(A) Two-photon fiberscope setup. Excitation light is delivered through fiber optics to a miniaturized microscope front piece that is attached to the head of a freely behaving animal. Two principle options for fluorescence detection are indicated. (B) Example for Option 1: Original design used by Helmchen et al. (2001). Excitation light is guided through a single-mode fiber. Scanning is achieved using resonant mechanical vibration of the fiber tip. Near-infrared excitation light (red) is passed through a long-pass dichroic beam splitter and fluorescent light (green) is detected by a small photomultiplier tube (PMT) at the front piece. (C) Example for Option 2: Recent design used by Sawinski et al. (2009). A nonresonant fiber scanner is used and near-infrared excitation light is collimated and deflected by a short-pass dichroic beam splitter. Fluorescence is collected through a large-core multimode fiber and detected remotely. (D) Resonant fiber scanning. (Left) Resonant vibration along a line is induced by driving a single piezoelectric bender, to which the fiber is glued, near the resonance frequency (fres). (Middle) Two-dimensional Lissajous scanning. Stiffening of the fiber end in one direction splits the resonance frequency in fX and fY. Simultaneous excitation of vibrations in x and y directions creates a Lissajous pattern, more or less homogeneously covering an area. (Right) Spiral scanning. A piezo tube with quadrant electrodes induces spiral scanning by driving x and y directions at the same resonance frequency but with a 90° phase shift. (E) Nonresonant fiber scanner. This “piezolever fiber scanner” achieves fiber tip deflections by leveraging the movements of two crossed pairs of piezo benders. The fiber is glued to the tips of the cross-connected piezo pairs at the cross point close to the clamped fiber base. Driving the piezo benders with sawtooth-like waveforms (fast and slow) at frequencies below the resonance frequency creates raster scanning similar to conventional two-photon microscopes. Offset voltages to the x and y piezo benders provide the unique option to shift the scanned field laterally. Moreover, free-line scanning is possible. (F) Fiber resonance characteristics. (Left) Resonance curves measured with a position-sensitive detector (PSD) for a fiber tip stiffened in one direction. Note the distinct resonance peaks for x and y directions. (Right) Resonance frequency of a 125-μm diameter single-mode fiber as a function of the free length of the fiber end.
