4788
OPTICS LETTERS / Vol. 39, No. 16 / August 15, 2014
Femtosecond pulse shaping enables detection of optical Kerr-effect (OKE) dynamics for molecular imaging Francisco E. Robles,* Martin C. Fischer, and Warren S. Warren Department of Chemistry, Duke University, Durham, North Carolina 27708, USA *Corresponding author: [email protected] Received June 5, 2014; accepted June 27, 2014; posted July 14, 2014 (Doc. ID 213533); published August 8, 2014 We apply femtosecond pulse shaping to generate optical pulse trains that directly access a material’s nonlinear refractive index (n2 ) and can thus determine time-resolved optical Kerr-effect (OKE) dynamics. Two types of static pulse trains are discussed: The first uses two identical fields delayed in time, plus a pump field at a different wavelength. Time-resolved OKE dynamics are retrieved by monitoring the phase of the interference pattern produced by the two identical fields in the Fourier-domain (FD) as a function of pump–probe–time–delay (where the probe is one of the two identical fields). The second pulse train uses three fields with equal time delays, but with the center field phase shifted by π∕2. In this pulse scheme, changes on a sample’s nonlinear refractive index produce a new frequency in the FD signal, which in turn yields background-free intensity changes in the conjugate (time) domain and provides superior signal-to-noise ratios. The demonstrated sensitivity improvements enable, for the first time to our knowledge, molecular imaging based on OKE dynamics. © 2014 Optical Society of America OCIS codes: (180.4315) Nonlinear microscopy; (300.6420) Spectroscopy, nonlinear; (120.3180) Interferometry; (170.3880) Medical and biological imaging; (190.3270) Kerr effect. http://dx.doi.org/10.1364/OL.39.004788
Nonlinear optical imaging techniques have proven immensely useful in the field of biology and medicine [1–4]. For example, the inherent cross-sectional ability and high sensitivity of multiphoton excitation fluorescence (MPEF) microscopy has enabled high-resolution imaging of intact biological tissues at unprecedented depths using exogenous agents [2]. Other groups, including ours, have used pump–probe (pu-pr) schemes to gain access to other nonlinear interactions (such as stimulated Raman scattering, excited state absorption, and ground-state bleaching) for highly specific molecular contrast of endogenous species in applications such as melanoma diagnosis [4–6]. In general, most nonlinear imaging techniques rely on interactions that generate new optical frequencies, or that result in attenuation/ amplification of a probe beam. Nonlinear interactions that only result in phase changes [self-phase modulation (SPM), and cross-phase modulation (XPM)] remain largely unexplored in microscopy and are the focus of this Letter. The gold standard for measuring SPM is the Z-scan technique [7]. In this method, a transparent material of interest is scanned along the focus of a femtosecond beam, and transmitted light is collected after passing through a small aperture. Changes in the amount of light passing through the aperture can be related to n2 , with high sensitivity. However, this approach is relatively slow, can only be used to assess bulk n2 properties, and is also rapidly degraded by scattering [8], making it ill-suited for imaging biological samples. To overcome these challenges Fischer and colleagues have developed a number of techniques based on femtosecond pulse shaping that measure n2 with the same inherent crosssectional capabilities as MPEF microscopy, at fast speeds and with high sensitivity [9–12]. These methods have been applied to, for example, monitoring chemically activated neurons [10] and imaging of various biological samples [11–13]. However, these methods are mostly sensitive to the instantaneous nonresonant electronic 0146-9592/14/164788-04$15.00/0
response, which, unlike the other aforementioned nonlinear interactions, has poor molecular specificity. Microscopy with molecular contrast based on a material’s ultrafast time-varying nonlinear phase response, i.e., time-resolved optical Kerr effect (OKE), which contains highly specific molecular information, has not been demonstrated. (The OKE dynamic response describes the time evolution of the optically induced third-order polarization anisotropy, which is highly dependent on a molecule’s structure and surrounding medium [14–16].) Ultimately, current nonlinear phase imaging techniques have shown limited utility due to their lack of molecular specificity. Recently, we showed that OKE dynamics could be ascertained from wavelength-dependent phase changes on a probe beam using a modified pu-pr system with a Michelson interferometer and Fourier-domain (FD) detection [17]. The approach used techniques similar to holography and enabled indirect measure of the OKE dynamics. In this Letter, we improve on our previous work by employing femtosecond pulse shaping for greater phase stability. We further leverage the flexibility of the pulse shaper to design different pulse trains that provide direct access to the OKE dynamics. Finally, we demonstrate imaging of a biological sample with endogenous molecular contrast based on OKE dynamics. The experimental system consists of a pu-pr set up with a regenerative amplifier (RegA, Coherent) laser source seeded by a Ti:sapphire oscillator (Vitesse, Coherent), with a repetition rate of 20 kHz and center wavelength of 808 nm. Eighty percent of the light is sent into an optical parametric amplifier (OPA, Coherent) to produce a pump beam at 690 nm. The other 20% of the RegA output is sent to 4-f pulse shaper [18] to generate two to three time-delayed replicas of the input field (i.e., pulse train). The pulse train and the pump beam are combined using a dichroic mirror, and are then focused onto a sample with a 10×, 0.25 NA microscope objective. All pulses are ∼75 fs, and the power of the pump is © 2014 Optical Society of America
August 15, 2014 / Vol. 39, No. 16 / OPTICS LETTERS
300 μW and that of the two-/three-field pulse train is
OPTICS LETTERS / Vol. 39, No. 16 / August 15, 2014
Femtosecond pulse shaping enables detection of optical Kerr-effect (OKE) dynamics for molecular imaging Francisco E. Robles,* Martin C. Fischer, and Warren S. Warren Department of Chemistry, Duke University, Durham, North Carolina 27708, USA *Corresponding author: [email protected] Received June 5, 2014; accepted June 27, 2014; posted July 14, 2014 (Doc. ID 213533); published August 8, 2014 We apply femtosecond pulse shaping to generate optical pulse trains that directly access a material’s nonlinear refractive index (n2 ) and can thus determine time-resolved optical Kerr-effect (OKE) dynamics. Two types of static pulse trains are discussed: The first uses two identical fields delayed in time, plus a pump field at a different wavelength. Time-resolved OKE dynamics are retrieved by monitoring the phase of the interference pattern produced by the two identical fields in the Fourier-domain (FD) as a function of pump–probe–time–delay (where the probe is one of the two identical fields). The second pulse train uses three fields with equal time delays, but with the center field phase shifted by π∕2. In this pulse scheme, changes on a sample’s nonlinear refractive index produce a new frequency in the FD signal, which in turn yields background-free intensity changes in the conjugate (time) domain and provides superior signal-to-noise ratios. The demonstrated sensitivity improvements enable, for the first time to our knowledge, molecular imaging based on OKE dynamics. © 2014 Optical Society of America OCIS codes: (180.4315) Nonlinear microscopy; (300.6420) Spectroscopy, nonlinear; (120.3180) Interferometry; (170.3880) Medical and biological imaging; (190.3270) Kerr effect. http://dx.doi.org/10.1364/OL.39.004788
Nonlinear optical imaging techniques have proven immensely useful in the field of biology and medicine [1–4]. For example, the inherent cross-sectional ability and high sensitivity of multiphoton excitation fluorescence (MPEF) microscopy has enabled high-resolution imaging of intact biological tissues at unprecedented depths using exogenous agents [2]. Other groups, including ours, have used pump–probe (pu-pr) schemes to gain access to other nonlinear interactions (such as stimulated Raman scattering, excited state absorption, and ground-state bleaching) for highly specific molecular contrast of endogenous species in applications such as melanoma diagnosis [4–6]. In general, most nonlinear imaging techniques rely on interactions that generate new optical frequencies, or that result in attenuation/ amplification of a probe beam. Nonlinear interactions that only result in phase changes [self-phase modulation (SPM), and cross-phase modulation (XPM)] remain largely unexplored in microscopy and are the focus of this Letter. The gold standard for measuring SPM is the Z-scan technique [7]. In this method, a transparent material of interest is scanned along the focus of a femtosecond beam, and transmitted light is collected after passing through a small aperture. Changes in the amount of light passing through the aperture can be related to n2 , with high sensitivity. However, this approach is relatively slow, can only be used to assess bulk n2 properties, and is also rapidly degraded by scattering [8], making it ill-suited for imaging biological samples. To overcome these challenges Fischer and colleagues have developed a number of techniques based on femtosecond pulse shaping that measure n2 with the same inherent crosssectional capabilities as MPEF microscopy, at fast speeds and with high sensitivity [9–12]. These methods have been applied to, for example, monitoring chemically activated neurons [10] and imaging of various biological samples [11–13]. However, these methods are mostly sensitive to the instantaneous nonresonant electronic 0146-9592/14/164788-04$15.00/0
response, which, unlike the other aforementioned nonlinear interactions, has poor molecular specificity. Microscopy with molecular contrast based on a material’s ultrafast time-varying nonlinear phase response, i.e., time-resolved optical Kerr effect (OKE), which contains highly specific molecular information, has not been demonstrated. (The OKE dynamic response describes the time evolution of the optically induced third-order polarization anisotropy, which is highly dependent on a molecule’s structure and surrounding medium [14–16].) Ultimately, current nonlinear phase imaging techniques have shown limited utility due to their lack of molecular specificity. Recently, we showed that OKE dynamics could be ascertained from wavelength-dependent phase changes on a probe beam using a modified pu-pr system with a Michelson interferometer and Fourier-domain (FD) detection [17]. The approach used techniques similar to holography and enabled indirect measure of the OKE dynamics. In this Letter, we improve on our previous work by employing femtosecond pulse shaping for greater phase stability. We further leverage the flexibility of the pulse shaper to design different pulse trains that provide direct access to the OKE dynamics. Finally, we demonstrate imaging of a biological sample with endogenous molecular contrast based on OKE dynamics. The experimental system consists of a pu-pr set up with a regenerative amplifier (RegA, Coherent) laser source seeded by a Ti:sapphire oscillator (Vitesse, Coherent), with a repetition rate of 20 kHz and center wavelength of 808 nm. Eighty percent of the light is sent into an optical parametric amplifier (OPA, Coherent) to produce a pump beam at 690 nm. The other 20% of the RegA output is sent to 4-f pulse shaper [18] to generate two to three time-delayed replicas of the input field (i.e., pulse train). The pulse train and the pump beam are combined using a dichroic mirror, and are then focused onto a sample with a 10×, 0.25 NA microscope objective. All pulses are ∼75 fs, and the power of the pump is © 2014 Optical Society of America
August 15, 2014 / Vol. 39, No. 16 / OPTICS LETTERS
300 μW and that of the two-/three-field pulse train is
