Simultaneous Spatial And Temporal Focusing In Nonlinear Microscopy

Abstract

Multiphoton microscopy (MPM) has become a powerful tool for imaging biological samples due to its ability to perform optical sectioning. MPM yields many advantages over standard one-photon imaging: a deeper penetration depth due to longer wavelength excitation, confinement of the focal volume, and reduced photodamage. These properties allow MPM to image samples non-invasively, acting as a form of optical biopsy for cancer research. Simultaneous spatial and temporal focusing (SSTF), when combined with nonlinear microscopy, can improve the axial excitation confinement of wide-field and line-scanning imaging. Because two-photon excited fluorescence depends inversely on the pulse width of the excitation beam, SSTF decreases the background excitation of the sample outside of the focal volume by broadening the pulse width everywhere but at the geometric focus of the objective lens. Also, SSTF can scan the temporal focal plane axially by adjusting the group-delay dispersion (GDD) in the excitation beam path. We further discuss this technique for axial-scanning multiphoton fluorescence fiber probes without any moving parts at the distal end. The temporal focusing effect in SSTF essentially replaces the focusing of one spatial dimension in conventional wide-field and line-scanning imaging. Although the best axial confinement achieved by SSTF cannot surpass that of a regular point-scanning system, this trade-off between spatial and temporal focusing can provide significant advantages in applications such as high-speed imaging and remote axial scanning in an endoscopic fiber probe. We also present two new techniques for tunable dispersion compensation that are low cost, high speed, broadband, capable of high intensities, and have a large tuning range. By rotating a cylindrical lens at the Fourier plane of a folded 4-f grating pair system, the group-delay dispersion can be tuned over a range greater than 105 fs2, sufficient for compensating the dispersion of several meters of optical fiber. We also show that a single-element piezo bimorph mirror can generate GDD in a folded 4-f grating pair setup. With a kilohertz sinusoidal drive voltage applied to the piezo bimorph, we demonstrate high-speed axial scanning in an SSTF setup at a rate of 2 kilohertz over a range of 100 microns.