The Fast mode for ZEISS LSM 880 with Airyscan: high-speed confocal imaging with super-resolution and improved signal-to-noise ratio

Huff, Joseph

doi:10.1038/nmeth.f.398

Cited by 28 publications

(22 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Imaging of FISH samples was performed at the Waitt Biophotonics Center at the Salk Institute. FISH samples were imaged on Zeiss Airyscan 880 microscopes using the Airyscan Fast mode (Huff 2016) at a resolution of 40 nm in the x and y axes. Z sections were imaged every 160 nm.…”

Section: Fluorescence In Situ Hybridization (Fish)mentioning

confidence: 99%

Upon microbial challenge, human neutrophils undergo rapid changes in nuclear architecture and chromatin folding to orchestrate an immediate inflammatory gene program

Denholtz

Zhu

et al. 2020

Genes Dev.

View full text Add to dashboard Cite

Differentiating neutrophils undergo large-scale changes in nuclear morphology. How such alterations in structure are established and modulated upon exposure to microbial agents is largely unknown. Here, we found that prior to encounter with bacteria, an armamentarium of inflammatory genes was positioned in a transcriptionally passive environment suppressing premature transcriptional activation. Upon microbial exposure, however, human neutrophils rapidly (<3 h) repositioned the ensemble of proinflammatory genes toward the transcriptionally permissive compartment. We show that the repositioning of genes was closely associated with the swift recruitment of cohesin across the inflammatory enhancer landscape, permitting an immediate transcriptional response upon bacterial exposure. We found that activated enhancers, marked by increased deposition of H3K27Ac, were highly enriched for cistromic elements associated with PU.1, CEBPB, TFE3, JUN, and FOSL2 occupancy. These data reveal how upon microbial challenge the cohesin machinery is recruited to an activated enhancer repertoire to instruct changes in chromatin folding, nuclear architecture, and to activate an inflammatory gene program.

show abstract

Section: Fluorescence In Situ Hybridization (Fish)mentioning

confidence: 99%

Upon microbial challenge, human neutrophils undergo rapid changes in nuclear architecture and chromatin folding to orchestrate an immediate inflammatory gene program

Denholtz

Zhu

et al. 2020

Genes Dev.

View full text Add to dashboard Cite

show abstract

“…To overcome the dual localization caveat, ACLSM was used in a superresolution fast mode to simultaneously track more than one channel at a time (Huff, 2016;Korobchevskaya et al, 2017). Despite the high resolution capacity of 2D SIM and TIRF-SIM, these platforms exhibit significant phototoxicity resulting in progressive photobleaching of fluorophores and cell damage after the time frames of observation used herein.…”

Section: Discussionmentioning

confidence: 99%

“…2D SIM was also used, but due to the configuration of the platform, it was possible to acquire images at the two channels sequentially with significant delays compared to ACLSM, where sequential imaging was done considerably faster. Moreover, owing to its detection principle, ACLSM allows the best photon collection even at suboptimal signal-to-noise ratios at approximately the same resolution as 2D SIM (Figures 1, 2; Huff, 2016).…”

Section: Detailed View On Map65-2 Colocalizing With Microtubulesmentioning

confidence: 99%

Complementary Superresolution Visualization of Composite Plant Microtubule Organization and Dynamics

et al. 2020

View full text Add to dashboard Cite

Microtubule bundling is an essential mechanism underlying the biased organization of interphase and mitotic microtubular systems of eukaryotes in ordered arrays. Microtubule bundle formation can be exemplified in plants, where the formation of parallel microtubule systems in the cell cortex or the spindle midzone is largely owing to the microtubule crosslinking activity of a family of microtubule associated proteins, designated as MAP65s. Among the nine members of this family in Arabidopsis thaliana, MAP65-1 and MAP65-2 are ubiquitous and functionally redundant. Crosslinked microtubules can form high-order arrays, which are difficult to track using widefield or confocal laser scanning microscopy approaches. Here, we followed spatiotemporal patterns of MAP65-2 localization in hypocotyl cells of Arabidopsis stably expressing fluorescent protein fusions of MAP65-2 and tubulin. To circumvent imaging difficulties arising from the density of cortical microtubule bundles, we use different superresolution approaches including Airyscan confocal laser scanning microscopy (ACLSM), structured illumination microscopy (SIM), total internal reflection SIM (TIRF-SIM), and photoactivation localization microscopy (PALM). We provide insights into spatiotemporal relations between microtubules and MAP65-2 crossbridges by combining SIM and ACLSM. We obtain further details on MAP65-2 distribution by single molecule localization microscopy (SMLM) imaging of either mEos3.2-MAP65-2 stochastic photoconversion, or eGFP-MAP65-2 stochastic emission fluctuations under specific illumination conditions. Time-dependent dynamics of MAP65-2 were tracked at variable time resolution using SIM, TIRF-SIM, and ACLSM and post-acquisition kymograph analysis. ACLSM imaging further allowed to track end-wise dynamics of microtubules labeled with TUA6-GFP and to correlate them with concomitant fluctuations of MAP65-2 tagged with tagRFP. All different microscopy modules examined herein are accompanied by restrictions in either the spatial resolution achieved, or in the frame rates of image acquisition. PALM imaging is compromised by speed of acquisition. This limitation was partially compensated by exploiting emission fluctuations of eGFP which allowed much higher photon counts at substantially smaller time series compared to mEos3.2. SIM, TIRF-SIM, and ACLSM

show abstract

“…This is repeated thousands of times. (Huff, 2016) and STED (12.5 Hz for 2.9×2.9 μm) (Yang et al, 2014), are more suitable for live-cell imaging. SIM is the most commonly used super-resolution imaging technique for live-cell imaging (Chen et al, 2018;Fiolka et al, 2012;Turcotte et al, 2019).…”

Section: Super-resolution Microscopymentioning

confidence: 99%

Imaging of the immune system – towards a subcellular and molecular understanding

Wen

Fan

Mikulski

et al. 2020

Journal of Cell Science

View full text Add to dashboard Cite

Immune responses involve many types of leukocytes that traffic to the site of injury, recognize the insult and respond appropriately. Imaging of the immune system involves a set of methods and analytical tools that are used to visualize immune responses at the cellular and molecular level as they occur in real time. We will review recent and emerging technological advances in optical imaging, and their application to understanding the molecular and cellular responses of neutrophils, macrophages and lymphocytes. Optical live-cell imaging provides deep mechanistic insights at the molecular, cellular, tissue and organism levels. Live-cell imaging can capture quantitative information in real time at subcellular resolution with minimal phototoxicity and repeatedly in the same living cells or in accessible tissues of the living organism. Advanced FRET probes allow tracking signaling events in live cells. Light-sheet microscopy allows for deeper tissue penetration in optically clear samples, enriching our understanding of the higher-level organization of the immune response. Super-resolution microscopy offers insights into compartmentalized signaling at a resolution beyond the diffraction limit, approaching single-molecule resolution. This Review provides a current perspective on live-cell imaging in vitro and in vivo with a focus on the assessment of the immune system.

show abstract

The Fast mode for ZEISS LSM 880 with Airyscan: high-speed confocal imaging with super-resolution and improved signal-to-noise ratio

Cited by 28 publications

References 6 publications

Upon microbial challenge, human neutrophils undergo rapid changes in nuclear architecture and chromatin folding to orchestrate an immediate inflammatory gene program

Upon microbial challenge, human neutrophils undergo rapid changes in nuclear architecture and chromatin folding to orchestrate an immediate inflammatory gene program

Complementary Superresolution Visualization of Composite Plant Microtubule Organization and Dynamics

Imaging of the immune system – towards a subcellular and molecular understanding

Contact Info

Product

Resources

About