Imaging sub-diffraction dynamics of neural nanostructures involved in behaviors such as learning and memory in a freely moving animal is not possible with existing techniques. Here, we present a solution in the form of a two-photon (2P), fiber-coupled, stimulated emission depletion microscope and demonstrate its capabilities by acquiring super-resolution imaging of mammalian cells. A polarization-maintaining fiber is used to transport both the 2P excitation light (915 nm) and the donut-shaped depletion beam (592 nm), which is constructed by adding two temporally incoherent and orthogonally polarized Hermite–Gaussian fiber modes. The fiber output is insensitive to bending or temperature changes and is the first demonstration toward deep tissue super-resolution imaging in awake behaving animals.
more »
« less
Note: When clicking on a Digital Object Identifier (DOI) number, you will be taken to an external site maintained by the publisher.
Some full text articles may not yet be available without a charge during the embargo (administrative interval).
What is a DOI Number?
Some links on this page may take you to non-federal websites. Their policies may differ from this site.
-
-
more » « less
Abstract We present results for a new type of fiber-coupled stimulated emission depletion (STED) microscope which uses a single fiber to transport STED and excitation light, as well as collect the fluorescence signal. Our method utilizes two higher-order eigenmodes of polarization maintaining (PM) fiber to generate the doughnut-shaped STED beam. The modes are excited with separate beams that share no temporal coherence, yielding output that is independent of fiber bending. We measured the resolution using 45 nm fluorescent beads and found a median bead image size of 116 nm. This resolution does not change as function of fiber bending radius, demonstrating robust operation. We report, for the first time, STED images of fixed biological samples collected in the epi-direction through fiber. Our microscope design shows promise for future use in super-resolution micro-endoscopes and
in vivo neural imaging in awake and freely-behaving animals.
