A schematic diagram of the experimental setup is shown in Fig. 1. A 720 to 950 nm tunable femto-second laser (pulse-width: 140 fs FWHM, repetition rate: 80 MHz, Chameleon, Coherence Inc., Santa Clara, CA) was used as the excitation source. A computer controlled attenuator including a wave-plate and a polarization beam splitter was installed directly at the beam exit of the laser to control the excitation power. The beam was scanned over tissue samples by the combination of a resonant scanner (CRS8, Cambridge Technology, Lexington, MA) as the fast axis and a galvonometer scanner (, Cambridge Technology, Lexington, MA) as the slow axis. The field of view (FOV) for imaging can be varied from μm2 to μm2. For spectral measurements of smaller region of interest (ROI), the laser beam can be scanned over smaller area down to μm2. The resonant scanner has a scanning frequency of 8 kHz, which enables real-time imaging ( per second).11 The emitted signal was separated from the excitation beam using a dichroic mirror (, Semrock, Lake Forest, Illinois). It was then split by a splitter (NT32-363, Edmund Optics, Barrington, NJ) into an imaging channel and a spectroscopy channel. The beam in the imaging channel was further split using a long pass dichroic (, Semrock, Lake Forest, Illinois) and collected by two photomultiplier tubes (PMT) for TPF and SHG imaging, respectively. Emission spectra are collected by a spectrometer (SpectraPro-150, Roper Scientific, Princeton, NJ) for EEM data accumulation. The exposure time for each emission spectrum was between 2 and 3 s. During this time period, the laser beam had scanned over the ROI for 24 to 36 times. Therefore, each emission spectrum represented the average emission signal (SHG or TPF) from the ROI. A specially designed fiber bundle was applied here to increase the collection area of emitted photons for a high signal-to-noise ratio in the spectral acquisition. The arrangement of fibers within the fiber bundle between the imaging system and the spectrometer has two different patterns (Fig. 1, inset a and inset b). The fiber bundle has 90 small fibers [single fiber core diameter: 100 μm; numerical aperture (NA): 0.12] arranged in a hexagon pattern at the input end to provide a much larger collection area than a single fiber. The output end (Fig. 1, inset b) has all of the 90 fibers arranged as 2 straight lines with 45 fibers in each line so that most of the collected light can be coupled into the narrow entrance slit of the spectrometer. The width of this line-shape bundle of fibers is 200 μm, leading to a spectral resolution of 4 nm. The -number () of the spectrograph system () has been matched with the NA of the fiber (0.12) as: for optimum performance. (Fig. 1).