Two-photon, fiber-coupled, super-resolution microscope for biological imaging

Brendan M. Heffernan,Mark E. Siemens,Juliet T. Gopinath,Peter S. Riley,Stephanie A. Meyer,Emily A. Gibson,Omkar D. Supekar,Diego Restrepo

doi:10.1063/5.0075012

Abstract

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.

Highlights

Two-photon (2P) excited fluorescence miniaturized fibercoupled microscopes offer an unprecedented view into the neural activity of awake behaving animals.1–7 There are two benefits that stem from using a 2P excitation process
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
We have presented a novel 2P fiber stimulated emission depletion (STED) microscope and demonstrated its ability to achieve resolutions down to 139 nm using fluorescent beads

Summary

INTRODUCTION

Two-photon (2P) excited fluorescence miniaturized fibercoupled microscopes offer an unprecedented view into the neural activity of awake behaving animals. There are two benefits that stem from using a 2P excitation process. Work on a 2P fiber STED endoscope used a double clad fiber and adjusted launch conditions of the depletion beam to selectively couple to the LP1,1 mode group.49 This approach is not robust due to the near degeneracy and destructive interference of modes within this mode group, which will result in a changing intensity and polarization profile when perturbed, degrading the resolution enhancement.. This approach is not robust due to the near degeneracy and destructive interference of modes within this mode group, which will result in a changing intensity and polarization profile when perturbed, degrading the resolution enhancement.50 Another possible solution is to use a wavelength selective phase or polarization plate to structure the depletion beam while leaving the excitation beam unchanged. Power throughput of the depletion laser from the laser source to microscope scan system is currently estimated to be 5%–10% but is not optimized and is limited by the chromatic aberration of our fiber coupling lens and our choice of SLM

Characterizing system resolution

Imaging mammalian cells

POLARIZATION CONSIDERATIONS

CONCLUSIONS