All images were acquired with a custom multimodal NLO platform built at the Laboratory for Biophotonics of the State University of Campinas, which was recently described in detail.11,17 However, for this work the platform was improved by adapting FLIM modality in an available side port of the microscope. Now, the system allows image acquisition as shown in Fig. 1. Briefly, the platform was built around an inverted microscope IX-81, equipped with an Olympus FV300 scanner. All signals were excited with a Mai Tai laser from Spectra-Physics. SHG and THG signals were collected in the transmission mode while TPEF and FLIM were collected in the backscattering mode. SHG and THG images were acquired one after the other due to the optical filters exchange, but simultaneously with the backward TPEF signal detected with FV300 scan head photomultiplier tubes (PMT) with the aperture widely open. A high-pass filter (HP) E-690-HP (Omega Filters) reflected the TPEF and FLIM signal to a fast photon-counting PMT (Becker & Hickl, PMH-100). A time correlated single photon-counting [TSPC] card electronics (Becker & Hickl, SPC-830) detector allowed the direct TPEF image acquisition by direct photocounting, which was later processed to obtain the FLIM images in the time domain. The instrument response function (IRF) of the overall system was . TPEF, SHG, and THG images were acquired with a PLANAPO 40X, N.A. 1.3 oil immersion objective (Olympus, Tokyo, Japan) and excited at 940 nm with a Ti:Sapphire Mai Tai HP Spectra-Physics (Irvine, USA) which provides 100 fs pulses with a repetition rate of 80 MHz and powers from 1 to 3.5 W, generating a SHG signal at 470 nm and a THG signal at 313 nm. All the other parameters such as detector gain, offset, and frame averaging were maintained constant between different samples to enable comparative analysis. Images were acquired with spatial resolution, using a pixel dwell time of 5 ms, with total scanning time of order of 3 s, after a 5 frames Kalman filtering. All ImageJ (NIH, available from http://rsb.info.nih.gov/ij) digital processing were performed on the unprocessed images to avoid artifacts. FLIM data were acquired with the 890 nm excitation (5 mW at the sample), which excites mainly the fluorescence of flavin adenine dinucleotide (FAD), and collected for 60 s with a frame size. Becker & Hickl supply two programs, one for capturing image data and a separate program for displaying a lifetime image. The imaging program (SPCImage Ver. 2.9, Becker & Hickl) determines the best exponential fit to the histograms at each pixel and displays lifetime data utilizing a color-mapping scheme.