**5.2 Phonon depletion CARS microscopy[132]**

As discussed above, successful methods of breaking through the theoretical diffraction limitation of CARS microscopy depend on the controllable emissive properties of the useful signals in the focus. But the above suggested methods for breaking through the diffraction limited resolution can only be used for dealing with the single bond signal. By researching the CARS process with quantum optics theory, we presented our method for breaking through the diffraction limitation, unlike the above methods, which is effective for ultrabroadband T-CARS microscopy.

In our theoretical model, all incident laser fields, generated signal field and the material system are all described with quantum mechanics theory. In the CARS process, the first light-matter

Ultra-Broadband Time-Resolved Coherent Anti-Stokes Raman Scattering

biology, medicine and life science in the near future.

Science, 1998, 280(5371): 1954-1955.

San Diego, CA: Academic, 1989.

Orlando, FL: Academic, 1984.

San Diego, CA: Academic, 1999.

tissue, Biophys. J, 2002, 82(1): 493-508.

93: 10763-10768.

1986, 11(2): 94-96.

1211.

microscopy, Science, 1990, 248(4951): 73-76.

surface microprobe, Opt. Lett., 1986, 11(2): 97-99.

Molecular Probes, 1996.

**7. References** 

Spectroscopy and Microscopy with Photonic Crystal Fiber Generated Supercontinuum 199

describe the mechanisms of Raman scattering and T-CARS process with the classical and quantum mechanical theory. The CARS signals with much stronger strength and welloriented direction originate from the coherent resonant enhancement between incident lights and molecular vibrations. In order to quickly and accurately distinguish different kinds of molecules in a complex, such as in a live cell, a method for simultaneously detecting the ultra-broadband CARS signals without the NRB noise has to be developed. On the basis of theoretical analysis and simulation of the SC generation with a PCF, a satisfied SC source can be achieved for obtaining the ultra-broadband even complete CARS spectra of the specimen by optimizing the parameters and the other experimental conditions of the PCF and ultra-short laser. At the same time, the NRB noise can be effectively suppressed in a broad spectral range with the time-resolved method. The method study for obtaining subdiffraction limited spatial resolution is still on the stage of theoretical research. Some original techniques are presented in this chapter. The PD-CARS technique provides a possible route to the realization of ultra-broadband T-CARS microscopy with sub-diffraction limited spatial resolution, which will probably become an attractive imaging method in

[1] R P Haugland, Handbook of Fluorescent Probes and Research Chemicals, Eugene, OR:

[2] R Y Tsien and A Miyawaki, Biochemical imaging: Seeing the Machinery of Live Cells,

[3] E Kohen and J G Hirschberg, Cell structure and function by microspectrofluorometry,

[6] W Denk, J H Strickler and W W Webb, Two-photon laser scanning fluorescence

[7] Xu C, W Zipfel, J B Shear, et al, Multiphoton fluorescence excitation: new spectral

[8] J Hoyland, Fluorescent and Luminescent Probes for Biological Activity, ed. W T Mason.

[9] G T Boyd, Y R Shen and T Hansch, Continuous-wave second-harmonic generation as a

[10] U Gauderon, P B Lukins, and C J R Sheppard, Three-dimensional second-harmonic

[11] J C Paul, C M Andrew, T Mark, et al, Three-dimensional high-resolution second-

[12] I Freund and M Deutsch, Second-harmonic microscopy of biological tissue, Opt. Lett.,

[13] R M Williams, W R Zipfel and W W Webb. Interpreting second-harmonic generation

imaes of collagen I fibers, Biophys. J., 2005, 88:1377-1386.

windows for biological nonlinear microscopy, Proc. Natl. Acad. Sci. U.S.A., 1996,

generation imaging with femtosecond laser pulses, Opt. Lett., 1998, 23(15): 1209-

harmonic generation imaging of endogenous structural proteins in biological

[4] J B Pawley, Handbook of Biological Confocal Microscopy, New York: Plenum, 1995. [5] T Wilson and C Sheppard, Theory and Practice of Scanning Optical Microscopy,

interaction process involves resonant enhancements of all active molecular vibrational modes, in which the frequency differences of pump and Stokes fields equal the inherent vibrational frequencies of the molecular bonds respectively. The resonantly enhanced molecular vibrations exist in quantized forms which are called the phonons. Their numbers are equal to the numbers of generated Stokes photons respectively. When a probe field propagates through the matter, the photons of the probe interact with the generated phonons. The photons with anti-Stokes frequencies are generated, and phonons are annihilated at the same time.

Based on the whole quantized picture of the CARS process, we presented a phonon depletion CARS (PD-CARS) technique by introducing an additional probe beam with the frequency different from the one of the probe beam in the center of the focus. When the pump and Stokes simultaneously reaches the focus, the phonons are generated. The additional probe beam, which is shaped into a doughnut profile at the focus with a phase mask, reaches the focus a little bit earlier than the probe beam in the center of the focus. Therefore the wavelengths of the generated anti-Stokes signals at the peripheral region differ from the ones at the center of the focus and can be easily separated with a proper interference filter. By this way, the spatial resolution of the ultra-broadband T-CARS microscopy can be improved greatly. The simulation result of PSF is defined as [132]:

$$
\Delta r = \sqrt{2} \frac{\lambda}{\pi n \sin \alpha} \frac{\lambda}{\sqrt{3 + \frac{I\_{p\_1}^{\text{max}}}{I\_{dep}}}} \approx \frac{0.9}{\sqrt{3 + \frac{I\_{p\_1}^{\text{max}}}{I\_{dep}}}} \cdot \frac{\lambda}{2n \sin \alpha} \,\tag{5.2}
$$

where Idep and max *<sup>P</sup>*<sup>1</sup> *I* are intensities of phonon field at the center of the focus and additional probe field for phonon depletion in the annual region respectively. From equation (5.2), we can know that the spatial resolution of CARS microscopy will be improved by increasing the intensity of additional probe beam. The simulation result of PSF is shown in figure 22. When max *<sup>P</sup>*<sup>1</sup> *I* is fiftyfold of Idet, the spatial resolution of the ultra-broadband T-CARS microscopy reaches 41nm.

Fig. 22. Simulation result of the PSF in the PD-CARS microscopy [132].

#### **6. Conclusions and prospects**

In this chapter, we mainly introduce a kind of noninvasive label-free imaging technique – the ultra-broadband T-CARS spectroscopy and microscopy with SC generated by PCF. We describe the mechanisms of Raman scattering and T-CARS process with the classical and quantum mechanical theory. The CARS signals with much stronger strength and welloriented direction originate from the coherent resonant enhancement between incident lights and molecular vibrations. In order to quickly and accurately distinguish different kinds of molecules in a complex, such as in a live cell, a method for simultaneously detecting the ultra-broadband CARS signals without the NRB noise has to be developed. On the basis of theoretical analysis and simulation of the SC generation with a PCF, a satisfied SC source can be achieved for obtaining the ultra-broadband even complete CARS spectra of the specimen by optimizing the parameters and the other experimental conditions of the PCF and ultra-short laser. At the same time, the NRB noise can be effectively suppressed in a broad spectral range with the time-resolved method. The method study for obtaining subdiffraction limited spatial resolution is still on the stage of theoretical research. Some original techniques are presented in this chapter. The PD-CARS technique provides a possible route to the realization of ultra-broadband T-CARS microscopy with sub-diffraction limited spatial resolution, which will probably become an attractive imaging method in biology, medicine and life science in the near future.
