molecular specificity (if two resonances fromthe same molecule are probed), which will becomeparticularly important when trying to discriminatespecific cells in the complex context of tissue imaging.

Acknowledgements Tyler Weeks acknowledges supportby the Lawrence Scholar Program from Lawrence Liver-more National Laboratory. Thomas Huser is grateful forsupport from the American Heart Association throughthe Grant-in-Aid program. Support by the National Insti-tutes of Health, National Cancer Institute, under grantnumber 1U54CA136465–01 is acknowledged. This workwas also supported in part by funding from the NationalScience Foundation. The Center for Biophotonics, an NSFScience and Technology Center, is managed by the Uni-versity of California, Davis, under Cooperative Agree-ment No. PHY 0120999. Support is also acknowledgedfrom the UCD Clinical Translational Science Center un-der grant number UL1 RR024146 from the National Cen-ter for Research Resources (NCRR).

Tyler Weeks received his BSin Physics from BrighamYoung University in Provo,Utah. He is currently aPh.D. candidate in the De-partment of Applied Scienceat the University of Califor-nia, Davis. Recently, Tylerbecame a Scholar in theLawrence Livermore Na-tional Laboratory LawrenceScholarship Program. His

research is performed at the NSF Center for Biopho-tonics in Sacramento, CA, where his interests includenonlinear optics and label-free imaging.

Iwan W Schie earned his Di-ploma in Biomedical Engi-neering from the Beuth Uni-versity of Applied Science inBerlin and is currently a grad-uate student in BiomedicalEngineering at the Universityof California, Davis. His ma-jor research interests arenonlinear microscopy techni-ques like CARS and SHGand their possible applica-tions to medical diagnostics.

Sebastian Wachsmann-Ho-giu is an Associate Professorin the Department ofPathology and LaboratoryMedicine at the Universityof California, Davis. He con-ducted his undergraduatestudies in Physics, with amajor in Biophysics at Bu-charest University, Roma-nia. In 2000 he received his

Ph.D. in Experimental Physics from Humboldt Univer-sity/Max-Born-Institute, Berlin, Germany. After a two-year postdoctoral appointment in the Chemistry De-partment at Carnegie-Mellon University he moved toCedars-Sinai Medical Center as Research Scientist I. InMarch 2005, he became faculty (assistant professor le-vel) in the Surgery Department, and Director of theAdvanced Optical Imaging Laboratory within the Mini-mally Invasive Surgical Technologies Institute. SinceApril 2007, he is also Facilities Director at the NSFCenter for Biophotonics, Science and Technology, UCDavis. His research interests include time-resolved Ra-man spectroscopy/imaging, nonlinear microscopy, andSERS-based biosensors.

Thomas Huser is an Associ-ate Professor in the Depart-ment of Internal Medicineat the University of Califor-nia, Davis. He also serves asChief Scientist for the Cen-ter for Biophotonics Scienceand Technology, and Co-Di-rector of the TranslationalWorking Group for the Clin-ical Translational ScienceCenter at UC Davis. Until

November 2005, Dr. Huser was a Group leader forBiophotonics and Nanospectroscopy at Lawrence Li-vermore National Laboratory in Livermore, CA. He

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worked at Lawrence Livermore National Laboratorysince joining LLNL in 1998 as a postdoctoral research-er and became a staff scientist in 2000. Dr. Huser ob-tained his Ph.D. in Physics from the University ofBasel, Switzerland, where he worked primarily onnear-field optical microscopy. At UC Davis he appliesRaman spectroscopy and imaging, and single moleculefluorescence microscopy to biological and medical pro-blems at the single cell level.

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