A Fiber-Coupled Stimulated Emission Depletion Microscope for Bend-Insensitive Through-Fiber Imaging

Brendan M Heffernan,Diego Restrepo,Mark E Siemens,Juliet T Gopinath,Emily A Gibson,Stephanie A Meyer

doi:10.1038/s41598-019-47319-w

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.

Highlights

Animal is crucial to further understanding the brain; combining stimulated emission depletion (STED) and a miniature head-mounted microscope would provide unprecedented resolution and groundbreaking new opportunities for neuroscience studies
The STED beam passes through a half-wave plate, and a polarizing cube beam splitter (PBS) separates it into two arms of a MachZehnder interferometer
A quarter-wave plate followed by a half-wave plate controls the polarization of the excitation beam, and a high-pass dichroic mirror (Chroma ZT561sprdc) combines it with the STED beam path

Summary

Introduction

Animal is crucial to further understanding the brain; combining STED and a miniature head-mounted microscope would provide unprecedented resolution and groundbreaking new opportunities for neuroscience studies. An all-fiber STED system based on this solution maintains high-purity modes suitable for STED even under extreme bending, with resolutions of 103 nm[23,24] Another solution uses double-clad fiber and two-photon excitation, and has demonstrated through-fiber imaging of fluorescent beads, but these experiments were performed at a relatively low numerical aperture (NA) of 0.35, resulting in a STED resolution of 310 nm[25]. We demonstrate a proof-of-concept fiber STED microscope that utilizes the PM01 and PM10 modes of a commercially available PM fiber to generate a doughnut STED beam By exciting these two fiber modes using two beams that are temporally incoherent with respect to each other, we establish robust operation with no loss of resolution due to fiber bending.

Methods

Results

Discussion

Conclusion