We compare the maximal two-photon fluorescence microscopy (TPM) imaging depth achieved with 775-nm excitation to that achieved with 1280-nm excitation through in vivo and ex vivo TPM of fluorescently-labeled blood vessels in mouse brain. We achieved high contrast imaging of blood vessels at approximately twice the depth with 1280-nm excitation as with 775-nm excitation. An imaging depth of 1 mm can be achieved in in vivo imaging of adult mouse brains at 1280 nm with approximately 1-nJ pulse energy at the sample surface. Blood flow speed measurements at a depth of 900 mum are performed.

We study and demonstrate the technique of simultaneous spatial and temporal focusing of femtosecond pulses, with the aim to improve the signal-to-background ratio in multiphoton imaging. This concept is realized by spatially separating spectral components of pulses into a "rainbow beam" and recombining these components only at the spatial focus of the objective lens. Thus, temporal pulse width becomes a function of distance, with the shortest pulse width confined to the spatial focus. We developed analytical expressions to describe this method and experimentally demonstrated the feasibility. The concept of simultaneous spatial and temporal focusing of femtosecond pulses has the great potential to significantly reduce the background excitation in multiphoton microscopy, which fundamentally limits the imaging depth in highly scattering biological specimens.

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We show theoretically and experimentally that simultaneous spatial and temporal focusing can scan the temporal focal plane axially by adjusting the group velocity dispersion in the excitation beam path. When the group velocity dispersion is small, the pulse width at the temporal focal plane is transform-limited, and the amount of shift depends linearly upon the dispersion. By adding a meter of large mode area fiber into the system, we demonstrate this axial scanning capability in a fiber delivery configuration. Because a transform-limited pulse width is automatically recovered at the temporal focal plane, simultaneous spatial and temporal focusing negates the need for any dispersion pre-compensation, further facilitating its integration into a fiber delivery system. A highly promising application for simultaneous spatial and temporal focusing is an axial scanning multiphoton fluorescence fiber probe without any moving parts at the distal end and without dispersion pre-compensation.

SIMULTANEOUS SPATIAL AND TEMPORAL FOCUSING IN NONLINEAR MICROSCOPYMichael Earle Durst, Ph. D. Cornell University 2009Multiphoton 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 10 5 fs 2 , 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.iii BIOGRAPHICAL SKETCH Michael Earle Durst grew up in Plant City, FL, the winter strawberry capital of the world. He claims Plant City as his hometown because it served as his home address for the longest amount of time. Michael was born in Ft. Myers, FL, went to kindergarten in Port Charlotte, FL, first through third grade in Alexandria, VA, fourth through part of sixth grade in Sarasota, FL, seventh and eighth grade in Tampa, FL, and all of high school in Plant City, FL.Durst enrolled at Georgetown University in Washington, DC in the fall of 1999. He had expressed his interest in physics as a major when he filled out the application...