29 research outputs found
Additive manufacturing of solid diffractive optical elements via near index matching
Diffractive optical elements (DOEs) have a wide range of applications in
optics and photonics, thanks to their capability to perform complex wavefront
shaping in a compact form. However, widespread applicability of DOEs is still
limited, because existing fabrication methods are cumbersome and expensive.
Here, we present a simple and cost-effective fabrication approach for solid,
high-performance DOEs. The method is based on conjugating two nearly refractive
index-matched solidifiable transparent materials. The index matching allows for
extreme scaling up of the elements in the axial dimension, which enables simple
fabrication of a template using commercially available 3D printing at
tens-of-micrometer resolution. We demonstrated the approach by fabricating and
using DOEs serving as microlens arrays, vortex plates, including for highly
sensitive applications such as vector beam generation and super-resolution
microscopy using MINSTED, and phase-masks for three-dimensional single-molecule
localization microscopy. Beyond the advantage of making DOEs widely accessible
by drastically simplifying their production, the method also overcomes
difficulties faced by existing methods in fabricating highly complex elements,
such as high-order vortex plates, and spectrum-encoding phase masks for
microscopy
Microsecond Single-Molecule Tracking (μsSMT)
Here we report on a method to track individual molecules on nanometer length and microsecond timescales using an optical microscope. Our method is based on double-labeling of a molecule with two spectrally distinct fluorophores and illuminating it with laser pulses of different wavelengths that partially overlap temporally. We demonstrate our method by using it to resolve the motion of short DNA oligomers in solution down to a timescale of 100 μs
SRpHi ratiometric pH biosensors for super-resolution microscopy
Fluorescence-based biosensors have become essential tools for modern biology, allowing real-time monitoring of biological processes within living cells. Intracellular fluorescent pH probes comprise one of the most widely used families of biosensors in microscopy. One key application of pH probes has been to monitor the acidification of vesicles during endocytosis, an essential function that aids in cargo sorting and degradation. Prior to the development of super-resolution fluorescence microscopy (nanoscopy), investigation of endosomal dynamics in live cells remained difficult as these structures lie at or below the ~250 nm diffraction limit of light microscopy. Therefore, to aid in investigations of pH dynamics during endocytosis at the nanoscale, we have specifically designed a family of ratiometric endosomal pH probes for use in live-cell STED nanoscopy