I. Introduction

Over the past decade, coherent anti-Stokes Raman scattering (CARS) microscopy has developed into a powerful label-free nonlinear optical imaging technique.1 With its intrinsic vibra- tional contrast, great sensitivity, and three-dimensional section- ing ability, CARS microscopy has become an important label- free chemical imaging tool in biology and material sciences.1-3 Most CARS microscopy techniques demonstrated so far have employed two synchronized picosecond laser pulses. Since vibrational response of typical organic molecules lasts a few picoseconds, laser pulses with similar time duration are optimal in the consideration of both spectral resolution and signal sensitivity.1,4,5 The use of picosecond laser pulses also reduces the nonresonant background and nonlinear photodamages sig- nificantly.4 This configuration of CARS microscopy, which we will call “narrow CARS” method from now on, has proven to be an excellent imaging tool for visualizing lipid-rich structures in cells and tissues when it detects CARS signals at 2840 cm-1.1

The vibrational fingerprint region (800-1800 cm-1) is an important frequency range where many molecular functional groups have unique vibrational resonances.6 In this frequency region, however, the vibrational spectrum is often crowded with multiple peaks, and a single vibrational frequency peak is not enough to identify local chemical structures in many situations.7-10 In addition, the ubiquitous nonresonant background interferes with relatively weak vibrationally resonant CARS signals to distort the frequency response of measured signals significantly at this frequency region. In the past decade, there have been numerous CARS methods developed to remove the effect of nonresonant background and extract vibrational CARS signals.1,11-15 Among them, the fast frequency modulation (FM) CARS technique has proven to be an effective way to eliminate the nonresonant background. It relies on the different spectral shapes of the resonant CARS signals and nonresonant backgrounds.13,15 The line width of vibrational peaks in the fingerprint region is typically 5-20 cm-1, while the nonresonant background is spectrally flat due to the instantaneous time response of an off- resonant electronic four-wave-mixing process. If one can

* Corresponding author. E-mail: shlim@mail.utexas.edu.

measure the difference between CARS signals at two vibrational excitation frequencies, the contribution of the nonresonant background in the measured signals can be removed, since the strength of the nonresonant background should not change with respect to the excitation frequency (i.e., the frequency difference between the pump and Stokes pulses). This FM-CARS technique has been demonstrated with narrow-band picosecond lasers by combination of fast frequency modulation of one laser and lock- in signal detection.13 In beam-scanning microscopy, sophisti- cated laser systems were required to perform the necessary fast frequency modulation, for example, the one with a novel optical parametric oscillator that can switch the output frequency at tens of megahertz.16

Recently, new CARS microscopy methods have emerged based on the so-called “spectral focusing” mechanism.14,15,17-21 These methods can excite a single vibrational level with high spectral resolution by “chirped broad-band” laser pulse pairs (pump and Stokes pulses) and the excitation vibrational frequency can be switched by the time delay between the pulse pair.22 Cell and tissue imaging with these methods have already been demonstrated.15,20,23 Since the vibrational excitation fre- quency can be modulated by the time delay (not by the actual frequency of the laser) in this method, the FM technique can be adopted in a relatively simple and low-cost setup. A FM spectral focusing CARS method with passive polarization optics has been demonstrated very recently.14,15

In this publication, we introduce a new active version of the FM spectral focusing CARS method that works in the vibrational fingerprint region. Our method allows the use of a laser with a high peak power and lower repetition rate (∼10 nJ at 2 MHz), and we can obtain vibrational images of good quality at the fingerprint region. We show that our FM technique not only removes the nonresonant background but also generates Raman- like CARS signals. It can perform both background-free CARS imaging and microspectroscopy in real time. We demonstrate its utility with various samples including edible oils, lipid membranes, skin tissues, and plant cell walls.

This paper is organized as follows. In section II, we present the time-domain picture of spectral focusing mechanism and explain fundamental aspects of this method.

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