May 1, 2015 / Vol. 40, No. 9 / OPTICS LETTERS
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Background-free 3D chemical imaging based on polarization coherent Raman holography Yonggang Lv,1 Ziheng Ji,1 Hong Yang,1 Kebin Shi,1,2,* and Qihuang Gong1,2 1
State Key Laboratory for Mesoscopic Physics, School of Physics, Peking University, Beijing 100871, China 2 Collaborative Innovation Center of Quantum Matter, Beijing, China *Corresponding author: [email protected] Received January 5, 2015; revised March 13, 2015; accepted April 7, 2015; posted April 8, 2015 (Doc. ID 231548); published April 30, 2015
We report on a holographic coherent anti-Stokes Raman scattering imaging by using polarization discrimination for nonresonant background suppression. With reduced polarization scrambling effect under weakly focused excitation, nonresonant background-free coherent Raman imaging is demonstrated. A fast chemically selective imaging is achieved in a three-dimensional volume of 70 μm × 70 μm × 100 μm in 2 s. © 2015 Optical Society of America OCIS codes: (190.4380) Nonlinear optics, four-wave mixing; (090.1995) Digital holography; (180.4315) Nonlinear microscopy. http://dx.doi.org/10.1364/OL.40.002095
Since its inception [1], holography has progressed significantly toward powerful imaging apparatuses [2,3], By using the scheme of wide-field detection and wave front phase retrieval, holographic imaging supports scanningless data acquisition in three-dimension (3D) and therefore possesses superior imaging speed than that in laser-scanning-based microscopes. Benefited from digitized holographic microscope (DHM) [4], holography has revived and found many applications in modern science such as optical manipulation [5] and particle tracking [6]. In most holographic systems, linearly scattered or deflected signals are generated for hologram recording. Holography imaging based on more chemically selective signal generation would be in interest of a broader scientific scope including material and biological sciences. Recent developments on fluorescence and secondharmonic generation (SHG)-based hologram have demonstrated promising applications of holography for chemically selective imaging. An inline holographic microscope [7,8] using wave-front modulation was proposed to achieve scanning-less 3D fluorescence imaging. Holographic SHG imaging modalities [9,10] were also proposed to obtain 3D bio-photonic imaging by recording holograms of harmonic signal generated from nanocrystals with broken center-symmetry or inherent nonsymmetric structures in bio-samples. In contrast to the holographic imaging based on labeling samples, a thirdorder nonlinear hologram utilizing inherent coherent anti-Stokes Raman scattering (CARS) signal has been demonstrated [11,12] to perform rapid nonlabeling chemical 3D imaging. Similar to the laser scanning CARS microscopy, a major limitation of CARS holography is the existence of the nondispersive nonresonant background, which originates from electronic contributions of the third-order susceptibility in the sample and solvent [13]. Since CARS holography employs wide field excitation, the generated nonresonant wide-field signal gives rise to even worse detection noise in hologram recording and consequential distortion in 3D imaging retrieval than that caused in laser scanning systems. Various mechanisms such as timeresolved technique [14,15], polarization discrimination 0146-9592/15/092095-04$15.00/0
[16–18], spectrally heterodyne detection [19,20], and phase-controlled superposition [21,22] have been proposed as promising candidates for suppressing non resonant background in CARS. Yet in CARS holography, the using of time-resolved scheme for nonresonant background suppression is usually limited since the broad spectral bandwidth of femtosecond sources would cause fringe distortions in hologram recording. Previously reported spectrally heterodyne detection [19,20] is also inaccessible under wide-field holographic recording scheme. In this work, we report on a back-ground-free CARS holographic imaging by using polarization discrimination between resonant and nonresonant CARS signals. The polarization discrimination in CARS essentially arises from the difference of depolarization ratio between generated resonant and nonresonant fields [16,17]. As shown in the inset picture in Fig. 1, an x-polarized pump beam and a Stokes beam polarized at angle ϕ to the x axis propagate along the z axis. By assuming a depolarization NR ratio χ NR 2112 ∕χ 1111 of 1/3 [23] for nonresonant field generation, we can obtain the nonresonant signal polarized at the angle α to the x axis with the relationship satisfying tan α 1∕3 tan ϕ[18] as shown in inserted in Fig. 1.
Fig. 1. Calculated focusing depolarization effect of a Gaussian beam: jEy max j∕jE x max j and jE z max j∕jEx max j2 as a function of numerical aperture; the inset picture shows the polarization CARS scheme. © 2015 Optical Society of America
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Without electronic resonance, depolarization ratio χ R2112 ∕χ R1111 of vibrational resonant signal is equal to the corresponding spontaneous Raman depolarization ratio [23]. Therefore the generated resonant field will be polarized at a different angle to the nonresonant signal as indicated in the inset picture in Fig. 1. As a result, we can suppress the nonresonant background by placing a detection polarizer perpendicular to the nonresonant field polarization. It is worth to mention that in practice there is always nonresonant background noise projected onto the detection polarization axis due to the birefringence of optical components and field depolarization in the foci region. While the birefringence can be suppressed by using highquality optics, the field depolarization would become the major mechanism reducing the signal-to-noise ratio (SNR) in polarization CARS detection. As shown in Fig. 1, we calculated the field depolarization effects relative to the focusing numerical aperture (NA) for an linearly xpolarized Gaussian beam [24]. jE x max j, jE y max j, and jE z max j is the maximum relative values for each individual field components polarized along x, y, and z directions, respectively, at the focal plane. In tightly focused scheme, which is employed in laser scanning CARS microscopes, one would observe noticeable polarization scrambling. However, in our wide-field holographic CARS imaging, SNR degradation caused by focusing depolarization becomes negligible since NA of 0.01 was used for excitation. It is also observed in Fig. 1 that the longitudinal component generated at foci is found one order of magnitude stronger than transversely depolarized component, suggesting a noteworthy contribution from longitudinal field at foci. Based on the arrangement and calculation shown in Fig. 1, we can estimate the degradation of SNR for different focusing schemes. We define that E py and E pz are the depolarized electric fields of the pump beam along y and z axes at foci respectively, and E sz is the electric field of the Stokes beam along z axis. Ignoring quadratic terms from depolarized fields and the spatial distribution for different depolarized components, the x and y projections of nonresonant field resulting from focusing depolarization can be written as
along the detection signal: r de
axis and total nonresonant
P de 2 NR r y P NR 3
p 10 − 1 2 rz 3
(4)
where P NR
P NR x 2 cos ϕ χ NR . 1111 E p E s cos α cos α
(5)
In a typical tightly focused scheme (NA of 0.95), the degradation factor is estimated at 13.97% by using the calculation shown in Fig. 1. On the contrary, our holographic CARS imaging employs weakly focused scheme with NA of 0.01, which only leads to a degradation factor at 1.02 × 10−5 , four orders of magnitudes smaller comparing with that in laser scanning systems. The improved tolerance to focusing depolarization make the polarization discrimination a more effective way for nonresonant background suppression in holographic CARS imaging. The schematic diagram of our experimental setup is shown in Fig. 2(a). A picosecond laser (Ekspla, PL2210 Series, λ 1064 nm, repetition rate: 1000 Hz, pulse duration: 25 ps) was employed as CARS pump beam. The frequency-doubled output at 532-nm wavelength from the laser pumped a tunable optical parametric amplifier (OPA) (Ekspla, PG500 Series) to produce an idler beam as the Stokes beam. A delay line is used in the pump beam path to ensure the temporal overlapping of pump and Stokes beams. Both beams were weakly focused onto the sample by a lens (focal length: 200 mm) to form a weakly focused wide-field excitation. The pulse energy of the pump and the Stokes pulses were 30 μJ and 10 μJ, respectively. The angle between them was about 6.2 deg. After CARS signal generation, a small iris was used to
NR NR P de x χ 1122 E p E py E s sin ϕχ 1212 E py E p E s sin ϕ NR NR χ NR 1331 E pz E pz E s cos ϕχ 1133 E p E pz E sz χ 1313 E pz E p E sz
(1) NR NR P de y χ 2121 E p E py E s cos ϕχ 2211 E py E p E s cos ϕ χ NR 2332 E pz E pz E s sin ϕ
(2)
NR NR NR NR NR where χ NR 2112 χ 1122 χ 1212 χ 1331 χ 1133 χ 1313 1 NR NR NR NR χ 2121 χ 2211 χ 2332 3 χ 1111 in isotropic materials [23]. Thus the nonresonant polarization projected along the detection direction can be written as de de P de NR P x cos90° − α − P y cos α.
(3)
By defining depolarization ratio r y E py ∕E px and r z E pz ∕E px E sz ∕E sx , we obtain the degradation factor r de defined as the ratio between nonresonant field
Fig. 2. (a) Schematic diagram of the experimental setup. L1: singlet lens, focal length 200 mm; L2: long-working-distance objective lens, focal length 10 mm; L3: singlet lens, focal length 750 mm; I: iris; SPF: short pass filter; P, polarizer; HW, half-wave plate. (b) A typical hologram of a polystyrene microsphere recorded on an EMCCD camera. (c) A typical CARS spectrum of a polystyrene microsphere. (d) The Raman spectrum of polystyrene microspheres. (e) Dependence of CARS signal on the power of the pump beam in logarithmic scale; (f) dependence of CARS signal on the power of the Stokes beam in logarithmic scale.
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block the transmitted pump and Stokes beams. A short pass filter (Chroma, HQ950-60) was used to filter the CARS signal. To suppress the nonresonant background, the polarization angle between the pump and the Stokes beams is set to 71.6 deg [18] to achieve the maximum SNR. A polarizer was applied before the detection to block the nonresonant background. The wide-field CARS signal generated from the sample was magnified by an imaging system consisting of a long-workingdistance objective lens (Edmund, numerical aperture: 0.42, focal length: 10 mm) and a lens (focal length: 750 mm). To record a hologram, the signal output of the OPA is used as the reference beam. The reference wave first passed through a half wave plate polarization adjustment and then was sent to a delay line to ensure its temporal overlapping with the generated CARS field. These two beams interfered at an angle 3.7 deg on an electronmultiplying charge-coupled device (EMCCD) camera (Andor, iXon X3). Figure 1(b) shows a typical CARS hologram. We used polystyrene microspheres (Bangs Laboratories, diameter: 10 μm) as the resonance sample. The spontaneous Raman spectrum of polystyrene is shown in Fig. 2(d), from which we can tell that the strongest excite molecular vibration of polystyrene is at around 1001 cm−1 . For resonant imaging, the wavelength of the Stokes beam was tuned to 1191 nm. We verified the power dependence by coupling output signal into a spectrograph (Acton 2500, Princeton Instruments) equipped with a liquid-nitrogen-cooled CCD detector (Spec-10, Princeton Instruments). The peak of the signal at 961.7 nm is shown in Fig. 2(c). Figure 2(e) shows the power dependence of the measured signal intensity at 961.7 nm on the average pulse energy of the pump beam in logarithmic scale. The measured data is fitted with a slope of 2.02, indicating quadratic dependence of signal on pump power. The linear power dependence of signal on Stokes beam power is also shown in Fig. 2(f). These results suggest that the signal generation is a third-order nonlinear process. The polarization CARS holography imaging experiments were carried by using a mixed sample consisting of resonant polystyrene microspheres (diameter: 10 μm) and nonresonant silicon dioxide microspheres (Tianjin Base-Line Chrom-Tech Research Center, diameter: 8 μm) sandwiched between a pair of microscope cover glasses (VWR No.1 cover glass). The microspheres were all suspending in water, and the edges of the cover glasses were sealed with high-vacuum grease (DOW CORNING). Figure 3(a) shows the bright field image of the sample, where a polystyrene microsphere (right) and a silicon dioxide microsphere (left) can be observed. We first carried the conventional CARS holography without placing the polarizer in front of CCD. The pump and Stokes wavelengths were 1064 nm and 1191 nm, respectively, to resonantly excite the 1001 cm−1 mode of polystyrene. Figure 3(b) shows the recorded CARS hologram of these two microspheres. The amplitude [Fig. 3(c)] and phase [Fig. 3(d)] of the CARS field without diffraction compensation are retrieved from the hologram. Since the complex CARS image field is captured, it can be back-propagated digitally [4] to realize the 3D imaging. Figure 3(e) shows the back propagated imaging. The silicon dioxide
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Fig. 3. (a) Bright-field imaging of a polystyrene microsphere (right) and a silicon dioxide microsphere (left) suspended in water. (b) CARS hologram of the microspheres without polarization detection; retrieved amplitude image (c) and phase image (d) of the CARS field from the hologram shown in (b); (e) reconstructed CARS intensity image based on the hologram (b). The inset picture in (e) is the reconstructed CARS intensity distribution along the arrow direction. (f) Polarization CARS hologram of the microspheres; retrieved amplitude image (g) and phase image (h) of the CARS field from the hologram shown in (f); (i) reconstructed CARS intensity image based on the hologram (f). The inset picture in (f) is the reconstructed CARS intensity distribution along the arrow direction.
microsphere is found to provide a considerable nonresonant contribution. Polarization CARS holography was then recorded by keeping the 71.6-deg angle between the pump and Stokes beams with a polarizer in front of the EMCCD to block the nonresonant background. We adjust the half wave plate in the reference path to record a hologram [Fig. 3(f)]. The amplitude [Fig. 3(g)] and phase [Fig. 3(h)] of the CARS field are also retrieved from the hologram. Figure 3(i) is the reconstructed image in polarization CARS holography scheme, in which only the polystyrene microspheres can be observed. The inserted cross-section shows a nearly zero floor from the position where the silicon dioxide microsphere locates, indicating a prominent nonresonant background suppression. One of the most important advantages of CARS holography is fast 3D imaging capability. We prepared a sample containing polystyrene microspheres (diameter: 10 μm) suspended in water at different depths. Figure 4(a) shows the recorded resonant CARS hologram from which the complex CARS image field amplitude [Fig. 4(b)] and phase [Fig. 4(c)] can be retrieved. The exposure time was 2 s. Then the CARS images at different axial planes can be realized through back-propagation method. Figures 4(d) and 4(e) show the CARS intensity distribution of propagated CARS field at z 15 μm and z −75 μm, respectively. Two individual polystyrene microspheres are digitally brought to focus, respectively. Comparing to previous report on holographic CARS [11], this result indicates that our polarization CARS
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Ministry of Science and Technology of China (National Basic Research Program of China under Grant No. 2013CB921904).
Fig. 4. (a) Holographic CARS imaging of two polystyrene microspheres suspended in water; retrieved amplitude intensity image (b) and phase image (c) of the CARS field from the hologram shown in (a) (without diffraction compensation); intensity distribution of digitally propagated CARS field at different planes: (d) z 15 μm; (e) z −75 μm; (f) reconstructed CARS intensity imaging of the two polystyrene microspheres when the Stokes wavelength apart from the polystyrene resonance (at 950 cm−1 ).
holography has achieved a larger depth of field (nearly 100 μm) due to the improved SNR. When we tuned the Stokes wavelength to apart from the polystyrene resonance (at 950 cm−1 ), a reconstructed CARS imaging of this sample is shown in Fig. 4(f), where no signal could be recorded. In summary, we have demonstrated a wide-field polarization CARS holographic imaging apparatus. Our results show that the polarization technique can suppress the nonresonant background and improve the chemical selectivity in CARS holography. By capturing both the amplitude and the phase of a complex CARS image field, CARS holography can perform fast imaging of a threedimensional volume of 70 μm × 70 μm × 100 μm in 2 s, making it promising imaging modality for fast nonlabeling 3D imaging [25]. Our results also indicate that the polarization discrimination, together with the heterodyne nature of hologram, can lead to a pure phase contrast imaging for nonlinear holography based on third-order nonlinearity. K. Shi acknowledges the funding support from the National Science Foundation of China (NSFC#11174019, 61322509, and 11121091) and the
References 1. D. Gabor, Nature 161, 777 (1948). 2. I. Yamaguchi and T. Zhang, Opt. Lett. 22, 1268 (1997). 3. E. Cuche, F. Bevilacqua, and C. Depeursinge, Opt. Lett. 24, 291 (1999). 4. J. M. Cowley and D. J. Walker, Ultramicroscopy 6, 71 (1981). 5. J. E. Curtis, B. A. Koss, and D. G. Grier, Opt. Commun. 207, 169 (2002). 6. W. Xu, M. H. Jericho, H. J. Kreuzer, and I. A. Meinertzhagen, Opt. Lett. 28, 164 (2003). 7. B. W. Schilling, T. C. Poon, G. Indebetouw, B. Storrie, K. Shinoda, Y. Suzuki, and M. H. Wu, Opt. Lett. 22, 1506 (1997). 8. J. Rosen and G. Brooker, Nat. Photonics 2, 190 (2008). 9. Y. Pu, M. Centurion, and D. Psaltis, Appl. Opt. 47, A103 (2008). 10. C. L. Hsieh, R. Grange, Y. Pu, and D. Psaltis, Opt. Express 17, 2880 (2009). 11. K. Shi, H. Li, Q. Xu, D. Psaltis, and Z. Liu, Phys. Rev. Lett. 104, 093902 (2010). 12. Q. Xu, K. Shi, H. Li, K. Choi, R. Horisaki, D. Brady, D. Psaltis, and Z. Liu, Opt. Express 18, 8213 (2010). 13. J. X. Cheng and X. S. Xie, J. Phys. Chem. B 108, 827 (2004). 14. S. Roy, T. R. Meyer, and J. R. Gord, Appl. Phys. Lett. 87, 264103 (2005). 15. Y.-G. Lu, Z.-J. Li, L.-Z. Wu, P. Wang, L.-M. Fu, and J.-P. Zhang, Acta Physico-Chimica Sinica 29, 1632 (2013). 16. S. A. Akhmanov, A. F. Bunkin, S. G. Ivanov, and N. I. Koroteev, Sov. JETP Lett. 25, 416 (1977). 17. J. L. Oudar, R. W. Smith, and Y. R. Shen, Appl. Phys. Lett. 34, 758 (1979). 18. J. X. Cheng, L. D. Book, and X. S. Xie, Opt. Lett. 26, 1341 (2001). 19. C. L. Evans, E. O. Potma, and X. S. N. Xie, Opt. Lett. 29, 2923 (2004). 20. E. O. Potma, C. L. Evans, and X. S. Xie, Opt. Lett. 31, 241 (2006). 21. G. Marowsky and G. Lupke, Appl. Phys. B 51, 49 (1990). 22. G. Lupke, G. Marowsky, and R. Steinhoff, Appl. Phys. B 49, 283 (1989). 23. Y. Yan, L. Cheng, K. Nelson, R. Clark, and R. Hester, Adv. Spectrosc. Series 6, 299 (1988). 24. B. Hecht, Principles of Nano-Optics (Cambridge University, 2012). 25. K. Shi, P. S. Edwards, J. Hu, Q. Xu, Y. Wang, D. Psaltis, and Z. Liu, Biomed. Opt. Express 3, 1744 (2012).
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Background-free 3D chemical imaging based on polarization coherent Raman holography Yonggang Lv,1 Ziheng Ji,1 Hong Yang,1 Kebin Shi,1,2,* and Qihuang Gong1,2 1
State Key Laboratory for Mesoscopic Physics, School of Physics, Peking University, Beijing 100871, China 2 Collaborative Innovation Center of Quantum Matter, Beijing, China *Corresponding author: [email protected] Received January 5, 2015; revised March 13, 2015; accepted April 7, 2015; posted April 8, 2015 (Doc. ID 231548); published April 30, 2015
We report on a holographic coherent anti-Stokes Raman scattering imaging by using polarization discrimination for nonresonant background suppression. With reduced polarization scrambling effect under weakly focused excitation, nonresonant background-free coherent Raman imaging is demonstrated. A fast chemically selective imaging is achieved in a three-dimensional volume of 70 μm × 70 μm × 100 μm in 2 s. © 2015 Optical Society of America OCIS codes: (190.4380) Nonlinear optics, four-wave mixing; (090.1995) Digital holography; (180.4315) Nonlinear microscopy. http://dx.doi.org/10.1364/OL.40.002095
Since its inception [1], holography has progressed significantly toward powerful imaging apparatuses [2,3], By using the scheme of wide-field detection and wave front phase retrieval, holographic imaging supports scanningless data acquisition in three-dimension (3D) and therefore possesses superior imaging speed than that in laser-scanning-based microscopes. Benefited from digitized holographic microscope (DHM) [4], holography has revived and found many applications in modern science such as optical manipulation [5] and particle tracking [6]. In most holographic systems, linearly scattered or deflected signals are generated for hologram recording. Holography imaging based on more chemically selective signal generation would be in interest of a broader scientific scope including material and biological sciences. Recent developments on fluorescence and secondharmonic generation (SHG)-based hologram have demonstrated promising applications of holography for chemically selective imaging. An inline holographic microscope [7,8] using wave-front modulation was proposed to achieve scanning-less 3D fluorescence imaging. Holographic SHG imaging modalities [9,10] were also proposed to obtain 3D bio-photonic imaging by recording holograms of harmonic signal generated from nanocrystals with broken center-symmetry or inherent nonsymmetric structures in bio-samples. In contrast to the holographic imaging based on labeling samples, a thirdorder nonlinear hologram utilizing inherent coherent anti-Stokes Raman scattering (CARS) signal has been demonstrated [11,12] to perform rapid nonlabeling chemical 3D imaging. Similar to the laser scanning CARS microscopy, a major limitation of CARS holography is the existence of the nondispersive nonresonant background, which originates from electronic contributions of the third-order susceptibility in the sample and solvent [13]. Since CARS holography employs wide field excitation, the generated nonresonant wide-field signal gives rise to even worse detection noise in hologram recording and consequential distortion in 3D imaging retrieval than that caused in laser scanning systems. Various mechanisms such as timeresolved technique [14,15], polarization discrimination 0146-9592/15/092095-04$15.00/0
[16–18], spectrally heterodyne detection [19,20], and phase-controlled superposition [21,22] have been proposed as promising candidates for suppressing non resonant background in CARS. Yet in CARS holography, the using of time-resolved scheme for nonresonant background suppression is usually limited since the broad spectral bandwidth of femtosecond sources would cause fringe distortions in hologram recording. Previously reported spectrally heterodyne detection [19,20] is also inaccessible under wide-field holographic recording scheme. In this work, we report on a back-ground-free CARS holographic imaging by using polarization discrimination between resonant and nonresonant CARS signals. The polarization discrimination in CARS essentially arises from the difference of depolarization ratio between generated resonant and nonresonant fields [16,17]. As shown in the inset picture in Fig. 1, an x-polarized pump beam and a Stokes beam polarized at angle ϕ to the x axis propagate along the z axis. By assuming a depolarization NR ratio χ NR 2112 ∕χ 1111 of 1/3 [23] for nonresonant field generation, we can obtain the nonresonant signal polarized at the angle α to the x axis with the relationship satisfying tan α 1∕3 tan ϕ[18] as shown in inserted in Fig. 1.
Fig. 1. Calculated focusing depolarization effect of a Gaussian beam: jEy max j∕jE x max j and jE z max j∕jEx max j2 as a function of numerical aperture; the inset picture shows the polarization CARS scheme. © 2015 Optical Society of America
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Without electronic resonance, depolarization ratio χ R2112 ∕χ R1111 of vibrational resonant signal is equal to the corresponding spontaneous Raman depolarization ratio [23]. Therefore the generated resonant field will be polarized at a different angle to the nonresonant signal as indicated in the inset picture in Fig. 1. As a result, we can suppress the nonresonant background by placing a detection polarizer perpendicular to the nonresonant field polarization. It is worth to mention that in practice there is always nonresonant background noise projected onto the detection polarization axis due to the birefringence of optical components and field depolarization in the foci region. While the birefringence can be suppressed by using highquality optics, the field depolarization would become the major mechanism reducing the signal-to-noise ratio (SNR) in polarization CARS detection. As shown in Fig. 1, we calculated the field depolarization effects relative to the focusing numerical aperture (NA) for an linearly xpolarized Gaussian beam [24]. jE x max j, jE y max j, and jE z max j is the maximum relative values for each individual field components polarized along x, y, and z directions, respectively, at the focal plane. In tightly focused scheme, which is employed in laser scanning CARS microscopes, one would observe noticeable polarization scrambling. However, in our wide-field holographic CARS imaging, SNR degradation caused by focusing depolarization becomes negligible since NA of 0.01 was used for excitation. It is also observed in Fig. 1 that the longitudinal component generated at foci is found one order of magnitude stronger than transversely depolarized component, suggesting a noteworthy contribution from longitudinal field at foci. Based on the arrangement and calculation shown in Fig. 1, we can estimate the degradation of SNR for different focusing schemes. We define that E py and E pz are the depolarized electric fields of the pump beam along y and z axes at foci respectively, and E sz is the electric field of the Stokes beam along z axis. Ignoring quadratic terms from depolarized fields and the spatial distribution for different depolarized components, the x and y projections of nonresonant field resulting from focusing depolarization can be written as
along the detection signal: r de
axis and total nonresonant
P de 2 NR r y P NR 3
p 10 − 1 2 rz 3
(4)
where P NR
P NR x 2 cos ϕ χ NR . 1111 E p E s cos α cos α
(5)
In a typical tightly focused scheme (NA of 0.95), the degradation factor is estimated at 13.97% by using the calculation shown in Fig. 1. On the contrary, our holographic CARS imaging employs weakly focused scheme with NA of 0.01, which only leads to a degradation factor at 1.02 × 10−5 , four orders of magnitudes smaller comparing with that in laser scanning systems. The improved tolerance to focusing depolarization make the polarization discrimination a more effective way for nonresonant background suppression in holographic CARS imaging. The schematic diagram of our experimental setup is shown in Fig. 2(a). A picosecond laser (Ekspla, PL2210 Series, λ 1064 nm, repetition rate: 1000 Hz, pulse duration: 25 ps) was employed as CARS pump beam. The frequency-doubled output at 532-nm wavelength from the laser pumped a tunable optical parametric amplifier (OPA) (Ekspla, PG500 Series) to produce an idler beam as the Stokes beam. A delay line is used in the pump beam path to ensure the temporal overlapping of pump and Stokes beams. Both beams were weakly focused onto the sample by a lens (focal length: 200 mm) to form a weakly focused wide-field excitation. The pulse energy of the pump and the Stokes pulses were 30 μJ and 10 μJ, respectively. The angle between them was about 6.2 deg. After CARS signal generation, a small iris was used to
NR NR P de x χ 1122 E p E py E s sin ϕχ 1212 E py E p E s sin ϕ NR NR χ NR 1331 E pz E pz E s cos ϕχ 1133 E p E pz E sz χ 1313 E pz E p E sz
(1) NR NR P de y χ 2121 E p E py E s cos ϕχ 2211 E py E p E s cos ϕ χ NR 2332 E pz E pz E s sin ϕ
(2)
NR NR NR NR NR where χ NR 2112 χ 1122 χ 1212 χ 1331 χ 1133 χ 1313 1 NR NR NR NR χ 2121 χ 2211 χ 2332 3 χ 1111 in isotropic materials [23]. Thus the nonresonant polarization projected along the detection direction can be written as de de P de NR P x cos90° − α − P y cos α.
(3)
By defining depolarization ratio r y E py ∕E px and r z E pz ∕E px E sz ∕E sx , we obtain the degradation factor r de defined as the ratio between nonresonant field
Fig. 2. (a) Schematic diagram of the experimental setup. L1: singlet lens, focal length 200 mm; L2: long-working-distance objective lens, focal length 10 mm; L3: singlet lens, focal length 750 mm; I: iris; SPF: short pass filter; P, polarizer; HW, half-wave plate. (b) A typical hologram of a polystyrene microsphere recorded on an EMCCD camera. (c) A typical CARS spectrum of a polystyrene microsphere. (d) The Raman spectrum of polystyrene microspheres. (e) Dependence of CARS signal on the power of the pump beam in logarithmic scale; (f) dependence of CARS signal on the power of the Stokes beam in logarithmic scale.
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block the transmitted pump and Stokes beams. A short pass filter (Chroma, HQ950-60) was used to filter the CARS signal. To suppress the nonresonant background, the polarization angle between the pump and the Stokes beams is set to 71.6 deg [18] to achieve the maximum SNR. A polarizer was applied before the detection to block the nonresonant background. The wide-field CARS signal generated from the sample was magnified by an imaging system consisting of a long-workingdistance objective lens (Edmund, numerical aperture: 0.42, focal length: 10 mm) and a lens (focal length: 750 mm). To record a hologram, the signal output of the OPA is used as the reference beam. The reference wave first passed through a half wave plate polarization adjustment and then was sent to a delay line to ensure its temporal overlapping with the generated CARS field. These two beams interfered at an angle 3.7 deg on an electronmultiplying charge-coupled device (EMCCD) camera (Andor, iXon X3). Figure 1(b) shows a typical CARS hologram. We used polystyrene microspheres (Bangs Laboratories, diameter: 10 μm) as the resonance sample. The spontaneous Raman spectrum of polystyrene is shown in Fig. 2(d), from which we can tell that the strongest excite molecular vibration of polystyrene is at around 1001 cm−1 . For resonant imaging, the wavelength of the Stokes beam was tuned to 1191 nm. We verified the power dependence by coupling output signal into a spectrograph (Acton 2500, Princeton Instruments) equipped with a liquid-nitrogen-cooled CCD detector (Spec-10, Princeton Instruments). The peak of the signal at 961.7 nm is shown in Fig. 2(c). Figure 2(e) shows the power dependence of the measured signal intensity at 961.7 nm on the average pulse energy of the pump beam in logarithmic scale. The measured data is fitted with a slope of 2.02, indicating quadratic dependence of signal on pump power. The linear power dependence of signal on Stokes beam power is also shown in Fig. 2(f). These results suggest that the signal generation is a third-order nonlinear process. The polarization CARS holography imaging experiments were carried by using a mixed sample consisting of resonant polystyrene microspheres (diameter: 10 μm) and nonresonant silicon dioxide microspheres (Tianjin Base-Line Chrom-Tech Research Center, diameter: 8 μm) sandwiched between a pair of microscope cover glasses (VWR No.1 cover glass). The microspheres were all suspending in water, and the edges of the cover glasses were sealed with high-vacuum grease (DOW CORNING). Figure 3(a) shows the bright field image of the sample, where a polystyrene microsphere (right) and a silicon dioxide microsphere (left) can be observed. We first carried the conventional CARS holography without placing the polarizer in front of CCD. The pump and Stokes wavelengths were 1064 nm and 1191 nm, respectively, to resonantly excite the 1001 cm−1 mode of polystyrene. Figure 3(b) shows the recorded CARS hologram of these two microspheres. The amplitude [Fig. 3(c)] and phase [Fig. 3(d)] of the CARS field without diffraction compensation are retrieved from the hologram. Since the complex CARS image field is captured, it can be back-propagated digitally [4] to realize the 3D imaging. Figure 3(e) shows the back propagated imaging. The silicon dioxide
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Fig. 3. (a) Bright-field imaging of a polystyrene microsphere (right) and a silicon dioxide microsphere (left) suspended in water. (b) CARS hologram of the microspheres without polarization detection; retrieved amplitude image (c) and phase image (d) of the CARS field from the hologram shown in (b); (e) reconstructed CARS intensity image based on the hologram (b). The inset picture in (e) is the reconstructed CARS intensity distribution along the arrow direction. (f) Polarization CARS hologram of the microspheres; retrieved amplitude image (g) and phase image (h) of the CARS field from the hologram shown in (f); (i) reconstructed CARS intensity image based on the hologram (f). The inset picture in (f) is the reconstructed CARS intensity distribution along the arrow direction.
microsphere is found to provide a considerable nonresonant contribution. Polarization CARS holography was then recorded by keeping the 71.6-deg angle between the pump and Stokes beams with a polarizer in front of the EMCCD to block the nonresonant background. We adjust the half wave plate in the reference path to record a hologram [Fig. 3(f)]. The amplitude [Fig. 3(g)] and phase [Fig. 3(h)] of the CARS field are also retrieved from the hologram. Figure 3(i) is the reconstructed image in polarization CARS holography scheme, in which only the polystyrene microspheres can be observed. The inserted cross-section shows a nearly zero floor from the position where the silicon dioxide microsphere locates, indicating a prominent nonresonant background suppression. One of the most important advantages of CARS holography is fast 3D imaging capability. We prepared a sample containing polystyrene microspheres (diameter: 10 μm) suspended in water at different depths. Figure 4(a) shows the recorded resonant CARS hologram from which the complex CARS image field amplitude [Fig. 4(b)] and phase [Fig. 4(c)] can be retrieved. The exposure time was 2 s. Then the CARS images at different axial planes can be realized through back-propagation method. Figures 4(d) and 4(e) show the CARS intensity distribution of propagated CARS field at z 15 μm and z −75 μm, respectively. Two individual polystyrene microspheres are digitally brought to focus, respectively. Comparing to previous report on holographic CARS [11], this result indicates that our polarization CARS
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OPTICS LETTERS / Vol. 40, No. 9 / May 1, 2015
Ministry of Science and Technology of China (National Basic Research Program of China under Grant No. 2013CB921904).
Fig. 4. (a) Holographic CARS imaging of two polystyrene microspheres suspended in water; retrieved amplitude intensity image (b) and phase image (c) of the CARS field from the hologram shown in (a) (without diffraction compensation); intensity distribution of digitally propagated CARS field at different planes: (d) z 15 μm; (e) z −75 μm; (f) reconstructed CARS intensity imaging of the two polystyrene microspheres when the Stokes wavelength apart from the polystyrene resonance (at 950 cm−1 ).
holography has achieved a larger depth of field (nearly 100 μm) due to the improved SNR. When we tuned the Stokes wavelength to apart from the polystyrene resonance (at 950 cm−1 ), a reconstructed CARS imaging of this sample is shown in Fig. 4(f), where no signal could be recorded. In summary, we have demonstrated a wide-field polarization CARS holographic imaging apparatus. Our results show that the polarization technique can suppress the nonresonant background and improve the chemical selectivity in CARS holography. By capturing both the amplitude and the phase of a complex CARS image field, CARS holography can perform fast imaging of a threedimensional volume of 70 μm × 70 μm × 100 μm in 2 s, making it promising imaging modality for fast nonlabeling 3D imaging [25]. Our results also indicate that the polarization discrimination, together with the heterodyne nature of hologram, can lead to a pure phase contrast imaging for nonlinear holography based on third-order nonlinearity. K. Shi acknowledges the funding support from the National Science Foundation of China (NSFC#11174019, 61322509, and 11121091) and the
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