Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging

Togo, Shinji; Yamagata, Kazuya; Kondo, Takefumi; Hayashi, Shigeo; Shitamukai, Atsunori; Konno, Daijiro; Matsuzaki, Fumio; Takayama, Jun; Onami, Shuichi; Nakayama, Hiroshi; Kosugi, Yasuhito; Watanabe, Tomonobu M.; Fujita, Katsumasa; Mimori-Kiyosue, Yuko

doi:10.1073/pnas.1216696110

Cited by 85 publications

(68 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This instrument bears a resemblance to a number of multifocal multiphoton configurations, whose major advantage is that they increase the image acquisition rate over that of point-scanning systems (12,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Most use widefield detection for parallelized detection of the multiple focal spots and this is important for 2P-MSIM for two critical reasons.…”

Section: Msim Excitation Was Originally Implemented With a Digital MImentioning

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.

112

View full text Add to dashboard Cite

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.

show abstract

Section: Msim Excitation Was Originally Implemented With a Digital MImentioning

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.

112

View full text Add to dashboard Cite

show abstract

“…1A), on a charge-coupled device (CCD) camera, thus increasing the data acquisition rate. The main drawback of SDCM is the cross-talk between pinholes; this can be circumvented by increasing the inter-pinhole distance or by decreasing the out-of-focus light by using two-photon excitation (Shimozawa et al, 2013). Both CLSM and SDCM have been ground-breaking for imaging in vitro as well as in vivo; for instance, relatively thin model systems (Fig.…”

Section: Optical Tweezers In Vivomentioning

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

View full text Add to dashboard Cite

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.

show abstract

“…IVM imaging of dynamic and fast events, such as blood flow or mobile immune cells, can be accomplished using resonant scanners, which allow imaging speeds up to 30 frames per second (Kirkpatrick et al, 2012;Matsumoto et al, 2010;Nguyen et al, 2001) or multibeam imaging, which can also reduce photodamage by spreading the excitation power over a number of focal spots (Niesner et al, 2007;Rinnenthal et al, 2013;Shimozawa et al, 2013).…”

Section: Improving Acquisition Speedmentioning

confidence: 99%

Molecular mobility and activity in an intravital imaging setting – implications for cancer progression and targeting

Nobis

Warren

Lucas

et al. 2018

Journal of Cell Science

View full text Add to dashboard Cite

Molecular mobility, localisation and spatiotemporal activity are at the core of cell biological processes and deregulation of these dynamic events can underpin disease development and progression. Recent advances in intravital imaging techniques in mice are providing new avenues to study real-time molecular behaviour in intact tissues within a live organism and to gain exciting insights into the intricate regulation of live cell biology at the microscale level. The monitoring of fluorescently labelled proteins and agents can be combined with autofluorescent properties of the microenvironment to provide a comprehensive snapshot of in vivo cell biology. In this Review, we summarise recent intravital microscopy approaches in mice, in processes ranging from normal development and homeostasis to disease progression and treatment in cancer, where we emphasise the utility of intravital imaging to observe dynamic and transient events in vivo. We also highlight the recent integration of advanced subcellular imaging techniques into the intravital imaging pipeline, which can provide in-depth biological information beyond the singlecell level. We conclude with an outlook of ongoing developments in intravital microscopy towards imaging in humans, as well as provide an overview of the challenges the intravital imaging community currently faces and outline potential ways for overcoming these hurdles.

show abstract

Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging

Cited by 85 publications

References 37 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

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

Molecular mobility and activity in an intravital imaging setting – implications for cancer progression and targeting

Contact Info

Product

Resources

About