A high-density 3D localization algorithm for stochastic optical reconstruction microscopy.
Hazen P. Babcock,Yaron M. Sigal,Xiaowei Zhuang +2 more
- Vol. 1, Iss: 1, pp 6
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TLDR
This work presents a method to analyze high-density super-resolution data in three dimensions, where the images of individual fluorophores not only overlap, but also have varying PSFs that depend on the z positions of the fluorophore.Abstract:
Stochastic optical reconstruction microscopy (STORM) and related methods achieves sub-diffraction-limit image resolution through sequential activation and localization of individual fluorophores. The analysis of image data from these methods has typically been confined to the sparse activation regime where the density of activated fluorophores is sufficiently low such that there is minimal overlap between the images of adjacent emitters. Recently several methods have been reported for analyzing higher density data, allowing partial overlap between adjacent emitters. However, these methods have so far been limited to two-dimensional imaging, in which the point spread function (PSF) of each emitter is assumed to be identical. In this work, we present a method to analyze high-density super-resolution data in three dimensions, where the images of individual fluorophores not only overlap, but also have varying PSFs that depend on the z positions of the fluorophores. This approach accurately analyzed data sets with an emitter density five times higher than previously possible with sparse emitter analysis algorithms. We applied this algorithm to the analysis of data sets taken from membrane-labeled retina and brain tissues which contain a high-density of labels, and obtained substantially improved super-resolution image quality.read more
Citations
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Spatially resolved, highly multiplexed RNA profiling in single cells
Kok Hao Chen,Alistair N. Boettiger,Jeffrey R. Moffitt,Siyuan Wang,Xiaowei Zhuang,Xiaowei Zhuang +5 more
TL;DR: This report reports multiplexed error-robust FISH (MERFISH), a single-molecule imaging method that allows thousands of RNA species to be imaged in single cells by using combinatorial FISH labeling with encoding schemes capable of detecting and/or correcting errors.
Journal ArticleDOI
Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells
Bogdan Bintu,Leslie J. Mateo,Jun-Han Su,Nicholas A Sinnott-Armstrong,Mirae Parker,Seon Kinrot,Kei Yamaya,Alistair N. Boettiger,Xiaowei Zhuang +8 more
TL;DR: A super-resolution chromatin tracing method that allows determination of both the structural features and their genomic coordinates with high resolution in single cells is reported, suggesting that cohesin is not required for the formation or maintenance of single-cell domain structures, but that their preferential boundary positions are influenced by cohes in-CTCF interaction.
Journal ArticleDOI
Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes.
Sang Hee Shim,Chenglong Xia,Guisheng Zhong,Guisheng Zhong,Hazen P. Babcock,Joshua C. Vaughan,Joshua C. Vaughan,Bo Huang,Xun Wang,Cheng Xu,Guo-Qiang Bi,Xiaowei Zhuang +11 more
TL;DR: This work identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes, and demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes.
Journal ArticleDOI
Quantitative evaluation of software packages for single-molecule localization microscopy
Daniel Sage,Hagai Kirshner,Thomas Pengo,Nico Stuurman,Junhong Min,Suliana Manley,Michael Unser +6 more
TL;DR: This work focuses on the computational aspects of super-resolution microscopy and presents a comprehensive evaluation of localization software packages, reflecting the various tradeoffs of SMLM software packages and helping users to choose the software that fits their needs.
Journal ArticleDOI
High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization
TL;DR: Advances in multiplexed error-robust fluorescence in situ hybridization (MERFISH) are reported that increase the measurement throughput by two orders of magnitude and allow gene expression profiling of ∼40,000 human cells in a single 18-h measurement, substantially extending the range of biological questions that can be addressed by MERFISH.
References
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Journal ArticleDOI
Imaging intracellular fluorescent proteins at nanometer resolution.
Eric Betzig,George H. Patterson,Rachid Sougrat,O. Wolf Lindwasser,Scott G. Olenych,Juan S. Bonifacino,Michael W. Davidson,Jennifer Lippincott-Schwartz,Harald F. Hess +8 more
TL;DR: This work introduced a method for optically imaging intracellular proteins at nanometer spatial resolution and used this method to image specific target proteins in thin sections of lysosomes and mitochondria and in fixed whole cells to image retroviral protein Gag at the plasma membrane.
Journal ArticleDOI
Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).
TL;DR: A high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores that can, in principle, reach molecular-scale resolution is developed.
Journal ArticleDOI
DAOPHOT: A Computer Program for Crowded-Field Stellar Photometry
TL;DR: The DAOPHOT program as mentioned in this paper performs stellar photometry in crowded fields using CCD images of stars in a crowded field, and shortcomings and possible improvements of the program are considered.
Journal ArticleDOI
Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy
TL;DR: A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit, and suggests a means to address a significant number of biological questions that had previously been limited by microscope resolution.
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Lapack Users' Guide
TL;DR: The third edition of LAPACK provided a guide to troubleshooting and installation of Routines, as well as providing examples of how to convert from LINPACK or EISPACK to BLAS.