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Experimental study on two-photon fluorescence and coherent anti-Stokes Raman scattering microscopy

## Experimental study on two-photon fluorescence and coherent anti-Stokes Raman scattering microscopy

Hou Guo-Hui, Luo Teng, Chen Bing-Ling, Liu Jie, Lin Zi-Yang, Chen Dan-Ni, Qu Jun-Le
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• #### Abstract

Two-photon excitation fluorescence (2PEF) and coherent anti-Stokes Raman scattering (CARS) are both third-order nonlinear optical processes, but for a long time, the true relationship and differences between them are not clearly understood. For decades, the second harmonic generation has been studied in conjunction with two-photon excitation fluorescence, so it was thought that the latter was a second-order nonlinear optical process. In order to make the two nonlinear interaction processes clear enough, the two nonlinear interaction processes are worthy to study at the same time. In this paper, firstly, we give the relationships between the 2PEF, CARS signal and their third-order nonlinear susceptibility, respectively; secondly, we use our own near infrared super-continuum CARS microscopy system to study both processes. In doing so, we describe the relationship between their third-order nonlinear susceptibility and the signal. The reconstructed images derived from CARS and those derived from 2PEF differ significantly when imaging the same 1.01 $\muup$m fluorescence polystyrene beads. If the lateral spatial resolution of the CARS imaging system is larger than the fluorescence polystyrene beads, the measured size cannot be used to calculate the real spatial resolution of the CARS system. However, the resolution of the 2PEF microscopy system can be obtained through the de-convolution of the 2PEF image, which is approximately equivalent to the current resolution of the CARS imaging system, which is measured using 280 nm polystyrene beads. The images of 280 nm polystyrene beads and 190 nm fluorescent polystyrene beads also exhibit differences between the two samples and the environment around them, respectively. This means that although CARS and 2PEF are both third-order nonlinear optical processes, they have their own properties. In particular, CARS is a third-order nonlinear optical oscillation process which is caused by the phasing match condition, but 2PEF is not influenced by the phasing match condition. The phase matching condition is responsible for the differences around the sample in the images of the 280 nm pure polystyrene beads, but not for the 190 nm fluorescent polystyrene beads. The de-convolution results for the 1.01 $\muup$m fluorescence polystyrene beads and the 280 nm pure polystyrene beads are very similar, so we can use the de-convolution results for 2PEF by the 1.01 $\muup$m fluorescence polystyrene beads to approximate the current measure condition and the resolution of the CARS imaging system. If we want to gain a more accurate resolution from the CARS imaging system, the spherical sample should be smaller than the lateral spatial resolution of this system.

#### Authors and contacts

###### Corresponding author: Chen Dan-Ni, danny@szu.edu.cn;jlqu@szu.edu.cn ; Qu Jun-Le, danny@szu.edu.cn;jlqu@szu.edu.cn
• Funds: Project supported by the Special Funds of the Major Scientific Instruments Equipment Development of China (Grant No. 2012YQ15009203), the National Natural Science Foundation of China (Grant No. 61235012), and the National Basic Research Program of China (Grant Nos. 2015CB352005, 2012CB825802).

#### References

 [1] Potma E O, Xie X S N 2008 Handbook of Biomedical Nonlinear Optical Microscopy (New York: Oxford University Press) pp412-435 [2] Potma E O, Xie X S N 2008 Handbook of Biomedical Nonlinear Optical Microscopy (New York: Oxford University Press) pp164-186 [3] Zhang D, Slipchenko M N, Cheng J X 2011 Phys. Chem. Lett. 2 1248 [4] Nestor J R 1978 Chem. Phys. 69 1778 [5] Göeppert-Mayer M 1931 Ann. Phys. 9 273 [6] So P T C, Dong C Y, Masters B R, Berland K M 2000 Ann. Rev. BioMed. Eng. 2 399 [7] Song J J, Eesley G L, Levenson M D 1976 Appl. Phys. Lett. 29 567 [8] Lotem H, Lynch R T J, Bloembergen N 1976 Phy. Rev. A 14 1748 [9] Oudar J L, Smith R W, Shen Y R 1979 Appl. Phys. Lett. 34 758 [10] Lee Y J, Cicerone M T 2008 Appl. Phys. Lett. 92 15 [11] Isobe K, Kawano H, Takeda T, Suda A, Kumagai A, Mizuno H, Miyawaki A, Midorikawa K 2012 Biomed. Opt. Express 3 1594 [12] Cheng J X, Volkmer A, Xie X S 2002 Opt. Soc. Am. B 19 1363

#### Cited By

•  [1] Potma E O, Xie X S N 2008 Handbook of Biomedical Nonlinear Optical Microscopy (New York: Oxford University Press) pp412-435 [2] Potma E O, Xie X S N 2008 Handbook of Biomedical Nonlinear Optical Microscopy (New York: Oxford University Press) pp164-186 [3] Zhang D, Slipchenko M N, Cheng J X 2011 Phys. Chem. Lett. 2 1248 [4] Nestor J R 1978 Chem. Phys. 69 1778 [5] Göeppert-Mayer M 1931 Ann. Phys. 9 273 [6] So P T C, Dong C Y, Masters B R, Berland K M 2000 Ann. Rev. BioMed. Eng. 2 399 [7] Song J J, Eesley G L, Levenson M D 1976 Appl. Phys. Lett. 29 567 [8] Lotem H, Lynch R T J, Bloembergen N 1976 Phy. Rev. A 14 1748 [9] Oudar J L, Smith R W, Shen Y R 1979 Appl. Phys. Lett. 34 758 [10] Lee Y J, Cicerone M T 2008 Appl. Phys. Lett. 92 15 [11] Isobe K, Kawano H, Takeda T, Suda A, Kumagai A, Mizuno H, Miyawaki A, Midorikawa K 2012 Biomed. Opt. Express 3 1594 [12] Cheng J X, Volkmer A, Xie X S 2002 Opt. Soc. Am. B 19 1363
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•  Citation:
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• Abstract views:  1167
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##### Publishing process
• Received Date:  12 January 2017
• Accepted Date:  13 March 2017
• Published Online:  05 May 2017

## Experimental study on two-photon fluorescence and coherent anti-Stokes Raman scattering microscopy

###### Corresponding author: Qu Jun-Le, danny@szu.edu.cn;jlqu@szu.edu.cn
• 1. Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China
Fund Project:  Project supported by the Special Funds of the Major Scientific Instruments Equipment Development of China (Grant No. 2012YQ15009203), the National Natural Science Foundation of China (Grant No. 61235012), and the National Basic Research Program of China (Grant Nos. 2015CB352005, 2012CB825802).

Abstract: Two-photon excitation fluorescence (2PEF) and coherent anti-Stokes Raman scattering (CARS) are both third-order nonlinear optical processes, but for a long time, the true relationship and differences between them are not clearly understood. For decades, the second harmonic generation has been studied in conjunction with two-photon excitation fluorescence, so it was thought that the latter was a second-order nonlinear optical process. In order to make the two nonlinear interaction processes clear enough, the two nonlinear interaction processes are worthy to study at the same time. In this paper, firstly, we give the relationships between the 2PEF, CARS signal and their third-order nonlinear susceptibility, respectively; secondly, we use our own near infrared super-continuum CARS microscopy system to study both processes. In doing so, we describe the relationship between their third-order nonlinear susceptibility and the signal. The reconstructed images derived from CARS and those derived from 2PEF differ significantly when imaging the same 1.01 $\muup$m fluorescence polystyrene beads. If the lateral spatial resolution of the CARS imaging system is larger than the fluorescence polystyrene beads, the measured size cannot be used to calculate the real spatial resolution of the CARS system. However, the resolution of the 2PEF microscopy system can be obtained through the de-convolution of the 2PEF image, which is approximately equivalent to the current resolution of the CARS imaging system, which is measured using 280 nm polystyrene beads. The images of 280 nm polystyrene beads and 190 nm fluorescent polystyrene beads also exhibit differences between the two samples and the environment around them, respectively. This means that although CARS and 2PEF are both third-order nonlinear optical processes, they have their own properties. In particular, CARS is a third-order nonlinear optical oscillation process which is caused by the phasing match condition, but 2PEF is not influenced by the phasing match condition. The phase matching condition is responsible for the differences around the sample in the images of the 280 nm pure polystyrene beads, but not for the 190 nm fluorescent polystyrene beads. The de-convolution results for the 1.01 $\muup$m fluorescence polystyrene beads and the 280 nm pure polystyrene beads are very similar, so we can use the de-convolution results for 2PEF by the 1.01 $\muup$m fluorescence polystyrene beads to approximate the current measure condition and the resolution of the CARS imaging system. If we want to gain a more accurate resolution from the CARS imaging system, the spherical sample should be smaller than the lateral spatial resolution of this system.

Reference (12)

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