Raman And Coherent Anti-Stokes Raman Scattering (CARS) Spectroscopy And .

Raman and Coherent anti-StokesRaman Scattering (CARS) Spectroscopyand ImagingSelected Topics in Biophotonics (EAD289)James Chan, Ph.D.Lawrence Livermore National LaboratoryNSF Center for Biophotonics Science and Technology (CBST)January 29, 2009

Outline ! Motivation – Why Raman? ! Background theory on Raman spectroscopy ! Spontaneous Raman spectroscopy and imaging ! Background theory on Coherent Anti-Stokes Raman Scattering (CARS) ! CARS Instrumentation ! Brief Introductions to F-CARS, E-CARS, M-CARS ! Application of CARS to cell imaging ! Applications ! Summary

Live cell imaging requires the development ofnew optical microscopy methods ! Specificity ! Sensitivity ! Dynamic live cell imaging ! Long term live cell imaging

Current state of conventional opticalmicroscopyPhase contrast( ) Low cost( ) Easy to use(-) No chemical information(-) Low 3D-resolutionFluorescence( ) Specific labeling( ) 3D information with confocal andmultiphoton microscopy(-) Photobleaching – no long term studies(-) Toxicity, cell fixation - perturbs cellfunction

Raman scattering is the interaction ofphotons and intrinsic molecular bondsC.V. Raman1930 Nobel PrizeMolecular vibration in sampleEs h!s!Ei h!i!Eas h!as!Scattered lightIncidentlightExcited state!i!Virtual state!s!!i!"1!Ground state"0!!s! !i!Stokes!as!!as!Wavelengthanti-StokesBoltzmann distribution

Polarizability induced dipole equationClassical picture of Raman and Rayleigh scattering with a diatomic moleculeE Eo cos (!i t) Electric field of incident light oscillating at frequencyµind "E " Eo cos (!i t)Induced dipole from this E-field" "o (r – req) (d" / dr) Molecular polarizability changes with bond lengthr – req rmax cos (!vib t) The bond length oscillates at vibrational frequency" "o (d" / dr)rmaxcos(!vib t)Polarizability oscillates at vibrationalfrequencyµind "oEocos(!it) (1/2)Eormax(d" / dr)[cos((!i !vib) t) cos((!i-!vib)t)]RayleighAnti-StokesStokes

Raman spectra of cells provide a wealth ofbiological informationSingle live human T cellk 1/# (cm-1) Wavenumber unitsRaman shift (1/#incident) – (1/#scattered)

Confocal Raman microscope formicrospectroscopy and imagingCCD cameraCCD chipSample onscanning stage100XMicroscopeobjectiveAPDSpatialfilter FilterDichroicbeamsplitterRaman signalExcitation CWlaser (mW)

Conventional single cell Raman spectroscopy isusually performed on surfacesEsposito et. al., Appl. Spec. 57 (7) 868 (2003)Spores dried on a surface

Optical trapping combined with Ramanspectroscopy simplifies the analysis of single cellsOptically trapped spore !Advantages:–!–!–!–!Rapid sampling of many particles in solutionReduces background signals from surfacesMaximizes Raman signalsEnables manipulation and sorting of particlesChan et. al., Analytical Chemistry, 76 599 (2004)

Optical trapping immobilizes aparticle within the laser focus ! ! ! !Tight focusing conditionHigh intensity gradients in both axial and lateral directionsStable 3-D optical trapping with a single laser beamTrapping of organelles and whole cells have been demonstrated

Pediatric leukemia: Normal and malignant cellscan be discriminated by their Raman fingerprintMean Raman spectraRaji BDifference spectraNormal TJurkat TNormal BJurkat TNormal BRaji BNormal TReproducible within group spectraChan et. al., Biophysical Journal 90 648-656 (2006)Cancer spectral markers

Multivariate statistical techniques areused to separate and classify cell dataPrincipal component analysis (PCA)

Raman images of formaldehyde fixed human cellsSingle, fixed peripheral bloodlymphocyte in bufferSingle, fixed lens epithelialcell in buffer2850 cm-1 symmetric CH2 protein vibration, 120 mW 657 nm laser 400 nm resolution, 1 hour acquisition timeUzunbajakava et. al., Biopolymers, V72, 1-9 (2003)

Raman mapping combined with fluorescencemicroscopy for multi-modal analysisRaman mapping of chemical components inS. pombe cellsLive S. pombe cell30 min per image632 nm laser1602 cm-1 signalcolocalizes withGFP signalHuang et. al., Biochemistry, V44, 10009-10019 (2005)GFP mitochondriaimage

Advantages and limitations of spontaneousRaman spectroscopy/imagingAdvantages ! Minimally invasive technique ! Non-photobleaching signal for live cell studies ! Works under different conditions (temperatures and pressures) ! Chemical imaging without exogenous tags ! Works with different wavelengthsLimitations ! Fluorescence interference ! Limited spatial resolution ! Weak signal – long integration timesRaman scattering is extremely inefficient (10-30 cm2 cross sections)1 in 108 incident photons are Raman scattered

Why develop CARS? ! More sensitive (stronger signals) than spontaneous Ramanmicroscopy – faster, more efficient imaging for real-time analysis ! Contrast signal based on vibrational characteristics, no need forfluorescent tagging. ! CARS signal is at high frequency (lower wavelength) – minimalfluorescence interference ! Higher resolution

CARS uses two laser frequencies to interactresonantly with a specific molecular vibration!vib !pump - !Stokes!!pump!Beating at!Stokes!!pump - !Stokes!Coherent vibration of specificmolecules resonant at !vib

CARS signals are generated at wavelengths shorterthan the excitation wavelengths (anti-Stokes)!AS 2!pump - "0!Anti-StokesEnergy diagram

CARS is a third order nonlinear optical process,requiring high intensity laser pulsesPolarizationP(t) (1) E(t) (2) E(t)2 (3) E(t)3 Higher order terms becomes important when peak powers are highFor CARS,PAS (3) Ep2 EsRequires high intensity, pulsed laser sources (ps, fs)IAS Ip2 IS [ sin (%kz/2) / (%kz/2) ] 2kSkPkASkPPhase matching conditions

First CARS microscope demonstrated in 1982 ! Drawbacks of this configuration for biological imaging–! Laser wavelengths at 565-700 nm–! Phase matching configuration difficult to implement practicallyDuncan et. al., Optics Letters, V7, 350-352 (1982)

Major improvements developed in 1999 forbiological imaging ! ! ! ! !Tight focusing conditions relax phase matching conditionsAdvancement in laser technologyNear IR light reduces potential laser damage to cells, tissueCollinear geometry makes it much easier to implement3-D sectioning, through cells, tissue

Tight focusing using a high NA objective is keyfor CARS microscopic imagingTrapped particle"Translationstage"Microscope coverslip"Intensity distribution of an opticalfield focused by a 1.4 NA objective ! Phase matching condition relaxed ! Tight focus generates highest intensity at laser focus ! CARS signal generated within focal volume ! 3-D sectioning capabilityCheng et. al., J. Phys. Chem. B. V108, 827-840 (2004)100X, 1.3 NA oilimmersionobjective"

Two synchronized Ti:Sapphire lasers provide twofrequencies for CARS excitationCW Nd:YVO4 532 nmpump laser, 10 WModelocked Ti:Sapphire laser @780-930 nm, 5ps, 80MHz, 600 mW,!pumpModelocked Ti:Sapphire laser @780-930 nm, 5ps, 80MHz, 600 mW,!StokesElectronicfeedback

Optical parametric oscillators are anothertype of system used for CARS microscopyModelocked Nd:Vanadate PumpLaser @ 1064 nm, 7ps, 76MHz,10W, !StokesIntracavity doubled sync-pumped OPO780 nm – 920 nm, 5ps, 76MHz, 2W,!pump!Optical delay linePulses areinherentlysynchronized

Picosecond or femtosecond pulses, whichis better? There are several tradeoffsFemtosecond pulses (80fs 70 cm-1)Picosecond pulses (5ps 3.6 cm-1)Raman bandstypically 10 cm-1WavenumberPs pulses focus all energy to a single Raman band to maximize coherent vibration,at expense of losing peak intensity and multiplex advantage with fs pulses

Key components in a CARS microscope setupForward CARSScan stage orscanning mirrorsEpi rDichroicbeamsplitter!p! !s!CARS signalTelescopest

First demonstration on 910 nm polystyrene beadsZumbusch et. al., Phys. Rev. Lett. V82, 4142 (1999)

Jitter between two laser trains affects thequality of the CARS image0.5 µm polystyrene beads0.3 mW, 0.1 mW pump, stokes22 seconds to acquire imageJones et. al., Rev. Sci. Instrum. V73, 2843 (2002)

Examples of live cell imaging853 nm (100 µW) and 1135 nm (100 µW) tuned toRaman shift of 2913 cm-1 C-H vibrationUnstained live bacterialcells. Signal due to cellmembranes.Zumbusch et. al., Phys. Rev. Lett. V82, 4142 (1999)Unstained live HeLa cells.Bright spots due tomitochondria.

Example : CARS image of protein, nucleic acid in asingle cellUnstained live human epithelial cellLaser powers - 2 and 1 mW, tuned to 1570 cm-1 (protein, nucleic acid)image acquired in 8 min, smallest feature 300 nmCheng et. al., J. Phys. Chem. B. V105,1277 (2001)

Example : CARS image of MSC-derived adipocytesrich in lipid structuresCARS tuned to2845 cm-1lipid mode10 µm!10 !mMSC-derivedadipocytes(fat cells)Courtesy: Iwan Schie, Tyler Weeks, Gregory McNerney"

Example : CARS imaging of bacterial spores!S 812 nm!!0 750 nm! CARS lasers tuned to1013 cm-1 vibrationRaman spectrum of bacterial spore5 µm!AS!CARS signal at 697 nmCARS image of spores on glass substrate

Long-term dynamic cell processes can bemonitored with CARS microscopyConversion of 3T3-L1 fibroblast cells to adipocyte (fat) cells0 hr48 hr60 hr192 hrImaging of triglyceride droplets at 2845 cm-1 (lipid vibration)Nan et. al., J. Lipid Res. V44, 2202 (2003)

Tracking trajectories of organelles inside singleliving cellsTwo photon fluorescence - CARS image of lipidmitochondriadroplets overlaid on TPFimageNan et. al., Biophysical Journal. V91, 728 (2006)Trajectory of droplets byrepeated CARS imaging

Radiation pattern in the forward and backwarddirections may not be symmetricalIncoherent microscopy : Radiation is symmetrical in both forward and backwarddirection (Fluorescence, 2 photon fluorescence, Spontaneous Raman)Coherent microscopy : Radiation pattern is not symmetrical (CARS, SHG, THG)Small scatter radiatesas a single dipoleBulk scatterers addconstructively in theforward directionF-CARS detects large scatters, E-CARS detects small scatterersVolkmer et. al., Phys. Rev. Lett. V87, 0239011 (2001)

Comparison of F-CARS and E-CARS imageSmall scatterers in cytoplasmvisible in E-CARSNIH 3T3 cellsC-H 2870 cm-1 lipid membraneDark image due to destructive interference in E-CARSNuclear membrane edge visible in F-CARS, large axial lengthCytoplasm overwhelmedby solvent signalCheng et. al., Biophys. Journal, V83, 502-509 (2002)

Detection of F-CARS and E-CARS using one detectoris possible by temporal separation of the two signalsSchie et. al., Optics Express, V16, 2168-2175 (2008)

Nonresonant background is amajor issue in CARS microscopyPolarization CARS (P-CARS) – Cheng et. al, Optics Letters, V26 1341 (2001)Epi-CARS (E-CARS) – suppression of bulk background solvent

Dual pump CARS microscopy can be used tosubtract nonresonant backgroundOn resonance!PumpOff resonance!Stokes!StokesDifferenceBurkacky et. al., Optics Letters, V31, 3656 (2006)

Autofluorescence (2-photon) from thesample may overwhelm the CARS signal ! Raman lifetimes ps ! Fluorescence lifetimes ns

Multiplexed CARS (M-CARS) has beendeveloped for CARS spectroscopyPopulation of multiple levelssimultaneously (ps-fs combination)Cheng et. al., J. Phys. Chem. B, V106, 8493-8498 (2002)CARS spectrum of DOPCvesicle in the C-H stretchingregion ( 150 cm-1)

Supercontinuum generation in a photonic crystalfiber can function as a broad source for M-CARSKano et. al., Appl. Phys. Lett, V86, 121113 (2005)Andresen et. al., J. Opt. Soc. Am. B, V22, 1934 (2005)Petrov et. al., Proc. SPIE, 6089, 60890E (2006)Supercontinuum generationCARS spectrum of bacterial spore –1 second acq. time

Application : CARS in-vivo imaging2845 cm-1 vibration C-H lipidAdipocytes of the dermisEvans et. al., PNAS, V102 16807 (2005)StratumcorneumAdipocytes of subcutaneous layer

Applications : Fiber-based CARS endoscopyFiber delivery of twoultrashort laser pulsesFocusing unitSingle mode fiber750 nm beads imagedin epi-directionLegare et. al., Opt. Express, V14 4427 (2006)