ABSTRACT
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.
ABSTRACT
Fast acquisition of depth information is crucial for accurate 3D tracking of moving objects. Snapshot depth sensing can be achieved by wavefront coding, in which the point-spread function (PSF) is engineered to vary distinctively with scene depth by altering the detection optics. In low-light applications, such as 3D localization microscopy, the prevailing approach is to condense signal photons into a single imaging channel with phase-only wavefront modulation to achieve a high pixel-wise signal to noise ratio. Here we show that this paradigm is generally suboptimal and can be significantly improved upon by employing multi-channel wavefront coding, even in low-light applications. We demonstrate our multi-channel optimization scheme on 3D localization microscopy in densely labelled live cells where detectability is limited by overlap of modulated PSFs. At extreme densities, we show that a split-signal system, with end-to-end learned phase masks, doubles the detection rate and reaches improved precision compared to the current state-of-the-art, single-channel design. We implement our method using a bifurcated optical system, experimentally validating our approach by snapshot volumetric imaging and 3D tracking of fluorescently labelled subcellular elements in dense environments.
Subject(s)
Algorithms , MicroscopyABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
An outstanding challenge in single-molecule localization microscopy is the accurate and precise localization of individual point emitters in three dimensions in densely labeled samples. One established approach for three-dimensional single-molecule localization is point-spread-function (PSF) engineering, in which the PSF is engineered to vary distinctively with emitter depth using additional optical elements. However, images of dense emitters, which are desirable for improving temporal resolution, pose a challenge for algorithmic localization of engineered PSFs, due to lateral overlap of the emitter PSFs. Here we train a neural network to localize multiple emitters with densely overlapping Tetrapod PSFs over a large axial range. We then use the network to design the optimal PSF for the multi-emitter case. We demonstrate our approach experimentally with super-resolution reconstructions of mitochondria and volumetric imaging of fluorescently labeled telomeres in cells. Our approach, DeepSTORM3D, enables the study of biological processes in whole cells at timescales that are rarely explored in localization microscopy.
Subject(s)
Deep Learning , Imaging, Three-Dimensional/methods , Single Molecule Imaging/methods , Biological Phenomena , Neural Networks, Computer , Telomere/ultrastructureABSTRACT
The structural organization and dynamics of DNA are known to be of paramount importance in countless cellular processes, but capturing these events poses a unique challenge. Fluorescence microscopy is well suited for these live-cell investigations, but requires attaching fluorescent labels to the species under investigation. Over the past several decades, a suite of techniques have been developed for labeling and imaging DNA, each with various advantages and drawbacks. Here, we provide an overview of the labeling and imaging tools currently available for visualizing DNA in live cells, and discuss their suitability for various applications.