An ensemble-averaged, cell density-based digital model of zebrafish embryo development derived from light-sheet microscopy data with single-cell resolution

AbstractWhile immunoassays and mass spectrometry are powerful single-cell protein analysis tools, bottlenecks remain in interfacing and throughput. Here, we introduce highly parallel, synchronous, three-dimensional single-cell immunoblots to detect both cytosolic and nuclear proteins. The novel threedimensional microfluidic device is a photoactive polyacrylamide gel with a high-density microwell array patterned on one face (x-y) for cell isolation and lysis. From each microwell, single-cell lysate is ‘electrophoretically projected’ into the 3rd dimension (z-axis), separated by size, and photo-captured for immunoprobing and three-dimensional interrogation by confocal/light sheet microscopy. Design guidelines for throughput and separation performance are informed by simulation, analyses, and deconvolution postprocessing based on physics of 3D diffusion. Importantly, separations are nearly synchronous, whereas serial analyses can impart hours of delay between the first and last cell. We achieve an electrophoresis throughput of >2.5 cells/s (70X faster than serial sampling) and perform 25 immunoblots/mm2 device area (>10X increase over previous immunoblots). A straightforward device for parallel single-cell immunoblotting, projection electrophoresis promises to advance integration of protein-level profiles into the emerging single-cell atlas of genomic and transcriptomic profiles.

Download Full-text

Reconstruction of cell lineages and behaviors underlying arthropod limb outgrowth with multi-view light-sheet imaging and tracking

10.1101/112623 ◽

2017 ◽

Cited By ~ 3

Author(s):

Carsten Wolff ◽

Jean-Yves Tinevez ◽

Tobias Pietzsch ◽

Evangelia Stamataki ◽

Benjamin Harich ◽

...

Keyword(s):

Cell Proliferation ◽

Single Cell ◽

Limb Development ◽

Light Sheet ◽

Limb Bud ◽

List Type ◽

Cell Behaviors ◽

Light Sheet Microscopy ◽

Organ Morphogenesis ◽

Parhyale Hawaiensis

SUMMARYDuring development coordinated cell behaviors orchestrate tissue and organ morphogenesis to suit the lifestyle of the organism. We have used here the crustacean Parhyale hawaiensis to study the cellular basis of limb development. Transgenic Parhyale embryos with fluorescently labeled nuclei were imaged at high spatiotemporal resolution with multi-view light-sheet fluorescence microscopy over several days of embryogenesis spanning appendage morphogenesis from early specification up to late differentiation stages. Cell tracking with a new tool called Massive Multi-view Tracker (MaMuT) enabled the reconstruction of the complete cell lineage of an outgrowing thoracic limb with single-cell resolution. In silico clonal analyses suggested that the limb primordium becomes subdivided from an early stage first into anterior-posterior and then into dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb bud formation is associated with the spatial modulation of cell proliferation, while limb elongation is also driven by the preferential orientation of division of epidermal cells along the proximal-distal axis of growth. Cellular reconstructions were predictive of the expression patterns of limb development genes including the Decapentaplegic (Dpp) morphogen.HIGHLIGHTSMulti-view light-sheet microscopy of crustacean embryos from species Parhyale hawaiensis are ideal for cellular-level analysis of organ morphogenesis.Lineages of 3-dimensional organs were reconstructed at single-cell resolution with the Fiji/ImageJ plugin Massive Multi-view Tracker.The Parhyale limb primordium undergoes early lineage restrictions associated with particular cell behaviors and patterns of gene expression.Differential rates of cell proliferation and oriented cell divisions guide appendage proximal-distal outgrowth.

Download Full-text

Recent Progress in Light Sheet Microscopy for Biological Applications

Applied Spectroscopy ◽

10.1177/0003702818778851 ◽

2018 ◽

Vol 72 (8) ◽

pp. 1137-1169 ◽

Cited By ~ 19

Author(s):

Krishnendu Chatterjee ◽

Feby Wijaya Pratiwi ◽

Frances Camille M. Wu ◽

Peilin Chen ◽

Bi-Chang Chen

Keyword(s):

Developmental Biology ◽

Fluorescence Microscopy ◽

Single Cell ◽

Single Molecule ◽

Super Resolution ◽

Light Sheet ◽

High Signal ◽

Spatiotemporal Resolution ◽

Light Sheet Microscopy ◽

Resolution Imaging

The introduction of light sheet fluorescence microscopy (LSFM) has overcome the challenges in conventional optical microscopy. Among the recent breakthroughs in fluorescence microscopy, LSFM had been proven to provide a high three-dimensional spatial resolution, high signal-to-noise ratio, fast imaging acquisition rate, and minuscule levels of phototoxic and photodamage effects. The aforementioned auspicious properties are crucial in the biomedical and clinical research fields, covering a broad range of applications: from the super-resolution imaging of intracellular dynamics in a single cell to the high spatiotemporal resolution imaging of developmental dynamics in an entirely large organism. In this review, we provided a systematic outline of the historical development of LSFM, detailed discussion on the variants and improvements of LSFM, and delineation on the most recent technological advancements of LSFM and its potential applications in single molecule/particle detection, single-molecule super-resolution imaging, imaging intracellular dynamics of a single cell, multicellular imaging: cell–cell and cell–matrix interactions, plant developmental biology, and brain imaging and developmental biology.

Download Full-text

Lineage recording in human cerebral organoids

Nature Methods ◽

10.1038/s41592-021-01344-8 ◽

2021 ◽

Author(s):

Zhisong He ◽

Ashley Maynard ◽

Akanksha Jain ◽

Tobias Gerber ◽

Rebecca Petri ◽

...

Keyword(s):

Single Cell ◽

Time Window ◽

Induced Pluripotent Stem Cell ◽

Light Sheet ◽

Human Organ ◽

Light Sheet Microscopy ◽

Induced Pluripotent ◽

Fate Restriction ◽

Gene Perturbation

AbstractInduced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR–Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.

Download Full-text

High-Resolution Imaging of Single-Cell Behaviors in 3D Bacterial Biofilms using Lattice-Light Sheet Microscopy and Deep Learning-Based Image Processing

Microscopy and Microanalysis ◽

10.1017/s1431927621010540 ◽

2021 ◽

Vol 27 (S1) ◽

pp. 3038-3040

Author(s):

Ji Zhang ◽

Yibo Wang ◽

Mingxing Zhang ◽

Alecia Achimovich ◽

Jie Wang ◽

...

Keyword(s):

Image Processing ◽

Deep Learning ◽

High Resolution ◽

Single Cell ◽

Light Sheet ◽

Bacterial Biofilms ◽

High Resolution Imaging ◽

Cell Behaviors ◽

Light Sheet Microscopy ◽

Resolution Imaging

Download Full-text

BigStitcher: Reconstructing high-resolution image datasets of cleared and expanded samples

10.1101/343954 ◽

2018 ◽

Cited By ~ 12

Author(s):

David Hörl ◽

Fabio Rojas Rusak ◽

Friedrich Preusser ◽

Paul Tillberg ◽

Nadine Randel ◽

...

Keyword(s):

High Resolution ◽

Single Cell ◽

Light Sheet ◽

Resolution Image ◽

Light Sheet Microscopy ◽

High Resolution Image ◽

Biological Objects ◽

New Methods ◽

Biological Studies ◽

Optical Effects

New methods for clearing and expansion of biological objects create large, transparent samples that can be rapidly imaged using light-sheet microscopy. Resulting image acquisitions are terabytes in size and consist of many large, unaligned image tiles that suffer from optical distortions. We developed the BigStitcher software that efficiently handles and reconstructs large multi-tile, multi-view acquisitions compensating all major optical effects, thereby making single-cell resolved whole-organ datasets amenable to biological studies.

Download Full-text