Philipp J. Keller

Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy

A long-standing goal of biology is to map the behavior of all cells during vertebrate embryogenesis. We developed digital scanned laser light sheet fluorescence microscopy and recorded nuclei localization and movement in entire wild-type and mutant zebrafish embryos over the first 24 hours of development. Multiview in vivo imaging at 1.5 billion voxels per minute provides “digital embryos,” that is, comprehensive databases of cell positions, divisions, and migratory tracks. Our analysis of global cell division patterns reveals a maternally defined initial morphodynamic symmetry break, which identifies the embryonic body axis. We further derive a model of germ layer formation and show that the mesendoderm forms from one-third of the embryos cells in a single event. Our digital embryos, with 55 million nucleus entries, are provided as a resource.

Whole-brain functional imaging at cellular resolution using light-sheet microscopy

Brain function relies on communication between large populations of neurons across multiple brain areas, a full understanding of which would require knowledge of the time-varying activity of all neurons in the central nervous system. Here we use light-sheet microscopy to record activity, reported through the genetically encoded calcium indicator GCaMP5G, from the entire volume of the brain of the larval zebrafish in vivo at 0.8 Hz, capturing more than 80% of all neurons at single-cell resolution. Demonstrating how this technique can be used to reveal functionally defined circuits across the brain, we identify two populations of neurons with correlated activity patterns. One circuit consists of hindbrain neurons functionally coupled to spinal cord neuropil. The other consists of an anatomically symmetric population in the anterior hindbrain, with activity in the left and right halves oscillating in antiphase, on a timescale of 20 s, and coupled to equally slow oscillations in the inferior olive.

Fast, high-contrast imaging of animal development with scanned light sheet–based structured-illumination microscopy

Recording light-microscopy images of large, nontransparent specimens, such as developing multicellular organisms, is complicated by decreased contrast resulting from light scattering. Early zebrafish development can be captured by standard light-sheet microscopy, but new imaging strategies are required to obtain high-quality data of late development or of less transparent organisms. We combined digital scanned laser light-sheet fluorescence microscopy with incoherent structured-illumination microscopy (DSLM-SI) and created structured-illumination patterns with continuously adjustable frequencies. Our method discriminates the specimen-related scattered background from signal fluorescence, thereby removing out-of-focus light and optimizing the contrast of in-focus structures. DSLM-SI provides rapid control of the illumination pattern, exceptional imaging quality and high imaging speeds. We performed long-term imaging of zebrafish development for 58 h and fast multiple-view imaging of early Drosophila melanogaster development. We reconstructed cell positions over time from the Drosophila DSLM-SI data and created a fly digital embryo.

Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy

Live imaging of large biological specimens is fundamentally limited by the short optical penetration depth of light microscopes. To maximize physical coverage, we developed the SiMView technology framework for high-speed in vivo imaging, which records multiple views of the specimen simultaneously. SiMView consists of a light-sheet microscope with four synchronized optical arms, real-time electronics for long-term sCMOS-based image acquisition at 175 million voxels per second, and computational modules for high-throughput image registration, segmentation, tracking and real-time management of the terabytes of multiview data recorded per specimen. We developed one-photon and multiphoton SiMView implementations and recorded cellular dynamics in entire Drosophila melanogaster embryos with 30-s temporal resolution throughout development. We furthermore performed high-resolution long-term imaging of the developing nervous system and followed neuroblast cell lineages in vivo. SiMView data sets provide quantitative morphological information even for fast global processes and enable accurate automated cell tracking in the entire early embryo.

Fast, accurate reconstruction of cell lineages from large-scale fluorescence microscopy data

The comprehensive reconstruction of cell lineages in complex multicellular organisms is a central goal of developmental biology. We present an open-source computational framework for the segmentation and tracking of cell nuclei with high accuracy and speed. We demonstrate its (i) generality by reconstructing cell lineages in four-dimensional, terabyte-sized image data sets of fruit fly, zebrafish and mouse embryos acquired with three types of fluorescence microscopes, (ii) scalability by analyzing advanced stages of development with up to 20,000 cells per time point at 26,000 cells min−1 on a single computer workstation and (iii) ease of use by adjusting only two parameters across all data sets and providing visualization and editing tools for efficient data curation. Our approach achieves on average 97.0% linkage accuracy across all species and imaging modalities. Using our system, we performed the first cell lineage reconstruction of early Drosophila melanogaster nervous system development, revealing neuroblast dynamics throughout an entire embryo.

Tandem fluorescent protein timers for in vivo analysis of protein dynamics

The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule–mediated protein degradation.

Imaging Morphogenesis: Technological Advances and Biological Insights

Background Our understanding of developmental processes relies fundamentally on their in vivo observation. Morphogenesis, the shaping of an organism by cell movements, cell-cell interactions, collective cell behavior, cell shape changes, cell divisions, and cell death, is a dynamic process on many scales, from fast subcellular rearrangements to slow structural changes at the whole-organism level. The ability to capture, simultaneously, the fast dynamic behavior of individual cells, as well as their system-level interactions over long periods of time, is crucial for an understanding of the underlying biological mechanisms. Arriving at a complete picture of morphogenesis requires not only observation of single-cell to tissue-level morphological dynamics, but also quantitative measurement of protein dynamics, changes in gene expression, and readouts of physical forces acting during development. Live-imaging approaches based on light microscopy are of key importance to obtaining such information at the system level and with high spatiotemporal resolution. Live imaging of embryonic development. Nuclei-labeled Drosophila (top) and zebrafish (bottom) embryos were imaged with a simultaneous multiview light-sheet microscope (SiMView). The embryos are shown at 3 and 22 hours postfertilization, respectively. Color indicates depth in the image. Scale bars: 50 µm. Advances Live imaging with light microscopy requires carefully balancing competing parameters, among which spatiotemporal resolution and light exposure of the living specimen are chief. To maximize the quantity and quality of information extracted from the specimen under observation, optimal use must be made of the limited number of photons that can be collected under physiological conditions. Emerging techniques for noninvasive in vivo imaging can record morphogenetic processes at temporal scales from seconds to days and at spatial scales from hundreds of nanometers to several millimeters, with minimal energy load on the specimen. These approaches are able to capture cellular dynamics in entire vertebrate and higher invertebrate embryos throughout their development. It has become possible to follow cell movements, cell shape dynamics, subcellular protein localization, and changes in gene expression simultaneously, for the entire undisturbed living system. The application of these methods to the study of morphogenesis in the fly, fish, and mouse has led to fundamental insights into the mechanisms underlying epithelial folding, the control of tissue morphogenesis by signaling pathways, and the role of physical forces in local and tissue-wide morphogenetic changes. Outlook Current efforts in microscopy technology development are aimed at advancing deep-tissue in vivo imaging, improving spatial resolution, and increasing temporal sampling. To unlock their full potential, these methods need to be matched with new computational approaches and physical models that help convert the resulting, highly complex image data sets into biological insights. With the availability of system-level data on cell behavior and gene expression, and the potential for a system-level analysis of biophysical tissue properties, we are reaching the point at which it will be feasible to develop computational approaches that incorporate these data into a single model capable of dissecting morphogenesis at the whole-organism level. Morphogenesis, the development of the shape of an organism, is a dynamic process on a multitude of scales, from fast subcellular rearrangements and cell movements to slow structural changes at the whole-organism level. Live-imaging approaches based on light microscopy reveal the intricate dynamics of this process and are thus indispensable for investigating the underlying mechanisms. This Review discusses emerging imaging techniques that can record morphogenesis at temporal scales from seconds to days and at spatial scales from hundreds of nanometers to several millimeters. To unlock their full potential, these methods need to be matched with new computational approaches and physical models that help convert highly complex image data sets into biological insights.

Whole-animal functional and developmental imaging with isotropic spatial resolution

Imaging fast cellular dynamics across large specimens requires high resolution in all dimensions, high imaging speeds, good physical coverage and low photo-damage. To meet these requirements, we developed isotropic multiview (IsoView) light-sheet microscopy, which rapidly images large specimens via simultaneous light-sheet illumination and fluorescence detection along four orthogonal directions. Combining these four views by means of high-throughput multiview deconvolution yields images with high resolution in all three dimensions. We demonstrate whole-animal functional imaging of Drosophila larvae at a spatial resolution of 1.1-2.5 μm and temporal resolution of 2 Hz for several hours. We also present spatially isotropic whole-brain functional imaging in Danio rerio larvae and spatially isotropic multicolor imaging of fast cellular dynamics across gastrulating Drosophila embryos. Compared with conventional light-sheet microscopy, IsoView microscopy improves spatial resolution at least sevenfold and decreases resolution anisotropy at least threefold. Compared with existing high-resolution light-sheet techniques, IsoView microscopy effectively doubles the penetration depth and provides subsecond temporal resolution for specimens 400-fold larger than could previously be imaged.

Visualizing Whole-Brain Activity and Development at the Single-Cell Level Using Light-Sheet Microscopy

The nature of nervous system function and development is inherently global, since all components eventually influence one another. Networks communicate through dense synaptic, electric, and modulatory connections and develop through concurrent growth and interlinking of their neurons, processes, glia, and blood vessels. These factors drive the development of techniques capable of imaging neural signaling, anatomy, and developmental processes at ever-larger scales. Here, we discuss the nature of questions benefitting from large-scale imaging techniques and introduce recent applications. We focus on emerging light-sheet microscopy approaches, which are well suited for live imaging of large systems with high spatiotemporal resolution and over long periods of time. We also discuss computational methods suitable for extracting biological information from the resulting system-level image data sets. Together with new tools for reporting and manipulating neuronal activity and gene expression, these techniques promise new insights into the large-scale function and development of neural systems.

Light sheet microscopy of living or cleared specimens.

Light sheet microscopy is a versatile imaging technique with a unique combination of capabilities. It provides high imaging speed, high signal-to-noise ratio and low levels of photobleaching and phototoxic effects. These properties are crucial in a wide range of applications in the life sciences, from live imaging of fast dynamic processes in single cells to long-term observation of developmental dynamics in entire large organisms. When combined with tissue clearing methods, light sheet microscopy furthermore allows rapid imaging of large specimens with excellent coverage and high spatial resolution. Even samples up to the size of entire mammalian brains can be efficiently recorded and quantitatively analyzed. Here, we provide an overview of the history of light sheet microscopy, review the development of tissue clearing methods, and discuss recent technical breakthroughs that have the potential to influence the future direction of the field.