Linear Compressible Continuum Generation For Multicolor Two-Photon Excited Fluorescence Microscopy

Synchronous monitoring dynamic characteristics and interactions of various cells in the microenvironment of intravital tissue, is important for several biological research, such as the evaluation of immunotherapy, drug efficacy in vivo. Two-photon excited fluorescence (TPEF) microscopy provides spectacular insights into visualization of cellular events within intravital tissue due to advantages of an inherent sectioning ability, the relatively deep optical penetration, and low optical damage. Thus, multicolor TPEF imaging provides excellent dynamic tracking ability of variety of cells and substance in the microenvironment of intravital tissue. Currently, the spectral band of100-fs Ti:Sapphire oscillators canot optimally excite multicolor fluorophores, which limits its applications in intravital research. Ideally, continuum pulses covering peak excitation wavelengths of the majority of fluorophores are desirable for multicolor TPEF microscopy. Due to advantages of simple and versatile add-on to an existing Ti:Sapphire oscillator, continuum generation in the highly nonlinear photonic crystal fibers (HNPCFs) allows broadband pulses for multicolor TPEF microscopy. However, current broadband fiber continuums canot be linearly compressed and their pulse durations are conventionally spread to several picoseconds, resulting in low signal level for multicolor TPEF imaging.To achieve simultaneous and effective excitation of multiple fluorophores for multicolor TPEF imaging, we devote to solving the key problem of "how to generate a linear compressible broadband continuum based on the100-fs pulses propagation in HNPCF". By formulating the propagation properties of fs pulses in nonlinear fiber-optic microscope, the following research work has been done:Firstly, how parameters of fibers, input pulses, and gratings influencing the optimum performance of the compressor for enhanced TPEF imaging have been investigated. Effects of the ZDWs, the length, and the transmitted power of the HNPCF on the spectral broadening, dispersion compensation, and signal level in the TPEF microscope are fully investigated. Both theoretical and experimental results show that the PCF having a ZDW around800-900nm and a fiber length of20-30mm is most suitable for pulse compression at given input pulses of-200mW,100fs at700-850nm. We then combine the compressor with a standard TPEF microscope. Pulse evolution and TEPF imaging in the compressor-based system have been characterized to demonstrate the application of such a compressor in enhanced TPEF imaging.Then, we enhance SPM effect to get a broadband linear compressible continuum for multicolor TPEF microscopy. After theoretically and experimentally characterizing the pulse evolution of100-fs pulses in the blue shifted ZDW HNPCFs, we found that the low dispersion by pumping in the vicinity of fiber ZDW delays the occurrence of soliton fission and enhances the SPM-dominated spectral broadening in a longer fiber length. A700-900nm continuum is compressed down to57fs by a grating pair. The signal enhancement of the compressed continuum has been demonstrated in3-color TPEF imaging.To futher enhance the TPEF excitation efficiency and achieve selective excitation with fiber continuum, we also investigate the coherent control of fiber continuum. The key for coherent control is how to accurately introduce a phase function. We calibrated a pulse shaper and got a preliminary results of measuring and compensating pulse phase distortion.