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Experimental study on multicolor two-photon excited fluorescence microscopy

Qiu Jun-Peng Liang Run-Fu Peng Xiao Li Ya-Hui Liu Li-Xin Yin Jun Qu Jun-Le Niu Han-Ben

Experimental study on multicolor two-photon excited fluorescence microscopy

Qiu Jun-Peng, Liang Run-Fu, Peng Xiao, Li Ya-Hui, Liu Li-Xin, Yin Jun, Qu Jun-Le, Niu Han-Ben
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  • Abstract views:  864
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Publishing process
  • Received Date:  22 August 2014
  • Accepted Date:  26 September 2014
  • Published Online:  05 February 2015

Experimental study on multicolor two-photon excited fluorescence microscopy

  • 1. Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China;
  • 2. School of Physics and Optoelectronic Engineering, Xidian University, Xi'an 710071, China
Fund Project:  Project supported by the National Basic Research Program of China (Grant No. 2012CB825802), the Special Funds of the Major Scientific Instruments Equipment Development of China (Grant No. 2012YQ150092), the Key Program of the National Natural Science Foundation of China (Grant No. 61235012), the National Natural Science Foundation of China (Grant Nos. 11204226, 61378091), the Natural Science Foundation of Shaanxi Province, China (Grant No. 2014JM8324), and the Fundamental Research Funds for the Central Universities, China (Grant Nos. K5051305002, NSIY051405).

Abstract: Two-photon excited fluorescence (TPEF) microscopy is a nonlinear optical microscopy technique. The advantages of TPEF microscopy include high temporal and spatial resolutions, high signal-to-noise ratio and inherent three-dimensional sectioning. In traditional TPEF microscopy, a wavelength tunable ultrashort pulsed laser is used as an excitation source. In practical applications, sample usually contains various fluorophores or unknown components. Therefore the excitation wavelength of the ultrafast laser has to be tuned to achieve optimal excitation efficiencies of various fluorophores. In order to acquire the fluorescent signals of different fluorophores simultaneously, we develop a multicolor TPEF microscope system based on a supercontinuum laser source. In experiments, TPEF images of Lily rhizome sample slide stained by two fluorescent dyes with different excitation and emission wavelengths are obtained without tuning the wavelength. Experimental results show that the high-contrast TPEF images of the sample with various fluorophores can be obtained simultaneously by using the multicolor TPEF microscope compared with by using traditional TPEF microscopy. The system is simple in structure, easy in operation, and can provide rich information about the sample, which allows it to be widely used in life and material sciences.

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