Image Scanning Microscopy

Müller, Claus B.; Enderlein, Jörg

doi:10.1103/physrevlett.104.198101

Cited by 442 publications

(360 citation statements)

References 11 publications

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“…SIM uses contributions from both the excitation and emission point spread functions (PSFs) to substantially improve the transverse resolution and is generally performed by illuminating the sample with a set of sharp light patterns and collecting fluorescence on a multipixel detector, followed by image processing to recover superresolution detail from the interaction of the light pattern with the sample. A related technique, image scanning microscopy (ISM), uses a scanned diffraction-limited spot as the light pattern (5,6). Multifocal SIM (MSIM) parallelizes ISM by using many excitation spots (7), and has been shown to produce optically sectioned images with ∼145-nm lateral and ∼400-nm axial resolution at depths up to ∼50 μm and at ∼1 Hz imaging frequency.…”

mentioning

confidence: 99%

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Ingaramo

York

Wawrzusin

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

109

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Multifocal structured illumination microscopy (MSIM) provides a twofold resolution enhancement beyond the diffraction limit at sample depths up to 50 μm, but scattered and out-of-focus light in thick samples degrades MSIM performance. Here we implement MSIM with a microlens array to enable efficient two-photon excitation. Two-photon MSIM gives resolution-doubled images with better sectioning and contrast in thick scattering samples such as Caenorhabditis elegans embryos, Drosophila melanogaster larval salivary glands, and mouse liver tissue.multiphoton | superresolution F luorescence microscopy is an invaluable tool for biologists.Protein distributions in cells have an interesting structure down to the nanometer scale, but features smaller than 200-300 nm are blurred by diffraction in widefield and confocal fluorescence microscopes. Superresolution techniques like photoactivated localization microscopy (1), stochastic optical reconstruction microscopy (2), or stimulated emission depletion (STED) (3) microscopy allow the imaging of details beyond the limit imposed by diffraction, but usually trade acquisition speed or straightforward sample preparation. And although STED can provide resolution down to 40 nm, STED-specific fluorophores are recommended and it often requires light intensities that are orders of magnitude above widefield and confocal microscopy. On the other hand, structured illumination microscopy (SIM) (4) gives twice the resolution of a conventional fluorescence microscope with light intensities on the order of widefield microscopes and can be used with most common fluorophores. SIM uses contributions from both the excitation and emission point spread functions (PSFs) to substantially improve the transverse resolution and is generally performed by illuminating the sample with a set of sharp light patterns and collecting fluorescence on a multipixel detector, followed by image processing to recover superresolution detail from the interaction of the light pattern with the sample. A related technique, image scanning microscopy (ISM), uses a scanned diffraction-limited spot as the light pattern (5, 6). Multifocal SIM (MSIM) parallelizes ISM by using many excitation spots (7), and has been shown to produce optically sectioned images with ∼145-nm lateral and ∼400-nm axial resolution at depths up to ∼50 μm and at ∼1 Hz imaging frequency. In MSIM, images are excited with a multifocal excitation pattern, and the resulting fluorescence in the multiple foci are pinholed, locally scaled, and summed to generate an image [multifocal-excited, pinholed, scaled, and summed (MPSS)] with root 2-improved resolution relative to widefield microscopy, and improved sectioning compared with SIM due to confocal-like pinholing. Deconvolution is applied to recover the final MSIM image which has a full factor of 2 resolution improvement over the diffraction limit.

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mentioning

confidence: 99%

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Ingaramo

York

Wawrzusin

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

109

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“…The currently developed super-resolution techniques may also benefit the computational image processing techniques in their respective applications, when the image data shares a similar digital nature through current or modified instrumentation. Improve the resolution with sparse deconvolution, and determine the fluorescence dipole orientation with modulation fitting 75 Axelrod, 1989;Hafi et al, 2014;Zhanghao et al, 2016 SIM Use Moiré interference (differential frequency) to bring the high frequency feature to low frequency 100 Gustafsson et al, 2008;Li et al, 2015;Yang et al, 2016b;Yu et al, 2016 ISM Spatial deconvolution of the obtained confocal PSF 150 Müller and Enderlein, 2010;Sheppard et al, 2013;Yang et al, 2016b;Yu et al, 2016 SOFI Calculate the high-order correlation of the random blinking statistics of the emitters 75 Dertinger et al, 2009;Geissbuehler et al, 2012 SMLM Localize the position of each single molecule for super-resolution imaging 10-20 Betzig et al, 2006;Hess et al, 2006;Rust et al, 2006;Yang et al, 2016b;Yu et al, 2016 3B Calculate the Bayesian statistics of the random blinking emitters 50 Cox et al, 2012 STED Employ stimulated emission to shrink the PSF down to subdiffraction size; the subsequent deconvolution process further enhances the resolution and contrast 20-30 Schoonderwoert et al, 2013 TRAM Achieves a high-resolution image from multiple deconvolved low-resolution translation images~5 0 Qiu et al, 2016 …”

Section: Resultsmentioning

confidence: 99%

“…Image scanning microscopy (ISM) (Müller and Enderlein, 2010;Sheppard et al, 2013) is an improved version of conventional confocal microscopy. ISM replaces the PMT pinhole with a CCD and by deconvolution, it could improve the resolution by a factor of 2.…”

Section: Image Scanning Microscopymentioning

confidence: 99%

“…Structured illumination microscopy (SIM) extends the optical transfer function (OTF) in the Fourier domain to enhance the spatial resolution (Gustafsson, 2000). Image scanning microscopy (ISM) replaces conventionally used photomultiplier tubes (PMT) detector with charge-coupled device (CCD), and achieves subdiffraction resolution through deconvolution (Müller and Enderlein, 2010). Super-resolution optical fluctuation imaging (SOFI) employs correlation analysis on the temporal fluctuation of the fluorescence to distinguish signals from independently fluctuating fluorophores (Dertinger et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

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Computational methods in super-resolution microscopy

Zeng

Xie

Chen

et al. 2017

Frontiers Inf Technol Electronic Eng

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Abstract:The broad applicability of super-resolution microscopy has been widely demonstrated in various areas and disciplines. The optimization and improvement of algorithms used in super-resolution microscopy are of great importance for achieving optimal quality of super-resolution imaging. In this review, we comprehensively discuss the computational methods in different types of super-resolution microscopy, including deconvolution microscopy, polarization-based super-resolution microscopy, structured illumination microscopy, image scanning microscopy, super-resolution optical fluctuation imaging microscopy, single-molecule localization microscopy, Bayesian super-resolution microscopy, stimulated emission depletion microscopy, and translation microscopy. The development of novel computational methods would greatly benefit super-resolution microscopy and lead to better resolution, improved accuracy, and faster image processing.

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“…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%

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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Image Scanning Microscopy

Cited by 442 publications

References 11 publications

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Computational methods in super-resolution microscopy

Post-processing strategies in image scanning microscopy

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