The Provenzano laboratory is both an engineering and (quantitative) biology laboratory, equipped for robust equipment for cell and molecular biology work as well as engineering testing and analysis.  In addition, the Provenzano lab has a custom-built multi-photon laser scanning microscope (Prairie Technologies / Bruker) that is equipped with a Mai Tai laser that produces 100fs pulse widths at an 80 MHz repetition rate to simultaneously generate multiphoton excitation (MPE) and second harmonic generation (SHG) at wavelengths between 690-1040nm.  This system is equipped from fluorescence lifetime imaging micrsocopy (FLIM) and phosphorescence lifetime imaging microscopy (PLIM) detection, photoactivation controls and a specially designed animal stage for rapid movement and intravital imaging experiments.  Multiphoton excitation (MPE) of fluorescence and second harmonic generation (SHG) are optical sectioning techniques that use ultra-short pulses t<100fs of very high repetition rate (f =80 MHz) of long wavelength light (near infrared, ~700-1040nm) such that two or more photons are absorbed to excite a fluorophore (i.e. MPE) or undergo wave mixing (i.e. SHG) and permit much deeper and less toxic tissue imaging than single photon confocal microscopies. In conjunction with the inherent optical sectioning of 2-photon events, less scattering in tissue, and a non-descanned detection schema, we rapidly acquire signals within tumors with high SNR with galvo or resonant scanning.  Piezo controlled z-scanning in conjunction with resonant scanning enables ultrafast z-stack acquisition.  This system was controlled using PrairieView software and MATLAB interfaces and the light focused on the sample using a universal M plan fluorite infinity corrected near-infrared 40X objective (NA=0.8) or 20X-60X near infrared lenses with NAs between 0.8-1.  Emission is simultaneously detected in 4-Channels such as the blue, green, red and far-red channels (though bandpass filters at either 440/20, 460/50, 525/50, 525/70, 595/50, 605/70, or 690/50; note filters are easily interchangeable providing robust flexibility in the detection) with either multi-Alkali (Hamamatsu) or GaAsP (Hamamatsu model 7422PA-40) PMTs.  For FLIM, mulitphoton FLIM is performed on this system using the GaAsP PMT connected to a time correlated single photon counting (TCSPC) system (Becker & Hickl).  The IRF was measured using urea crystal to generate second harmonic signal. Each image of 256x256 or 512x512 is acquired using an integration time of 60 to 180s, with stable photon count rates during image acquisition to confirm that photobleaching did not occur.  The photon counts from each pixel are used to determine fluorescence lifetime after application of a deconvolution algorithm of the data and a known or estimated instrument response function. Analysis is performed with SPCImage (Becker & Hickl) with fluorescence decay modeled as a single- or multi-exponential with determined by the minimized Chivalue.

Optical Setup
Multiphoton Microscopy