An Integrated Platform for High-Throughput Nanoscopy

Diffraction-unlimited single-molecule switching (SMS) nanoscopy techniques like STORM /(F)PALM enable three-dimensional (3D) fluorescence imaging at 20-80 nm resolution and are invaluable to investigate sub-cellular organization. They suffer, however, from low throughput, limiting the output of a da...

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Main Authors Barentine, Andrew E S, Lin, Yu, Courvan, Edward M, Kidd, Phylicia, Liu, Miao, Balduf, Leonhard, Phan, Timy, Rivera-Molina, Felix, Grace, Michael R, Marin, Zach, Lessard, Mark, Rios-Chen, Juliana, Wang, Siyuan, Neugebauer, Karla M, Bewersdorf, Joerg, Baddeley, David
Format Paper
LanguageEnglish
Published Cold Spring Harbor Cold Spring Harbor Laboratory Press 20.04.2022
Cold Spring Harbor Laboratory
Edition1.3
Subjects
Biophysics
Data acquisition
Data processing
Mammalian cells
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Abstract Diffraction-unlimited single-molecule switching (SMS) nanoscopy techniques like STORM /(F)PALM enable three-dimensional (3D) fluorescence imaging at 20-80 nm resolution and are invaluable to investigate sub-cellular organization. They suffer, however, from low throughput, limiting the output of a day's worth of imaging to typically a few tens of mammalian cells. Here we develop an SMS imaging platform that combines high-speed 3D single-molecule data acquisition with an automated, fully integrated, high-volume data processing pipeline. We demonstrate 2-color 3D super-resolution imaging of over 10,000 mammalian cell nuclei in about 26 hours, connecting the traditionally low-throughput super-resolution community to the world of omics approaches. Competing Interest Statement ificant financial interest in Bruker Corp. and Hamamatsu Photonics Footnotes * Added new biological application of method.
AbstractList Diffraction-unlimited single-molecule switching (SMS) nanoscopy techniques like STORM /(F)PALM enable three-dimensional (3D) fluorescence imaging at 20-80 nm resolution and are invaluable to investigate sub-cellular organization. They suffer, however, from low throughput, limiting the output of a day's worth of imaging to typically a few tens of mammalian cells. Here we develop an SMS imaging platform that combines high-speed 3D single-molecule data acquisition with an automated, fully integrated, high-volume data processing pipeline. We demonstrate 2-color 3D super-resolution imaging of over 10,000 mammalian cell nuclei in about 26 hours, connecting the traditionally low-throughput super-resolution community to the world of omics approaches. Competing Interest Statement ificant financial interest in Bruker Corp. and Hamamatsu Photonics Footnotes * Added new biological application of method.
Diffraction-unlimited single-molecule techniques like STORM and (F)PALM enable three-dimensional (3D) fluorescence imaging at tens of nanometer resolution and are invaluable to investigate sub-cellular organization. The multitude of camera frames required to reconstruct a super-resolved image limits the typical throughput of these techniques to tens of cells per day, rendering these methods incompatible with large-scale cell biological or clinical application. STORM acquisition rates can be increased by over an order of magnitude, however the data volumes of about 40 TB a day and concomitant analysis burdens exceed the capacity of existing workflows. Here we present an integrated platform which transforms SMLM from a trick-pony technique into a work horse for cell biology. We leverage our developments in microscopy-specific data compression, distributed storage, and distributed analysis to automatically perform real-time localization analysis, which enable SMLM at throughputs of 10,000 cells a day. We implemented these advances in a fully-integrated environment that supports a highly-flexible architecture for distributed analysis, enabling quickly- and graphically-reconfigurable analyses to be automatically initiated from the microscope during acquisition, remotely executed, and even feedback and queue new acquisition tasks on the microscope. We demonstrate the utility of this framework by imaging hundreds of cells per well in multi-well sample formats. Our platform, the PYthon-Microscopy Environment (PYME), is easily configurable for hardware control on custom microscopes, and includes a plugin framework so users can readily extend all components of their imaging, visualization, and analysis pipeline. PYME is cross-platform, open source, and efficiently puts high-caliber visualization and analysis tools into the hands of both microscope developers and users.
Author Bewersdorf, Joerg
Barentine, Andrew E S
Marin, Zach
Neugebauer, Karla M
Grace, Michael R
Phan, Timy
Lin, Yu
Rios-Chen, Juliana
Kidd, Phylicia
Balduf, Leonhard
Lessard, Mark
Wang, Siyuan
Baddeley, David
Liu, Miao
Courvan, Edward M
Rivera-Molina, Felix
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Competing Interest Statement: ificant financial interest in Bruker Corp. and Hamamatsu Photonics
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Snippet Diffraction-unlimited single-molecule switching (SMS) nanoscopy techniques like STORM /(F)PALM enable three-dimensional (3D) fluorescence imaging at 20-80 nm...
Diffraction-unlimited single-molecule techniques like STORM and (F)PALM enable three-dimensional (3D) fluorescence imaging at tens of nanometer resolution and...
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Data acquisition
Data processing
Mammalian cells
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