Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy

Membrane remodeling is an essential part of transferring components to and from the cell surface and membrane-bound organelles and for changes in cell shape, which are particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging and mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and noninvasive illumination of the newly developed lattice light-sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and found that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and was relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light-sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and multicellular assemblies.

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Tracking cells in epithelial acini by light sheet microscopy reveals proximity effects in breast cancer initiation

eLife ◽

10.7554/elife.54066 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 4

Author(s):

Ashna Alladin ◽

Lucas Chaible ◽

Lucia Garcia del Valle ◽

Reither Sabine ◽

Monika Loeschinger ◽

...

Keyword(s):

Single Cells ◽

Image Data ◽

Integrated Approach ◽

Light Sheet ◽

Mammary Epithelial ◽

Proximity Effects ◽

Transformed Cells ◽

Light Sheet Microscopy ◽

Primary Mouse ◽

Longitudinal Imaging

Cancer clone evolution takes place within tissue ecosystem habitats. But, how exactly tumors arise from a few malignant cells within an intact epithelium is a central, yet unanswered question. This is mainly due to the inaccessibility of this process to longitudinal imaging together with a lack of systems that model the progression of a fraction of transformed cells within a tissue. Here, we developed a new methodology based on primary mouse mammary epithelial acini, where oncogenes can be switched on in single cells within an otherwise normal epithelial cell layer. We combine this stochastic breast tumor induction model with inverted light-sheet imaging to study single-cell behavior for up to four days and analyze cell fates utilizing a newly developed image-data analysis workflow. The power of this integrated approach is illustrated by us finding that small local clusters of transformed cells form tumors while isolated transformed cells do not.

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Augmin accumulation on long-lived microtubules drives amplification and kinetochore-directed growth

The Journal of Cell Biology ◽

10.1083/jcb.201805044 ◽

2019 ◽

Vol 218 (7) ◽

pp. 2150-2168 ◽

Cited By ~ 23

Author(s):

Ana F. David ◽

Philippe Roudot ◽

Wesley R. Legant ◽

Eric Betzig ◽

Gaudenz Danuser ◽

...

Keyword(s):

Spindle Assembly ◽

Light Sheet ◽

Spindle Pole ◽

Directional Bias ◽

Fiber Assembly ◽

Light Sheet Microscopy ◽

Sister Chromatids ◽

Spindle Dynamics ◽

Dividing Cells ◽

Microtubule Growth

Dividing cells reorganize their microtubule cytoskeleton into a bipolar spindle, which moves one set of sister chromatids to each nascent daughter cell. Early spindle assembly models postulated that spindle pole–derived microtubules search the cytoplasmic space until they randomly encounter a kinetochore to form a stable attachment. More recent work uncovered several additional, centrosome-independent microtubule generation pathways, but the contributions of each pathway to spindle assembly have remained unclear. Here, we combined live microscopy and mathematical modeling to show that most microtubules nucleate at noncentrosomal regions in dividing human cells. Using a live-cell probe that selectively labels aged microtubule lattices, we demonstrate that the distribution of growing microtubule plus ends can be almost entirely explained by Augmin-dependent amplification of long-lived microtubule lattices. By ultrafast 3D lattice light-sheet microscopy, we observed that this mechanism results in a strong directional bias of microtubule growth toward individual kinetochores. Our systematic quantification of spindle dynamics reveals highly coordinated microtubule growth during kinetochore fiber assembly.

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Rapid Whole Cell Imaging Reveals A Calcium-APPL1-Dynein Nexus That Regulates Cohort Trafficking of Stimulated EGF Receptors

10.1101/481796 ◽

2018 ◽

Author(s):

H M York ◽

A Patil ◽

U K Moorthi ◽

A Kaur ◽

A Bhowmik ◽

...

Keyword(s):

Signal Processing ◽

Cell Imaging ◽

Light Sheet ◽

Endosomal Trafficking ◽

Egf Receptors ◽

Whole Cell ◽

Cellular Processes ◽

Light Sheet Microscopy ◽

Signaling Receptors ◽

Cell Surface Signaling

ABSTRACTMulticellular life processes such as proliferation and differentiation depend on cell surface signaling receptors that bind ligands generally referred to as growth factors. Recently, it has emerged that the endosomal system provides rich signal processing capabilities for responses elicited by these factors [1-3]. At the single cell level, endosomal trafficking becomes a critical component of signal processing, as exemplified by the epidermal growth factor (EGF) receptors of the receptor tyrosine kinase family. EGFRs, once activated by EGF, are robustly trafficked to the phosphatase-enriched peri-nuclear region (PNR), where they are dephosphorylated [4-8]. However, the details of the mechanisms regulating the movements of stimulated EGFR in time and space, i.e., towards the PNR, are not known. What endosomal regulators provide specificity to EGFR? Do modifications to the receptor upon stimulation regulate its trafficking? To understand the events leading to EGFR translocation, and especially the early endosomal dynamics that immediately follow EGFR internalization, requires the real-time, long-term, whole-cell imaging of multiple elements. Here, exploiting the advantages of lattice light-sheet microscopy [9], we show that the binding of EGF by its receptor, EGFR, triggers a transient calcium increase that peaks by 30 s, causing the desorption of APPL1 from pre-existing endosomes within one minute, the rebinding of liberated APPL1 to EGFR within three minutes, and the dynein-dependent translocation of APPL1-EGF-bearing endosomes to the PNR within five minutes. The novel, cell spanning, fast acting network that we reveal integrates a cascade of events dedicated to the cohort movement of activated EGFR receptors. Our findings support the intriguing proposal that certain endosomal pathways have shed some of the stochastic strategies of traditional trafficking, and have evolved behaviors whose predictability is better suited to signaling [10, 11]. Work presented here demonstrates that our whole cell imaging approach can be a powerful tool in revealing critical transient interactions in key cellular processes such as receptor trafficking.

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Reflective imaging improves resolution, speed, and collection efficiency in light sheet microscopy

10.1101/154807 ◽

2017 ◽

Author(s):

Yicong Wu ◽

Abhishek Kumar ◽

Corey Smith ◽

Evan Ardiel ◽

Panagiotis Chandris ◽

...

Keyword(s):

High Resolution ◽

High Speed ◽

Collection Efficiency ◽

Single Cells ◽

Numerical Aperture ◽

Light Sheet ◽

Three Dimensions ◽

Spatiotemporal Resolution ◽

Light Sheet Microscopy ◽

Simultaneous Acquisition

AbstractLight-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, gentle imaging of live biological specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of multiple views, obtaining 4 complementary views in 250 ms, half the period it would otherwise take to collect only two views in symmetric dual-view selective plane illumination microscopy (diSPIM). We also report a modified deconvolution algorithm that removes the associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to < 300 nm in all three dimensions) by applying our method to a new asymmetric diSPIM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture (NA). We demonstrate the broad applicability of our method in a variety of samples of moderate (< 50 μm) thickness, studying mitochondrial, membrane, Golgi, and microtubule dynamics in single cells and calcium activity in nematode embryos.

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Proteomic, biomechanical and functional analyses define neutrophil heterogeneity in systemic lupus erythematosus

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-218338 ◽

2020 ◽

pp. annrheumdis-2020-218338

Author(s):

Kathleen R Bashant ◽

Angel M Aponte ◽

Davide Randazzo ◽

Paniz Rezvan Sangsari ◽

Alexander JT Wood ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

Cell Surface ◽

Lupus Erythematosus ◽

Light Sheet ◽

Normal Density ◽

Two Dimensional ◽

Biophysical Properties ◽

Systemic Lupus ◽

Light Sheet Microscopy ◽

Functional Analyses

ObjectivesLow-density granulocytes (LDGs) are a distinct subset of proinflammatory and vasculopathic neutrophils expanded in systemic lupus erythematosus (SLE). Neutrophil trafficking and immune function are intimately linked to cellular biophysical properties. This study used proteomic, biomechanical and functional analyses to further define neutrophil heterogeneity in the context of SLE.MethodsProteomic/phosphoproteomic analyses were performed in healthy control (HC) normal density neutrophils (NDNs), SLE NDNs and autologous SLE LDGs. The biophysical properties of these neutrophil subsets were analysed by real-time deformability cytometry and lattice light-sheet microscopy. A two-dimensional endothelial flow system and a three-dimensional microfluidic microvasculature mimetic (MMM) were used to decouple the contributions of cell surface mediators and biophysical properties to neutrophil trafficking, respectively.ResultsProteomic and phosphoproteomic differences were detected between HC and SLE neutrophils and between SLE NDNs and LDGs. Increased abundance of type 1 interferon-regulated proteins and differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs. The cell surface of SLE LDGs was rougher than in SLE and HC NDNs, suggesting membrane perturbances. While SLE LDGs did not display increased binding to endothelial cells in the two-dimensional assay, they were increasingly retained/trapped in the narrow channels of the lung MMM.ConclusionsModulation of the neutrophil proteome and distinct changes in biophysical properties are observed alongside differences in neutrophil trafficking. SLE LDGs may be increasingly retained in microvasculature networks, which has important pathogenic implications in the context of lupus organ damage and small vessel vasculopathy.

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Acoustic light-sheet microscopy

10.1101/2021.08.20.457051 ◽

2021 ◽

Author(s):

Stefan Wunderl ◽

Ayumu Ishijima ◽

Etsuo Susaki ◽

Zihui Xu ◽

Hong Song ◽

...

Keyword(s):

Light Propagation ◽

High Spatial Resolution ◽

Single Cells ◽

Acoustic Pulse ◽

Light Sheet ◽

Wide Field ◽

Axial Resolution ◽

3D Objects ◽

Light Sheet Microscopy ◽

The Brain

Light-sheet imaging of 3D objects with high spatial resolution remains an open challenge because of the trade-off between field-of-view (FOV) and axial resolution originating from the diffraction of light. We developed acoustic light-sheet microscopy (acoustic LSM), which actively manipulates the light propagation inside a large sample to obtain wide-field microscopic images deep inside a target. By accurately coupling a light-sheet illumination pulse into a planar acoustic pulse, the light-sheet can be continuously guided over large distances. We imaged a fluorescence-labeled transparent mouse brain for the FOVs of 19.3 x 12.4 mm2 and 9.7 x 5.9 mm2 with resolved microstructures and single cells deep inside the brain. Acoustic LSM creates new opportunities for the application of light-sheet in the field of industry to basic science.

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Accelerating iterative deconvolution and multiview fusion by orders of magnitude

10.1101/647370 ◽

2019 ◽

Cited By ~ 4

Author(s):

Min Guo ◽

Yue Li ◽

Yijun Su ◽

Talley Lambert ◽

Damian Dalle Nogare ◽

...

Keyword(s):

Software Design ◽

Single Cells ◽

Light Sheet ◽

Mouse Tissue ◽

3D Image ◽

Light Sheet Microscopy ◽

Point Spread ◽

Spread Function ◽

Gpu Processing ◽

Spatially Varying

AbstractWe describe theoretical and practical advances in algorithm and software design, resulting in ten to several thousand-fold faster deconvolution and multiview fusion than previous methods. First, we adapt methods from medical imaging, showing that an unmatched back projector accelerates Richardson-Lucy deconvolution by at least 10-fold, in most cases requiring only a single iteration. Second, we show that improvements in 3D image-based registration with GPU processing result in speedups of 10-100-fold over CPU processing. Third, we show that deep learning can provide further accelerations, particularly for deconvolution with a spatially varying point spread function. We illustrate the power of our methods from the subcellular to millimeter spatial scale, on diverse samples including single cells, nematode and zebrafish embryos, and cleared mouse tissue. Finally, we show that our methods facilitate the use of new microscopes that improve spatial resolution, including dual-view cleared tissue light-sheet microscopy and reflective lattice light-sheet microscopy.

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Cell fate coordinates mechano-osmotic forces in intestinal crypt morphogenesis

10.1101/2020.05.13.094359 ◽

2020 ◽

Cited By ~ 3

Author(s):

Qiutan Yang ◽

Shi-Lei Xue ◽

Chii Jou Chan ◽

Markus Rempfler ◽

Dario Vischi ◽

...

Keyword(s):

Cell Fate ◽

Single Cells ◽

Light Sheet ◽

Volume Reduction ◽

Biophysical Model ◽

Cell Swelling ◽

Specific Cell ◽

Light Sheet Microscopy ◽

Lumen Volume ◽

Sodium Glucose Cotransporter

AbstractIntestinal organoids derived from single cells undergo complex crypt-villus patterning and morphogenesis. However, the nature and coordination of the underlying forces remains poorly characterized. Through light-sheet microscopy and mechanical perturbations, we demonstrate that organoid crypt formation coincides with stark lumen volume reduction, which works synergistically with actomyosin-generated crypt apical and villus basal tension to drive morphogenesis. We analyse these mechanical features in a quantitative 3D biophysical model and detect a critical point in actomyosin tensions, above which crypt becomes robust to volume changes. Finally, via single-cell RNA sequencing and pharmacological perturbations, we show that enterocyte-specific expressed sodium/glucose cotransporter modulates lumen volume reduction via promoting cell swelling. Altogether, our study reveals how cell fate-specific changes in osmotic and actomyosin forces coordinate robust organoid morphogenesis.One Sentence SummaryEmergence of region-specific cell fates drive actomyosin patterns and luminal osmotic changes in organoid development

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The ‘super-resolution’ revolution

Biochemical Society Transactions ◽

10.1042/bst0371042 ◽

2009 ◽

Vol 37 (5) ◽

pp. 1042-1044 ◽

Cited By ~ 12

Author(s):

Ilan Davis

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Super Resolution ◽

Light Sheet ◽

Live Cell ◽

Stimulated Emission ◽

Structured Illumination ◽

Light Sheet Microscopy ◽

Resolution Imaging ◽

New Instruments

We are currently in the midst of an exciting revolution in microscopy. In many ways, this has been happening for several decades, but it is the rate of development of new methods that has increased recently. The last few years have seen an impressive proliferation of new instruments for imaging at higher resolution, imaging single molecules and faster and more sensitive multidimensional live cell imaging. These include light sheet microscopy, stimulated emission depletion, structured illumination and live cell imaging on the OMX (optical microscopy experimental) platform. However, new probes and image analysis methods have also been crucial for the development of these revolutionary methods.

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