STED Simulation and Analysis by the Enhanced Geometrical Ray Tracing Method
Alexander Brodsky, Natan Kaplan, and Karen Goldberg* Holo/Or Ltd., 13B Einstein Street, Science Park, Ness Ziona, 7403617 Israel
*
karen@holoor.co.il
Abstract: Stimulated emission depletion (STED) microscopy is a powerful tool for the study of sub-micron samples, especially in biology. In this article, we show a simple, straightforward method for modeling a STED microscope using Zemax geometrical optics ray tracing principles, while still achieving a realistic spot size. This method is based on scattering models, and while it employs fast ray-tracing simulations, its output behavior is consistent with the real, diffractive behavior of a STED de-excitation spot. The method can be used with Zemax Black Boxes of real objectives for analysis of their performance in a STED setup and open perspective of modeling,
tolerancing performance, and then building advanced
microscope systems from scratch. In an example setup, we show an analysis method for both XY and Z excitation and de-excitation intensity distributions. This method can simplify the design of custom STED setups by proper modeling of the resolution performance.
Keywords: Zemax OpticStudio® , stimulated emission depletion
microscopy (STED), super-resolution microscopy, diffractive optical elements (DOE), lasers
Introduction Stimulated emission depletion (STED) microscopy is
a well-known technique for achieving super resolution in microscopy, that is, resolving details that are smaller than the diffraction limit of an optical system. Developed to bypass the diffraction limit of light microscopy, which is the main limit to the resolution of traditional light microscopes, it cre- ates super-resolution images by illumination of fluorophores in a ring-like (donut) pattern that depletes the fluorescence from the outside area of the donut, thus minimizing the area of illumination at the sample focal point and enhancing the achievable resolution for a given system. By using the non- linear response of fluorophores, STED forces the excited fluo- rophores at the donut profile to emit at a longer wavelength that is then optically filtered out. Only the fluorescence from a small, sub-diffraction limited region is leſt, enabling super resolution. Designing and setting up a STED microscope system
is a complex task that requires multidisciplinary knowledge in the fields of laser design, laser optics, general geometrical and physical optics concepts, laser beam shaping, mechan- ics, electronics, analysis soſtware, and more. Holo/Or Ltd. (established 1989), an Israel-based manufacturer, designs and manufactures diffractive optical elements (DOE) and micro- optical elements. Holo/Or offers a vast variety of standard and custom diffractive optics and micro-optics products, including multi-channel beam splitters, spatial beam shapers (flat-top, homogenizers/diffusers, ring/donut shapers), focal beam shapers (multi-focal, elongated focus), and more. Our DOE are integrated in laser systems in various application fields including science, microscopy, medicine, aesthetics,
60 doi:10.1017/S1551929521000687
material processing, metrology, and many others. Holo/Or has a long history of successful cooperation with leading research institutes around the world, providing high-quality optical elements and technical support for research and aca- demic excellence. Tis article provides details and methods used by Holo/
Or Ltd. that simplify the optical design part of the complex design and setup of a STED microscope. Te use of Zemax OpticStudio®
and the objective black box will be discussed.
Overview of STED Microscopes Typical STED systems include two independent optical
channels: one for the long wavelength (red) depletion laser and another for the short wavelength (green) excitation laser. Both channels are combined into the same optical path by dichroic mirrors and are then focused by an objective on a sample. Fluorescent light reflected from the sample goes to a detec- tor. Figure 1 describes this setup schematically. Te excitation channel is focused by the objective, while the depletion chan- nel is propagated through a spiral phase plate (SPP), otherwise known as a vortex lens (VL) DOE, before it is focused by the objective. Te VL DOE adds a spiral wavefront to the incident Gaussian beam to convert it into a donut-shaped beam at the focal plane of the objective. Te resulting donut-shaped beam at the focal plane shares the same optical properties as a Gauss- Laguerre 01 laser mode. DOE are micro-optical window-like phase elements designed to modify the phase of
the light that propagates
through them to create various shaping functions, the main ones being multichannel splitting, spatial shaping, and focal shaping. In diffractive optics fabrication processes, simi- lar methods to those used in the semiconductor industry are applied, giving DOE perfect angular accuracy with extremely low manufacturing tolerances. Tey are flat, thin, and easy to integrate into any opto-mechanical design. In many cases, DOE present a much more cost-effective beam shaping method than their refractive counterparts that frequently demand complex electro-opto-mechanics, making DOE a more robust solution when it comes to lifetime value. Te main production processes for DOE consist of several
repeating steps including photoresist wafer coating followed by direct UV lithography and repeated etching directly into the fused silica substrate. Tis creates a binary pattern microstruc- ture at the surface of the millimeter-scale thick optical win- dow. To achieve optimal optical efficiency, up to 4 lithography steps are oſten applied to create 16 levels of microstructures. Figure 2 shows an actual diffractive structure of a 16-level vor- tex lens measured by an optical profilometer. Te last step in the DOE manufacturing process is deploying an anti-reflective coating layer. DOE made of fused silica have an outstanding
www.microscopy-today.com • 2021 May
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