Radial polarization CARS microscopy with annular aperature detection_APL_2009
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Improved contrast radially polarized coherent anti-Stokes Raman scattering microscopy using annular aperture detectionJian Lin, Fake Lu, Haifeng Wang, Wei Zheng, Colin JR Sheppard et al.Citation: Appl. Phys. Lett. 95, 133703 (2009); doi: 10.1063/1.3240874View online: /10.1063/1.3240874View Table of Contents: /resource/1/APPLAB/v95/i13Published by the American Institute of Physics.Related ArticlesAnnular solid-immersion lenslet array super-resolution optical microscopyJ. Appl. Phys. 112, 083110 (2012)New ProductsRev. Sci. Instrum. 83, 109501 (2012)Fast image reconstruction for fluorescence microscopyAIP Advances 2, 032174 (2012)Spectrally resolved fluorescence lifetime imaging microscope using tunable bandpass filtersRev. Sci. Instrum. 83, 093705 (2012)Holographic microrefractometerAppl. Phys. Lett. 101, 091102 (2012)Additional information on Appl. Phys. Lett.Journal Homepage: /Journal Information: /about/about_the_journalTop downloads: /features/most_downloadedInformation for Authors: /authorsImproved contrast radially polarized coherent anti-Stokes Raman scattering microscopy using annular aperture detectionJian Lin,1Fake Lu,1Haifeng Wang,2Wei Zheng,1Colin JR Sheppard,1andZhiwei Huang1,a͒1Optical Bioimaging Laboratory,Department of Bioengineering,Faculty of Engineering,National University of Singapore,Singapore117576,Singapore2Data Storage Institute,A*STAR,Singapore117608,Singapore͑Received15August2009;accepted9September2009;published online1October2009͒We propose a unique annular aperture detection scheme in radially polarized coherent anti-StokesRaman scattering͑RP-CARS͒microscopy for significantly removing nonresonant background forhigh contrast vibrational imaging.Ourfinite-difference time-domain calculations show that themaximum radiation patterns of RP-CARS signals from the scatterers vary with the scatterer’s sizes,which are different from nonresonant CARS radiation from surrounding water.By applyingappropriate sizes of annular stop apertures in the detection path,the nonresonant background fromwater can be effectively suppressed,yielding over110-fold improvements in signal-to-backgroundratio for the forward-detected RP-CARS,while over50-fold improvements for the backwardRP-CARS detection.©2009American Institute of Physics.͓doi:10.1063/1.3240874͔Coherent anti-Stokes Raman scattering͑CARS͒micros-copy has received much interest for imaging tissue and cells owing to its outstanding capabilities of high biochemi-cal selectivity and sensitivity,as well as its intrinsic three-dimensional͑3D͒optical sectioning ability with high spatial and spectral resolutions.1–3However,CARS micro-copy is not background free;the strong nonresonant signal arising from the electronic contributions of the surrounding water and other media in tissue and cells degrades the vibrational contrast and spectral selectivity in CARS imaging.3To tackle this problem,various CARS techniques, such as polarization-sensitive CARS,2epi-detected CARS,3 heterodyne-detected CARS,4–6and near-field CARS,7–9have been developed to suppress the nonresonant background;but most work is centered on using the linearly polarized excita-tion scheme for CARS imaging.Recently,radially polarizedlaser beams have attracted increasing attention because oftheir unique light distribution properties͑e.g.,a very stronglongitudinalfield component and a tighter focal spot size͒atthe focal point by using a high numerical aperture͑NA͒objective.10The unique radial focalfield distribution thushas potential applications in high-resolution3D microscopyimaging,such as confocal microscopy,11second harmonicgeneration,12third harmonic generation,13and CARSmicroscopy.14In this letter,we propose a unique annularaperture detection scheme in radially polarized CARS ͑RP-CARS͒microscopy under tightly focused radially polarized pump and Stokes light excitation to effectively re-move the nonresonant background for high contrast vibra-tional imaging.Thefinite-difference time-domain͑FDTD͒method7,15is employed to investigate the effects of the scat-terers’sizes and annular aperture diameters on the signal-to-background ratio͑SBR͒of the forward-and backward-detected RP-CARS microscopy.Figure1illustrates the schematic of an annular aperturedetected RP-CARS microscopy for both the forward-and backward-CARS detection.The radially polarized pump ͓E͑r,p͔͒and Stokes͓E͑r,s͔͒lightfields are tightly fo-cused onto a spherical scatterer through a high NA objective for CARS generation.Under tight focusing of the radially polarized lightfield,the longitudinal electricfield component ͑E z͒and the radial component͑E͒at the focal point can be expressed as10a͒Author to whom correspondence should be addressed.Electronic mail: biehzw@.sg.Tel.:ϩ65-65168856.FAX:ϩ65-68723069.FIG. 1.͑Color online͒Illustration of the annular-aperture detected RP-CARS microscopy.The radially polarized pump and Stokes lightfields are tightly focused into a scatterer͑i.e.,polystyrene bead͒by a high NA objective for CARS generation.The annular aperture detection in the for-ward and backward directions uses aperture stops to block the central part of the objective’s aperture.The pump beam wavelengthp is selected at750 nm;whereas the Stokes beams is852nm.The CARS radiation wavelengthCARS is at670nm,representing the monosubstituted benzene rings stretch vibration at1600cm−1of polystyrene beads.Note that the dashed box represents the FDTD computation space that is divided into cubic cells ofp/40at each step for simulations.The NAs of the focusing and collecting objectives are assumed to be1.2͑water immersion͒and1.42͑oil immer-sion͒,respectively.The refractive indices of water and polystyrene beads are 1.33and1.59,respectively.APPLIED PHYSICS LETTERS95,133703͑2009͒0003-6951/2009/95͑13͒/133703/3/$25.00©2009American Institute of Physics95,133703-1E z͑,z͒=2iA͵0␣cos1/2͑͒sin2͑͒l͑͒J0͑ksin͒e ikz cosd,͑1a͒E͑,z͒=2A͵0␣cos1/2͑͒sin͑2͒l͑͒J1͑ksin͒e ikz cosd,͑1b͒where A is a constant;␣=arcsin͑N A/n͒;k=2/is the wave vector;J0and J1denote Bessel functions of thefirst kind with orders0and1;l͑͒is the pupil function of a Bessel–Gaussian beam:l͑͒=expͫ−02ͩsincos␣ͪ2ͬJ1ͩ20sinsin␣ͪ,͑2͒where0=3/2is the ratio of the pupil radius to the beam waist.The induced third-order nonlinear polarization at the anti-Stokes frequency,as=2p−s with the phase matching condition͉⌬k͉DӶ͓D is the size of the scatterer;⌬k=k as −͑2k p−k s͔͒,can be written3P i͑3͒͑r,as͒=3͚jklijkl͑3͒E j͑r,p͒E k͑r,p͒E lء͑r,s͒,͑3͒whereijkl͑3͒is the third-order nonlinear susceptibility;i,j,k,and l run over x,y,z,respectively,of the three components in a Cartesian coordinate system.According to Green’s func-tion,the CARS radiation in the far-field can be expressed as16E as͑R,⌰,⌽͒=−as2c2exp͑ik as͉R͉͉͒R͉͵dV expͩ−ik as R·r͉R͉ͪϫ΄000cos⌰cos⌽cos⌰sin⌽−sin⌰−sin⌽cos⌽0΅ϫ΄P x͑3͒͑r͒P y͑3͒͑r͒P z͑3͒͑r͒΅iˆR iˆ⌰iˆ⌽,͑4͒where iˆR,iˆ⌰,and iˆ⌽denote the spherical components of theCARSfield͑inset of Fig.1͒.The collected CARS radiationpower͑I CARS͒can be calculated by integrating the Poynting vector over the spherical surface of radius R within the coneangle of the collection objective as follows:I CARS=n as c8͵⌰1⌰2d⌰͵⌽1⌽2d⌽͉E as͑R͉͒2R2sin⌰,͑5͒where the acceptance cone angles⌰1and⌰2are determined by the diameter of the annular aperture and the NA of the objective used.We apply the FDTD technique7,15to solve Maxwell’s equations directly in time domain through the leap-frogging scheme7,9for computing thefield distributions,e.g.,pump field͓E͑r,p͔͒,Stokesfield͓E͑r,s͔͒,and CARSfield ͓E͑r,as͔͒of the tightly focused radially polarized pumpand Stokesfields in the focal region of the high NA micro-scope objective,as well as the far-field CARS radiations col-lected by incorporating Eqs.͑1a͒,͑1b͒,and͑2͒–͑5͒into the FDTD program developed.7Figure2͑a͒shows an example of the far-field RP-CARS radiation pattern from a scatterer ͑D=0.5p͒centered at the focus under tightly focused radi-ally polarized pump and Stokefields excitation with a water immersion objective͑NA of1.2͒.It is observed that the RP-CARS radiation pattern is doughnut-shaped,with the ra-diation confined within the cone angles depending on the scatterers’sizes and the objective NA used.14Figure2͑b͒shows the comparison of RP-CARS radiation patterns gen-erated from the scatterers at different diameters͑D=0.1p, 0.5p,1.0p,and2.0p͒and the nonresonant background from water.For the forward RP-CARS detection using the oil immersion objective͑NA=1.42͒,the nonresonant back-ground from water increases with the acceptance cone anglesranging from0°to20°͑maximum͒,and then rapidly fallswhen further increasing the acceptance cone angles͑20°to70°͒.The RP-CARS radiation patterns from the scattererswith a small diameter͑Յ0.1p͒or a large diameter͑Ն2.0p, exceeding the focal volume of light beam͒are similar to thatof the nonresonant background radiation from water.How-ever,the RP-CARS radiation from those scatterers with sizesof between0.1p and2.0p is much stronger than the non-resonant background within the acceptance cone angles rang-ing from50°to70°for the forward-detected direction,whereas this is the case for the acceptance cone angles from115°to150°for the backward-detected direction͓Fig.2͑b͔͒.Therefore,by incorporating the suitable sizes of annular stopapertures into CARS detection paths,the nonresonantback-FIG.2.͑Color online͒͑a͒Far-field RP-CARS radiation pattern from a scat-terer͑D=0.5p͒centered at the focus under the tightly focused radially polarized pump and Stokefields excitation with a water immersion objective of NA1.2.The x,y and z axes are in arbitrary units.Only the radiation in the range−/2Յ⌽Յis displayed for clarity.͑b͒Far-field RP-CARS radia-tion patterns generated from the scatterers at different diameters͑D=0.1p, 0.5p,1.0p,and2.0p͒as well the nonresonant background from water. The vertically dashed lines in thefigure indicate the maximum acceptance cone angles of the microscope objectives for both the forward͑NA=1.2͒and backward͑NA=1.42͒CARS detection,respectively.ground from water can be significantly removed for high contrast RP-CARS imaging.To evaluate the efficacy of applying the annular aperture detection scheme in RP-CARS microscopy for improving image contrast,we employ the FDTD method to study the changes of forward-detected RP-CARS ͑F-CARS ͒intensities and the corresponding SBRs with different diameters of an-nular stop apertures ͑Fig.3͒.Although the F-CARS radiation from the scatterers with different diameters ͑i.e.,0.1p to 2.0p ͒decreases by four to five orders in intensity with the increased diameters ͑i.e.,0to 0.9͒of annular stop apertures used,the lowest F-CARS intensity level is still comparable to that in backward-detected RP-CARS ͑i.e.,E-CARS ͒with-out using any annular aperture ͓Fig.4͑a ͒at D S /D A =0͔;and the corresponding SBR of F-CARS radiation from the scat-terers with diameters of 0.1p to 2.0p increases remarkably,particularly when the annular stop aperture is larger than 0.4͓Fig.3͑b ͔͒.For instance,an SBR of 90can be achieved for F-CARS radiation from the scatterer with a diameter of 1.0p when applying an annular stop aperture of 0.9in di-ameter ͑acceptance cone angles ranging from 55°to 70°͒.We also calculate backward-detected RP-CARS ͑E-CARS ͒in-tensities and the corresponding SBR with different diameters of annular stop apertures placed in the detection path ͑Fig.4͒.The E-CARS radiation from the scatterers with differentdiameters ͑0.1p to 2.0p ͒decreases by one to two orders in intensity with the increased diameters ͑0to 0.9͒of annular stop apertures used ͓Fig.4͑a ͔͒.The corresponding SBR of E-CARS radiation from the scatterers with diameters of 0.1p to 2.0p increases gradually with the increased diam-eters of annular stop apertures of up to 0.8,͑corresponding to acceptance cone angles ranging from 115°to 120°͒,at which the maximum SBR of ϳ1.2ϫ104can be achieved for E-CARS radiation from the scatterer with a diameter of 1.5p ͓Fig.4͑b ͔͒.One notes that the SBRs of both F-CARS and E-CARS radiation from very small scatterers ͑e.g.,Յ0.1p ͒or large scatterers ͑e.g.,Ն2p ͒under annular aper-ture detection are quite limited,which are only in the range of 1.2–3for very small scatterers,or of 1.5–45for large scatterers.This indicates that when applying the aperture de-tection scheme into RP-CARS microscopy for imaging bio-logical systems,there are variations in image contrast im-provements among different intercellular and/or intracellular structures and organelles due to their different physical sizes and shapes in tissue and cells.In summary,compared with RP-CARS radiation without applying annular apertures ͓Figs.3͑b ͒and 4͑b ͒at D S /D A =0͔,the annular aperture detection can improve the SBR of up to 115times for forward-detected RP-CARS imaging,and of ϳ55times for backward-detected RP-CARS microscopy.This work demonstrates that the annular aperture detection scheme can effectively remove nonresonant background for high contrast vibrational imaging in RP-CARS microscopy.This research was supported by the Academic Research Fund from the Ministry of Education,the Biomedical Re-search Council,the National Medical Research Council,and the Faculty Research Fund from the National University of Singapore.1A.Zumbush,G.R.Holtom,and X.S.Xie,Phys.Rev.Lett.82,4142͑1999͒.2F.Lu,W.Zheng,and Z.Huang,Opt.Lett.33,2842͑2008͒.3A.V olkmer,J.X.Cheng,and X.S.Xie,Phys.Rev.Lett.87,023901͑2001͒.4F.Lu,W.Zheng,C.Sheppard,and Z.Huang,Opt.Lett.33,602͑2008͒.5E.O.Potma,C.L.Evans,and X.S.Xie,Opt.Lett.31,241͑2006͒.6F.Lu,W.Zheng,and Z.Huang,Appl.Phys.Lett.92,123901͑2008͒.7J.Lin,H.Wang,W.Zheng,F.Lu,C.Sheppard,and Z.Huang,Opt.Express 17,2423͑2009͒.8T.Ichimura,N.Hayazawa,M.Hashimoto,Y .Inouye,and S.Kawata,Phys.Rev.Lett.92,220801͑2004͒.9C.Liu,Z.Huang,F.Lu,W.Zheng,D.W.Hutmacher,and C.Sheppard,Opt.Express 15,4118͑2007͒.10H.Wang,L.Shi,B.Lukyanchuk,C.Sheppard,and C.T.Chong,Nat.Photonics 2,501͑2008͒.11L.Novotny,M.R.Beversluis,K.S.Youngworth,and T.G.Brown,Phys.Rev.Lett.86,5251͑2001͒.12E.Y .S.Yew and C.J.R.Sheppard,mun.275,453͑2007͒.13S.Yang,Q.Zhan,and J.Opt,Pure Appl.Opt.10,125103͑2008͒.14F.Lu,W.Zheng,and Z.Huang,Opt.Lett.34,1870͑2009͒.15K.S.Yee,IEEE Trans.Antennas Propag.14,302͑1966͒.16J.X.Cheng,A.V olkmer,and X.S.Xie,J.Opt.Soc.Am.B 19,1363͑2002͒.FIG.3.͑Color online ͒͑a ͒Calculated forward-detected RP-CARS ͑F-CARS ͒intensities of different scatterers ͑D =0.1p ,0.5p ,1.0p ,and 2.0p ͒using annular stop apertures with different diameters.͑b ͒Relationship of the SBR of F-CARS with different diameters of annular stop apertures.Note that the diameter of the annular stop aperture placed along the detection path is defined as the ratio of the diameter of the annular stop aperture ͑D S ͒to the diameter of the objective’s aperture ͑D A ͒.FIG. 4.͑Color online ͒͑a ͒Calculated backward-detected RP-CARS ͑E-CARS ͒intensities of different scatterers ͑D =0.1p ,0.5p ,1.0p ,and 2.0p ͒using annular stop apertures with different diameters.͑b ͒Relation-ship of the SBR of E-CARS with different diameters of annular stop apertures.。
