Instant super-resolution imaging in live cells and embryos via analog image processing

York, Andrew G.; Chandris, Panagiotis; Nogare, Damian Dalle; Head, Jeffrey; Wawrzusin, Peter; Fischer, R.; Chitnis, Ajay B.; Shroff, Hari

doi:10.1038/nmeth.2687

Cited by 366 publications

(374 citation statements)

References 35 publications

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“…When using a periodic multi-spot excitation array, a single scan mirror can suffice. This was demonstrated in (York et al 2013) which appeared after our manuscript was accepted for publication. Rescanning can also be performed with an electronically synchronised second scan mirror (or system of mirrors) or especially in the case of multi-spot illumination the rear side of the scan mirror can be used for rescanning.…”

Section: Discussionmentioning

confidence: 68%

Optical photon reassignment microscopy (OPRA)

et al. 2013

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To enhance the resolution of a confocal laser scanning microscope the additional information of a pinhole plane image taken at every excitation scan position can be used (Sheppard 1988). This photon reassignment principle is based on the fact that the most probable position of an emitter is at half way between the nominal focus of the excitation laser and the position corresponding to the (off centre) detection position. Therefore, by reassigning the detected photons to this place, an image with enhanced detection efficiency and resolution is obtained. Here we present optical photon reassignment microscopy (OPRA) which realizes this concept in an all-optical way obviating the need for image-processing. With the help of an additional intermediate optical beam expansion between descanning and a further rescanning of the detected light, an image with the advantages of photon reassignment can be acquired. However, just as in computational photon reassignment, a loss in confocal sectioning performance is caused by working with relatively open pinholes. The OPRA system shares properties such as flexibility and ease of use with a confocal laser scanning microscope, and is therefore expected to be of use for future biomedical routine research.

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Section: Discussionmentioning

confidence: 68%

Optical photon reassignment microscopy (OPRA)

et al. 2013

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“…3E), and the chromosomal rearrangement in a Drosophila syncytium with a three-to fivefold increase in resolution and an image acquisition speed that captured the total volume of the embryo within 1 s (Gao et al, 2012). Furthermore, multifocal plane illumination SIM was used to image the dynamics of the endoplasmic reticulum in cultured cells at 100 frames per second, or of the blood flow in zebrafish at 37 frames per second (York et al, 2013).…”

Section: Structured Illumination Microscopy (Sim)mentioning

confidence: 99%

Seeing is believing: multi-scale spatio-temporal imaging towards in vivo cell biology

Follain

Mercier

Osmani

et al. 2016

Journal of Cell Science

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Life is driven by a set of biological events that are naturally dynamic and tightly orchestrated from the single molecule to entire organisms. Although biochemistry and molecular biology have been essential in deciphering signaling at a cellular and organismal level, biological imaging has been instrumental for unraveling life processes across multiple scales. Imaging methods have considerably improved over the past decades and now allow to grasp the inner workings of proteins, organelles, cells, organs and whole organisms. Not only do they allow us to visualize these events in their most-relevant context but also to accurately quantify underlying biomechanical features and, so, provide essential information for their understanding. In this Commentary, we review a palette of imaging (and biophysical) methods that are available to the scientific community for elucidating a wide array of biological events. We cover the most-recent developments in intravital imaging, light-sheet microscopy, superresolution imaging, and correlative light and electron microscopy. In addition, we illustrate how these technologies have led to important insights in cell biology, from the molecular to the whole-organism resolution. Altogether, this review offers a snapshot of the current and state-of-the-art imaging methods that will contribute to the understanding of life and disease.

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“…This approach offers an improved signal to noise ratio and increased resolution with relatively little modification to the existing hardware of a laser--scanning microscope. There are now several different implementations of ISM, underpinned by the concept of 'pixel reassignment' [1--3], achieved either by optical (ISIM [4,5], OPRA [6]) or computational means (MSIM [7] & spinning disk ISM [8]. This manuscript will explore the potential of computational approaches utilizing methods drawn from single molecule localization microscopy for a posteriori pixel reassignment.…”

Section: Introductionmentioning

confidence: 99%

“…Each pixel on the camera can be considered as a 'micropinhole' [4], displaced by some distance from the excitation axis. Like a pinhole camera, each pixel 'micropinhole' detects an image of the emitted fluorescence; the smaller the pinhole, the sharper the image.…”

Section: Introductionmentioning

confidence: 99%

“…The processes involved in ISM are summarised in figure 2. [2] It is possible to scale each image around the excitation focus optically by descanning the emitted light and demagnifying the image of each excitation focus [4,6] or doubling the spacing between excitation foci [5,10]. This has the advantage of performing the scaling step in real time rather than at the post--processing stage.…”

Section: Introductionmentioning

confidence: 99%

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Post-processing strategies in image scanning microscopy

McGregor

Mitchell

Hartell

2015

Methods

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Image scanning microscopy (ISM) coupled with pixel reassignment offers a resolution improvement of 1.41 over standard widefield imaging. By scanning point--wise across the specimen and capturing an image of the fluorescent signal generated at each scan position, additional information about specimen structure is recorded and the highest accessible spatial frequency is doubled. Pixel reassignment can be achieved optically in real time or computationally a posteriori and is frequently combined with the use of a physical or digital pinhole to reject out of focus light. Here, we simulate an ISM dataset using a test image and apply standard and non--standard processing methods to address problems typically encountered in computational pixel reassignment and pinholing. We demonstrate that the predicted improvement in resolution is achieved by applying standard pixel reassignment to a simulated dataset and explore the effect of realistic displacements between the reference and true excitation positions. By identifying the position of the detected fluorescence maximum using localisation software and centring the digital pinhole on this co--ordinate before scaling around translated excitation positions, we can recover signal that would otherwise be degraded by the use of a pinhole aligned to an inaccurate excitation reference. This strategy is demonstrated using experimental data from a multiphoton ISM instrument. Finally we investigate the effect that imaging through tissue has on the positions of excitation foci at depth and observe a global scaling with respect to the applied reference grid. Using simulated and experimental data we explore the impact of a globally scaled reference on the ISM image and by pinholing around the detected maxima, recover the signal across the whole field of view.

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Instant super-resolution imaging in live cells and embryos via analog image processing

Cited by 366 publications

References 35 publications

Optical photon reassignment microscopy (OPRA)

Optical photon reassignment microscopy (OPRA)

Seeing is believing: multi-scale spatio-temporal imaging towards in vivo cell biology

Post-processing strategies in image scanning microscopy

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