An Integrated Platform for High-Throughput Nanoscopy

Barentine, Andrew E.S.; Lin, Yu; Liu, Miao; Kidd, P.; Balduf, Leonhard; Grace, Michael R.; Wang, Siyuan; Bewersdorf, Joerg; Baddeley, David

doi:10.1101/606954

Cited by 22 publications

(9 citation statements)

References 45 publications

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“…We then changed both the intensity, camera exposure time and effective laser exposure time to either 24 kW/cm 2 , 9 ms and 7.45 ms or 61 kW/cm 2 , 3 ms and 1.25 ms, respectively. Note that the latter condition (shown in Figure 1e,f ) does not fit our otherwise applied standard of keeping the product of effective laser exposure time and intensity constant, but was instead chosen to effectively fit the image acquisition conditions of a recent study by Barentine et al 5 . Other than this, the same procedure was applied as described above.…”

Section: Methodsmentioning

confidence: 99%

Optimizing imaging speed and excitation intensity for single-molecule localization microscopy

et al. 2020

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High laser powers are common practice in single molecule localization microscopy (SMLM) to speed up data acquisition. Here, we systematically quantified how excitation intensity influences localization precision and labeling density, the two main factors determining data quality. We found a strong trade-off between imaging speed and quality and present optimized imaging protocols for high-throughput, multi-color and 3D SMLM with greatly improved resolution and effective labeling efficiency.

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Section: Methodsmentioning

confidence: 99%

Optimizing imaging speed and excitation intensity for single-molecule localization microscopy

et al. 2020

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“…Of course, various strategies have been demonstrated to implement all these aspects, showing that the time span between data generation and final result can be considerably reduced. 205 This is highly beneficial, and not only to save time: If the data can be analyzed in or close to real time, the experimental parameters can be tuned flexibly, improving the quality of the generated data and the science learned, and throughput can be significantly improved.…”

Section: Perspectives On Methods and Applicationsmentioning

confidence: 99%

Super-resolution Microscopy with Single Molecules in Biology and Beyond–Essentials, Current Trends, and Future Challenges

2020

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Single-molecule super-resolution microscopy has developed from a specialized technique into one of the most versatile and powerful imaging methods of the nanoscale over the past two decades. In this perspective, we provide a brief overview of the historical development of the field, the fundamental concepts, the methodology required to obtain maximum quantitative information, and the current state of the art. Then, we will discuss emerging perspectives and areas where innovation and further improvement are needed. Despite the tremendous progress, the full potential of single-molecule super-resolution microscopy is yet to be realized, which will be enabled by the research ahead of us.

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“…The instrument control computer itself, if equipped with an appropriate SSD and HDD, is therefore adequate during early and intermediate stages of use. Additional solutions in the form of compression and distributed storage, either specific to localisation microscopy data 12 or more general, may be helpful as the user base of the instrument grows.…”

Section: Computer Infrastructure and Data Storagementioning

confidence: 99%

Implementation of a 4Pi-SMS super-resolution microscope

et al. 2020

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This protocol describes how to assemble a 4Pi single-molecule switching (SMS or SMLM) super-resolution microscope. Detailed instructions for beam-path alignment, testing, application to cellular samples, and troubleshooting are provided.TWEET New Protocol for setting up a 4Pi super-res microscope from @MicronOxford @GurdonInstitute @JonasRies and @bewersdorflab COVER TEASER Implementing a 4Pi super-resolution microscope ABSTRACTThe development of single-molecule switching (SMS) fluorescence microscopy (also called singlemolecule localisation microscopy (SMLM)), over the last decade has enabled researchers to image cell biological structures at unprecedented resolution. Using two opposing objectives in a so-called 4Pi geometry doubles the available numerical aperture, and coupling this with interferometric detection, has demonstrated three-dimensional (3D) resolution down to 10 nm over entire cellular volumes. The aim of this protocol is to enable interested researchers to establish 4Pi-SMS super-resolution microscopy in their laboratories. We describe in detail how to assemble the optomechanical components of a 4Pi-SMS instrument, align its optical beampath and test its performance. The protocol further provides instructions on how to prepare test samples of fluorescent beads, operate this instrument to acquire images of whole cells and how to analyse the raw image data to reconstruct super-resolution 3D data sets. Furthermore, we provide a trouble-shooting guide and present examples of anticipated results. An experienced optical instrument builder will require approximately 12 months from the start of ordering hardware components to acquiring high-quality biological images.Optical overview. The microscope presented here is centred around two opposing 100X/1.35 NA silicone oil immersion objective lenses (Olympus, UPLSAPO 100XS), OBJ0 and OBJ1 in Figure 1. Critically, these objectives must have closely matched magnification (within 0.3%) to allow for alignment of the two imaged fields of view for interference. Each objective lens is immediately followed by a quarterwave plate (Figure 1a, QWP0-1) set at 45 degrees with respect to the plane of the setup 2,4 . The quarterwave plates ensure that the emitted light is split in both beam paths into s-and p-polarisation components of equal intensity, independent of the initial polarisation orientation. Each objective's back pupil plane is imaged onto a deformable mirror (Figure 1a, lenses L1 and L3 for Objective OBJ0 and lenses L2 and L4 for OBJ1). The deformable mirrors (DMs) serve three functions: 1) introduce astigmatism to assist in emitter z-position determination, 2) correct for system aberrations

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An Integrated Platform for High-Throughput Nanoscopy

Cited by 22 publications

References 45 publications

Optimizing imaging speed and excitation intensity for single-molecule localization microscopy

Optimizing imaging speed and excitation intensity for single-molecule localization microscopy

Super-resolution Microscopy with Single Molecules in Biology and Beyond–Essentials, Current Trends, and Future Challenges

Implementation of a 4Pi-SMS super-resolution microscope

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