19 research outputs found
DeepSTORM3D: dense three dimensional localization microscopy and point spread function design by deep learning
Localization microscopy is an imaging technique in which the positions of
individual nanoscale point emitters (e.g. fluorescent molecules) are determined
at high precision from their images. This is the key ingredient in
single/multiple-particle-tracking and several super-resolution microscopy
approaches. Localization in three-dimensions (3D) can be performed by modifying
the image that a point-source creates on the camera, namely, the point-spread
function (PSF). The PSF is engineered using additional optical elements to vary
distinctively with the depth of the point-source. However, localizing multiple
adjacent emitters in 3D poses a significant algorithmic challenge, due to the
lateral overlap of their PSFs. Here, we train a neural network to receive an
image containing densely overlapping PSFs of multiple emitters over a large
axial range and output a list of their 3D positions. Furthermore, we then use
the network to design the optimal PSF for the multi-emitter case. We
demonstrate our approach numerically as well as experimentally by 3D STORM
imaging of mitochondria, and volumetric imaging of dozens of
fluorescently-labeled telomeres occupying a mammalian nucleus in a single
snapshot.Comment: main text: 9 pages, 5 figures, supplementary information: 29 pages,
20 figure
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
VIPR: vectorial implementation of phase retrieval for fast and accurate microscopic pixel-wise pupil estimation
Quantifying cell cycle dependent chromatin dynamics during interphase by live 3D tracking
The study of cell cycle progression and regulation is important to our understanding of fundamental biophysics, aging, and disease mechanisms. Local chromatin movements are generally considered to be constrained and relatively consistent during all interphase stages, although recent advances in our understanding of genome organization challenge this claim. Here, we use high spatiotemporal resolution, 4D (x, y, z and time) localization microscopy by point-spread-function (PSF) engineering and deep learning-based image analysis, for live imaging of mouse embryonic fibroblast (MEF 3T3) and MEF 3T3 double Lamin A Knockout (LmnaKO) cell lines, to characterize telomere diffusion during the interphase. We detected varying constraint levels imposed on chromatin, which are prominently decreased during G0/G1. Our 4D measurements of telomere diffusion offer an effective method to investigate chromatin dynamics and reveal cell-cycle-dependent motion constraints, which may be caused by various cellular processes
3D printable diffractive optical elements by liquid immersion
Diffractive optical elements (DOEs) are used to shape the wavefront of incident light. This can be used to generate practically any pattern of interest, albeit with varying efficiency. A fundamental challenge associated with DOEs comes from the nanoscale-precision requirements for their fabrication. Here we demonstrate a method to controllably scale up the relevant feature dimensions of a device from tens-of-nanometers to tens-of-microns by immersing the DOEs in a near-index-matched solution. This makes it possible to utilize modern 3D-printing technologies for fabrication, thereby significantly simplifying the production of DOEs and decreasing costs by orders of magnitude, without hindering performance. We demonstrate the tunability of our design for varying experimental conditions, and the suitability of this approach to ultrasensitive applications by localizing the 3D positions of single molecules in cells using our microscale fabricated optical element to modify the point-spread-function (PSF) of a microscope
Fluorescence Microscopy: a statistics-optics perspective
Fundamental properties of light unavoidably impose features on images
collected using fluorescence microscopes. Modeling these features is ever more
important in quantitatively interpreting microscopy images collected at scales
on par or smaller than light's wavelength. Here we review the optics
responsible for generating fluorescent images, fluorophore properties,
microscopy modalities leveraging properties of both light and fluorophores, in
addition to the necessarily probabilistic modeling tools imposed by the
stochastic nature of light and measurement