Development of line-scanning two-photon microscopy based on spatial and temporal focusing for autofluorescence imaging

Abstract

Two-photon microscopy(TPM) is noninvasive optical sectioning fluorescence microscopy based on nonlinear excitation. Thus, it is good for in vivo tissue imaging with minimal photo-damage and high imaging depth. Line-scanning TPM is one of high-speed TPM and it is based on spatial and temporal focusing. It makes depth-resolved excitation by changing the width of excitation laser pulse, so depth resolution is maintained even with line-scanning.TPM imaging is possible based on intrinsic fluorephores such as NAD(P)H, Riboflavin, Pyridoxine, Folic acid and collagen(SHG) molecules, but autofluorescence level is very small to obtain images. Also, it needs too much time to make autofluorescence images. For overcoming this problem, deep-ultraviolet(UV) microscope excite the intrinsic fluorophore in the UV spectral region where tryptophan is the major fluorophore which is the predominant fluorophore in tissue and emit strong fluorescence.In the thesis, we developed line-scanning TPM based on spatial and temporal focusing for autofluorescence imaging by exciting the tryptophan. Laser source for this system is optical parametric oscillator(OPO) and it makes near 570nm femtosecond pulse laser. It has 200fs pulse width and 1.72nm bandwidth, so we can predict achievable depth resolution is 2.41μm and field of view(FOV) is 10.8μm. For comparing the depth resolution, we obtain the full-width half maximum(FWHM) by imaging the blue microsphere(1μm, 365/415) and FOV by imaging the L-tryptophan(5mM) solution. From the characterization, our system has 3.0μm depth resolution and 12.3μm FOV.Developed line-scanning two-photon microscopy is applied to obtain high-speed two-photon excitation fluorescence images of fixed lymphocyte without extrinsic fluorophore. Although our system has lower resolution than point-scanning TPM, it could be improved by using multiple prism compressor. Thus, it is potentially applicable for monitoring inflammatory celluar reactions in humans.