scholarly journals Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

2018 ◽  
Author(s):  
Tsung-Li Liu ◽  
Srigokul Upadhyayula ◽  
Daniel E. Milkie ◽  
Ved Singh ◽  
Kai Wang ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
High Speed ◽  
Developmental Stages ◽  
Phenotypic Diversity ◽  
Metastatic Cancer ◽  
Light Sheet ◽  
Intrinsic Variability ◽  
Spatiotemporal Resolution ◽  
Light Sheet Microscopy

AbstractTrue physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution without inducing undue stress on either. We combined lattice light sheet microscopy with two-channel adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages, and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.One Sentence SummaryCombining lattice light sheet microscopy with adaptive optics enables high speed, high resolution in vivo 3D imaging of dynamic processes inside cells under physiological conditions within their parent organisms.

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

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.

scholarly journals Dual-view light-sheet imaging through tilted glass interface using a deformable mirror

2020 ◽  
Author(s):  
N Vladimirov ◽  
F Preusser ◽  
J Wisniewski ◽  
Z Yaniv ◽  
RA Desai ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
High Speed ◽  
Microfluidic Channel ◽  
Microfluidic Devices ◽  
Light Sheet ◽  
Stochastic Gradient Descent ◽  
Deformable Mirror ◽  
Optical Aberrations ◽  
Light Sheet Microscopy ◽  
Gradient Descent Algorithm

AbstractLight-sheet microscopy has become one of the primary tools for imaging live developing organisms because of its high speed, low phototoxicity, and optical sectioning capabilities. Detection from multiple sides (multi-view imaging) additionally allows nearly isotropic resolution via computational merging of the views. However, conventional light-sheet microscopes require that the sample is suspended in a gel to allow optical access from two or more sides. At the same time, the use of microfluidic devices is highly desirable for many experiments, but geometric constrains and strong optical aberrations caused by the coverslip titled relative to objectives make the use of multi-view lightsheet challenging for microfluidics.In this paper we describe the use of adaptive optics (AO) to enable multi-view light-sheet microscopy in such microfluidic setup by correcting optical aberrations introduced by the tilted coverslip. The optimal shape of deformable mirror is computed by an iterative stochastic gradient-descent algorithm that optimizes PSF in two orthogonal planes simultaneously. Simultaneous AO correction in two optical arms is achieved via a knife-edge mirror that splits excitation path and combines the detection path.We characterize the performance of this novel microscope setup and, by dual-view light-sheet imaging of C.elegans inside a microfluidic channel, demonstrate a drastic improvement of image quality due to AO and dual-view reconstruction. Our microscope design allows multi-view light-sheet microscopy with microfluidic devices for precisely controlled experimental conditions and high-content screening.

scholarly journals Light-Sheet Microscopy in Neuroscience

2019 ◽  
Vol 42 (1) ◽  
pp. 295-313 ◽  
Author(s):  
Elizabeth M.C. Hillman ◽  
Venkatakaushik Voleti ◽  
Wenze Li ◽  
Hang Yu
Keyword(s):  
High Speed ◽  
Light Sheet ◽  
Mammalian Brain ◽  
Diverse Range ◽  
Volumetric Imaging ◽  
Two Photon Microscopy ◽  
Light Sheet Microscopy ◽  
Noise Benefits ◽  
Longitudinal Imaging

Light-sheet microscopy is an imaging approach that offers unique advantages for a diverse range of neuroscience applications. Unlike point-scanning techniques such as confocal and two-photon microscopy, light-sheet microscopes illuminate an entire plane of tissue, while imaging this plane onto a camera. Although early implementations of light sheet were optimized for longitudinal imaging of embryonic development in small specimens, emerging implementations are capable of capturing light-sheet images in freely moving, unconstrained specimens and even the intact in vivo mammalian brain. Meanwhile, the unique photobleaching and signal-to-noise benefits afforded by light-sheet microscopy's parallelized detection deliver the ability to perform volumetric imaging at much higher speeds than can be achieved using point scanning. This review describes the basic principles and evolution of light-sheet microscopy, followed by perspectives on emerging applications and opportunities for both imaging large, cleared, and expanded neural tissues and high-speed, functional imaging in vivo.

scholarly journals Optical Sectioning of Live Mammal with Near-Infrared Light Sheet

2018 ◽  
Author(s):  
Feifei Wang ◽  
Hao Wan ◽  
Jingying Yue ◽  
Mingxi Zhang ◽  
Zhuoran Ma ◽  
...  
Keyword(s):  
Near Infrared ◽  
Optical Excitation ◽  
Light Sheet ◽  
Infrared Light ◽  
Optical Sectioning ◽  
Spatiotemporal Resolution ◽  
Near Infrared Light ◽  
Light Sheet Microscopy ◽  
Non Invasive

AbstractDeep-tissue three-dimensional optical imaging of live mammals in vivo with high spatiotemporal resolution in non-invasive manners has been challenging due to light scattering. Here, we developed near-infrared (NIR) light sheet microscopy (LSM) with optical excitation and emission wavelengths up to ~ 1320 nm and ~ 1700 nm respectively, far into the NIR-II (1000-1700 nm) region for 3D optical sectioning through live tissues. Suppressed scattering of both excitation and emission photons allowed one-photon optical sectioning at ~ 2 mm depth in highly scattering brain tissues. NIR-II LSM enabled non-invasive in vivo imaging of live mice, revealing never-before-seen dynamic processes such as highly abnormal tumor microcirculation, and 3D molecular imaging of an important immune checkpoint protein, programmed-death ligand 1 (PD-L1) receptors at the single cell scale in tumors. In vivo two-color near-infrared light sheet sectioning enabled simultaneous volumetric imaging of tumor vasculatures and PD-L1 proteins in live mammals.

scholarly journals Cell-accurate optical mapping across the entire developing heart

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Michael Weber ◽  
Nico Scherf ◽  
Alexander M Meyer ◽  
Daniela Panáková ◽  
Peter Kohl ◽  
...  
Keyword(s):  
High Speed ◽  
Cell Function ◽  
Three Dimensional ◽  
Light Sheet ◽  
Tissue Level ◽  
In Vivo Studies ◽  
Light Sheet Microscopy ◽  
Developing Heart ◽  
And Function

Organogenesis depends on orchestrated interactions between individual cells and morphogenetically relevant cues at the tissue level. This is true for the heart, whose function critically relies on well-ordered communication between neighboring cells, which is established and fine-tuned during embryonic development. For an integrated understanding of the development of structure and function, we need to move from isolated snap-shot observations of either microscopic or macroscopic parameters to simultaneous and, ideally continuous, cell-to-organ scale imaging. We introduce cell-accurate three-dimensional Ca2+-mapping of all cells in the entire electro-mechanically uncoupled heart during the looping stage of live embryonic zebrafish, using high-speed light sheet microscopy and tailored image processing and analysis. We show how myocardial region-specific heterogeneity in cell function emerges during early development and how structural patterning goes hand-in-hand with functional maturation of the entire heart. Our method opens the way to systematic, scale-bridging, in vivo studies of vertebrate organogenesis by cell-accurate structure-function mapping across entire organs.

scholarly journals In vivo NIR-II structured-illumination light-sheet microscopy

2021 ◽  
Vol 118 (6) ◽  
pp. e2023888118
Author(s):  
Feifei Wang ◽  
Zhuoran Ma ◽  
Yeteng Zhong ◽  
Felix Salazar ◽  
Chun Xu ◽  
...  
Keyword(s):  
Light Scattering ◽  
Molecular Imaging ◽  
Single Cell ◽  
Light Sheet ◽  
Single Cell Level ◽  
Structured Illumination ◽  
Cell Level ◽  
Spatiotemporal Resolution ◽  
Light Sheet Microscopy

Noninvasive optical imaging with deep tissue penetration depth and high spatiotemporal resolution is important to longitudinally studying the biology at the single-cell level in live mammals, but has been challenging due to light scattering. Here, we developed near-infrared II (NIR-II) (1,000 to 1,700 nm) structured-illumination light-sheet microscopy (NIR-II SIM) with ultralong excitation and emission wavelengths up to ∼1,540 and ∼1,700 nm, respectively, suppressing light scattering to afford large volumetric three-dimensional (3D) imaging of tissues with deep-axial penetration depths. Integrating structured illumination into NIR-II light-sheet microscopy further diminished background and improved spatial resolution by approximately twofold. In vivo oblique NIR-II SIM was performed noninvasively for 3D volumetric multiplexed molecular imaging of the CT26 tumor microenvironment in mice, longitudinally mapping out CD4, CD8, and OX40 at the single-cell level in response to immunotherapy by cytosine-phosphate-guanine (CpG), a Toll-like receptor 9 (TLR-9) agonist combined with OX40 antibody treatment. NIR-II SIM affords an additional tool for noninvasive volumetric molecular imaging of immune cells in live mammals.

scholarly journals Developmental adaptations of trypanosome motility to the tsetse fly host environments unravel a multifaceted in vivo microswimmer system

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sarah Schuster ◽  
Timothy Krüger ◽  
Ines Subota ◽  
Sina Thusek ◽  
Brice Rotureau ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
High Speed ◽  
Developmental Stages ◽  
Light Sheet ◽  
Infection Process ◽  
Tsetse Fly ◽  
Complex Life Cycle ◽  
3D Analysis ◽  
Cell Architecture

The highly motile and versatile protozoan pathogen Trypanosoma brucei undergoes a complex life cycle in the tsetse fly. Here we introduce the host insect as an expedient model environment for microswimmer research, as it allows examination of microbial motion within a diversified, secluded and yet microscopically tractable space. During their week-long journey through the different microenvironments of the fly´s interior organs, the incessantly swimming trypanosomes cross various barriers and confined surroundings, with concurrently occurring major changes of parasite cell architecture. Multicolour light sheet fluorescence microscopy provided information about tsetse tissue topology with unprecedented resolution and allowed the first 3D analysis of the infection process. High-speed fluorescence microscopy illuminated the versatile behaviour of trypanosome developmental stages, ranging from solitary motion and near-wall swimming to collective motility in synchronised swarms and in confinement. We correlate the microenvironments and trypanosome morphologies to high-speed motility data, which paves the way for cross-disciplinary microswimmer research in a naturally evolved environment.

Light-Sheet Microscopy and Its Potential for Understanding Developmental Processes

2019 ◽  
Vol 35 (1) ◽  
pp. 655-681 ◽  
Author(s):  
Yinan Wan ◽  
Katie McDole ◽  
Philipp J. Keller
Keyword(s):  
Developmental Biology ◽  
Single Molecule ◽  
Light Sheet ◽  
Experimental Investigations ◽  
Biological Processes ◽  
Spatiotemporal Resolution ◽  
Developmental Processes ◽  
Light Sheet Microscopy ◽  
Good Spatial Resolution

The ability to visualize and quantitatively measure dynamic biological processes in vivo and at high spatiotemporal resolution is of fundamental importance to experimental investigations in developmental biology. Light-sheet microscopy is particularly well suited to providing such data, since it offers exceptionally high imaging speed and good spatial resolution while minimizing light-induced damage to the specimen. We review core principles and recent advances in light-sheet microscopy, with a focus on concepts and implementations relevant for applications in developmental biology. We discuss how light-sheet microcopy has helped advance our understanding of developmental processes from single-molecule to whole-organism studies, assess the potential for synergies with other state-of-the-art technologies, and introduce methods for computational image and data analysis. Finally, we explore the future trajectory of light-sheet microscopy, discuss key efforts to disseminate new light-sheet technology, and identify exciting opportunities for further advances.

scholarly journals Spatiotemporal sensitivity of embryonic heart specification to FGFR signaling in Drosophila

2020 ◽  
Author(s):  
V. Yadav ◽  
N. Tolwinski ◽  
T. E. Saunders
Keyword(s):  
Cell Fate ◽  
Fibroblast Growth Factor Receptor ◽  
Early Stage ◽  
Growth Factor Receptor ◽  
Light Sheet ◽  
Heart Cells ◽  
Spatiotemporal Resolution ◽  
Light Sheet Microscopy ◽  
Spatiotemporal Regulation

ABSTRACTDevelopment of the Drosophila embryonic mesoderm is controlled through both internal and external inputs to the mesoderm. One such factor is Heartless (Htl), a Fibroblast Growth Factor Receptor (FGFR) expressed in the mesoderm. Htl is involved in shaping the mesoderm at both early and later stages during embryogenesis. How Htl expression levels and timing of signaling affect mesoderm morphogenesis after spreading remains elusive. We have developed an optogenetic tool (Opto-htl) to control the activation of Htl signaling with precise spatiotemporal resolution in vivo. We find that the embryo is most sensitive to Htl over-activation within a developmental window of ~4 hours ranging from late stage 10 until early stage 13, which corresponds to early stages of heart morphogenesis. Opto-htl restores heart cells in htl mutants upon light activation, independent of its role in early mesoderm shaping events. We also successfully generated spatially distinct regions of Htl activity in the mesoderm using light-sheet microscopy. The developing tissue was unable to correct for the ensuing asymmetries in cell fate. Overall, Opto-htl is a powerful tool for studying spatiotemporal regulation of Htl signaling during embryogenesis.

scholarly journals Cell-accurate optical mapping across the entire developing heart

2017 ◽  
Author(s):  
Michael Weber ◽  
Nico Scherf ◽  
Peter Kohl ◽  
Jan Huisken
Keyword(s):  
High Speed ◽  
Cell Function ◽  
Three Dimensional ◽  
Light Sheet ◽  
Tissue Level ◽  
Light Sheet Microscopy ◽  
Functional Maturation ◽  
Developing Heart ◽  
And Function

AbstractOrganogenesis depends on orchestrated interactions between individual cells and morphogenically relevant cues at the tissue level. This is true for the heart, whose function critically relies on well-ordered communication between neighbouring cells, which is established and fine-tuned during development. For an integrated understanding of the development of structure and function, we need to move from isolated snap-shot observations of either microscopic or macroscopic parameters to simultaneous and, ideally continuous, cell-to-organ scale imaging. We introduce cell-accurate three-dimensional Ca2+-mapping of all cells in the entire heart during the looping stage in live embryonic zebrafish, using high-speed light sheet microscopy and tailored image processing and analysis. We show how myocardial region-specific heterogeneity in cell function emerges during early development and how structural patterning goes hand-in-hand with functional maturation of the entire heart. Our method opens the way to systematic, scale-bridging, in vivo studies of vertebrate organogenesis by cell-accurate structure-function mapping across entire organs.

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