Figure 1 shows a schematic of our multimodal multiphoton nonlinear optical microspectroscopy imaging system based on a small GRIN lens probe. This multimodal multiphoton microendoscope imaging modality was modified and developed from a previously described system.23–25 The laser system used a home-made Ti:sapphire femtosecond oscillator pumped by a green laser source (Verdi V5, Coherent). The output beam from the oscillator generates a 120 fs, 56.1 MHz pulse train, and a 460 mW average output power with a center wavelength of 800 nm. Our laser system is described in greater detail in our previous paper.25 The laser beam passed through an optical isolator (IO-5-NIR-LP, Thorlabs) and was divided into two beams (3:1) by a beam splitter (BP133, Thorlabs). The reflected beam was transmitted via a series of optical components for spatial filtering, and a “pump beam” in a narrow band for the CARS process, with a pulse of 0.8 nm and center wavelength of 800 nm, was generated by combining two filters (LL01-808-12.5, Semrock and custom-made filter from Andover Corporation). The transmitted pulse, in contrast, was coupled into the photonic crystal fiber (FemtoWHITE 800, NKT Photonics) via an objective lens to make ultrabroadband “Stokes pulses.” Both the narrowband pump and broadband Stokes pulses were spatially and temporally overlapped by a long-wavelength pass filter (BLP01-785R-25, Semrock), and the two beams were passed through galvanometric mirrors and microendoscope. We demonstrated multiphoton microendoscopy by using a GRIN lens [GT-MO-080-018-810, 0.8 numerical aperture (NA), , WD: , GRINTECH], which was fixed under the microscope by a clamp (VK250, Micro V-Clamps, Thorlabs) attached to a three-axis stage. The objective lens (MPlanFLN, 0.3 NA, , Olympus) was used to inject light into the GRIN lens, and the total magnification of the microendoscope was . We used a comparable microscope objective lens (CFI Plan Fluor, 0.8 NA, , W, Nikon) to compare optical images obtained by an objective lens and endoscopy. The backwards-scattered nonlinear signals were collected by an epi-detection configuration. They were separated from the excitation beams by a dichroic mirror (custom-made mirror from CVI) and passed through the short-pass filter (FF01-775/SP-25, Semrock). The CARS, TPEF, and SHG signals were spectrally separated by dichroic mirrors and bandpass filters (CARS: HQ650/20 m, Chroma, TPEF: FF01-550/88, SHG: FF01-390/18, Semrock) to obtain multimodal nonlinear images. All of their signals were simultaneously recorded by two different photomultiplier tubes, one for CARS and the other for TPEF and SHG (H-8249-102, H-7827-012, Hamamatsu). A monochromator (DM500i, DongWoo) was used to obtain the CARS spectrum, and a CCD camera (iDus DV401A-BV, Andor) having an effective spectral resolution of was used to record it. To convert the signals to nonlinear optical images, a data acquisition program written in LabVIEW 8.6 was used. Typically, powers of 20 mW for the pump and 3 mW for the Stokes pulses were used at the sample for imaging.