Multi-Color Two-Photon Microscopic Imaging Based on a Single-Wavelength Excitation
Wei Yan,
Yangrui Huang,
Luwei Wang,
Jin Li,
Yong Guo,
Zhigang Yang,
Junle Qu
Affiliations
Wei Yan
Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China
Yangrui Huang
Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China
Luwei Wang
Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China
Jin Li
Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China
Yong Guo
Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China
Zhigang Yang
Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China
Junle Qu
Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China
Two-photon probes with broad absorption spectra are beneficial for multi-color two-photon microscopy imaging, which is one of the most powerful tools to study the dynamic processes of living cells. To achieve multi-color two-photon imaging, multiple lasers and detectors are usually required for excitation and signal collection, respectively. However, one makes the imaging system more complicated and costly. Here, we demonstrate a multi-color two-photon imaging method with a single-wavelength excitation by using a signal separation strategy. The method can effectively solve the problem of spectral crosstalk by selecting a suitable filter combination and applying image subtraction. The experimental results show that the two-color and three-color two-photon imaging are achieved with a single femtosecond laser. Furthermore, this method can also be combined with multi-photon imaging technology to reveal more information and interaction in thick biological tissues.
