Nonlinear optical imaging was performed using a modified system based on a commercial microscope (Fluoview 1000, Olympus, Japan), as described previously.29 It is reported that the two-photon absorption of the major intrinsic fluorophores such as NADH and FAD decreases as the excitation wavelength increases in the range of 700 to 900 nm.21 We tuned the excitation wavelength in the 730 to 890 nm range and found that 750 nm was the optimal wavelength to give the best signal-to-noise ratio in our microscopic system. A tunable Ti:Sapphire laser (Mai Tai, Spectra-Physics) operating at 750 nm was used for excitation, except where otherwise noted. A water-immersion objective ( water-immersion, Olympus) was employed for focusing the excitation beam into tissue samples and collecting the backscattered intrinsic nonlinear optical signals. The average laser power on the surface of the sample was about 15 mW. The scanning rate was . The emission signals are detected by three channels in the nondescanned mode. A narrow bandpass filter (FF01-380/14-25, Semrock) was used for the detection of SHG signals (; blue). Another two emission filters (FF01-445/45-25 and ; Semrock) were used to extract the intrinsic TPEF signals for the differentiation of acini, PSCs, and other nonacinar cells. The TPEF signals were detected with the same system parameters, including the voltage, gain, and offset of the photomultiplier tubes. The nonlinear optical signals were presented by the pseudocolor images. Blue, green, and red color-coded images correspond to SHG signals, TPEF signals between 423 and 467 nm, and TPEF signals between 509 and 551 nm, respectively.