The customized two-photon microscope (
Fig. 1B) uses a femtosecond laser (Chameleon-XR; Coherent Inc., Santa Clara, CA) and a scanning system (modified Tissue Surgeon; Rowiak GmbH, Hannover, Germany), which is coupled to the laser port of an inverted microscope (Axio Observer; Carl Zeiss, Jena, Germany) with a long-distance water-immersion objective (LD C-Apochromat 40×, NA 1.1; Carl Zeiss Meditec, Jena, Germany). The laser was tuned to a central wavelength of 800 nm for SHG and to 750 nm for the two-photon excitation of NAD(P)H. The fluorescent blue light and the backward SHG at 400 nm were separated from laser light through a dichroic mirror (HC 670/SP; Semrock, Rochester, NY) and a laser blocker (HC 680/SP; Semrock) and color filtered with a blue bandpass filter (BrightLine HC 390/40; Semrock). Finally the filtered light was collected onto a photomultiplier tube (
Fig. 1B, PMT 1; R6357; Hamamatsu Photonics, Herrsching, Germany). Forward SHG light was collected by a plano convex lens and filtered and detected by an equivalent setup (
Fig. 1B; PMT 2). The
z-coordinate of the slices was addressed with a piezo drive for the objective (nanoMIPOS 400; Piezosystem Jena GmbH, Jena, Germany). Mosaicing of larger images was performed via motorized
x–y translation of the microscope stage (Proscan H117; Prior Scientific Instruments GmbH, Jena, Germany). Automatic control and data acquisition of the microscope were programmed in commercial software (LabVIEW; National Instruments Germany GmbH, Munich, Germany). Images were processed offline with ImageJ (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at
http://rsb.info.nih.gov/ij/index.html), and ray-casting was performed with Voreen software.
39 In addition to the multiphoton microscope, an fs-CLSM channel (PMT 3 in
Fig. 1B) collected the descanned reflected fs-laser light, which was picked with an 8% reflective beamsplitter (BP108; Thorlabs, Dachau, Germany) and focused on a confocal pinhole. The acquisition of one image took 0.7 seconds, and an image stack of 600 images took approximately 10 minutes. For multimodal imaging, two successive image stacks at two different excitation wavelengths (750 and 800 nm) were taken, resulting in a total acquisition time of 20 minutes. To mirror physiological conditions as closely as possible, NAD(P)H detection at 750 nm was performed first.