Advances In Light-sheet Microscopy Research Articles

O-040 Investigating why early embryonic mitoses are highly error prone by real time imaging

Abstract Maintenance of genomic integrity during early embryogenesis is essential for faithful embryonic development. However, mitotic errors during preimplantation development are surprisingly common and despite the fundamental importance of the cell divisions at the beginning of life, the mechanisms that control the correct progression through mitosis during pre-implantation development are not well characterized. The sensitivity of mammalian embryos to light and its demanding in vitro culture requirements have limited real-time imaging during the first embryonic divisions in the past. Today, advances in light-sheet microscopy allow in toto imaging of preimplantation mouse development from zygote to blastocyst at single chromosome resolution and enable us to study spindle assembly, chromosome restructuring and segregation. This presentation will discuss the insights we have gained by real-time imaging of the first cell divisions in mammalian life, and the role of the key molecular mechanisms that regulate mitosis during the critical first stages of embryo development.

Light-sheet engineering using the Field Synthesis theorem

Recent advances in light-sheet microscopy have enabled sensitive imaging with high spatiotemporal resolution. However, the creation of thin light-sheets for high axial resolution is challenging, as the thickness of the sheet, field of view and confinement of the excitation need to be carefully balanced. Some of the thinnest light-sheets created so far have found little practical use as they excite too much out-of-focus fluorescence. In contrast, the most commonly used light-sheet for subcellular imaging, the square lattice, has excellent excitation confinement at the cost of lower axial resolving power. Here we leverage the recently discovered Field Synthesis theorem to create light-sheets where thickness and illumination confinement can be continuously tuned. Explicitly, we scan a line beam across a portion of an annulus mask on the back focal plane of the illumination objective, which we call C-light-sheets. We experimentally characterize these light-sheets and demonstrate their application on biological samples.

Deconvolution of Sustained Neural Activity From Large-Scale Calcium Imaging Data.

Recent technological advances in light-sheet microscopy make it possible to perform whole-brain functional imaging at the cellular level with the use of Ca2+ indicators. The outstanding spatial extent and resolution of this type of data open unique opportunities for understanding the complex organization of neuronal circuits across the brain. However, the analysis of this data remains challenging because the observed variations in fluorescence are, in fact, noisy indirect measures of the neuronal activity. Moreover, measuring over large field-of-view negatively impact temporal resolution and signal-to-noise ratio, which further impedes conventional spike inference. Here we argue that meaningful information can be extracted from large-scale functional imaging data by deconvolving with the calcium response and by modeling moments of sustained neuronal activity instead of individual spikes. Specifically, we characterize the calcium response by a linear system of which the inverse is a differential operator. This operator is then included in a regularization term promoting sparsity of activity transients through generalized total variation. Our results illustrate the numerical performance of the algorithm on simulated signals; i.e., we show the firing rate phase transition at which our model outperforms spike inference. Finally, we apply the proposed algorithm to experimental data from zebrafish larvæ. In particular, we show that, when applied to a specific group of neurons, the algorithm retrieves neural activation that matches the locomotor behavior unknown to the method.

Light-Sheet Microscopy and Its Potential for Understanding Developmental Processes.

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.

Light sheet microscopes: Novel imaging toolbox for visualizing life’s processes

Capturing dynamic processes in live samples is a nontrivial task in biological imaging. Although fluorescence provides high specificity and contrast compared to other light microscopy techniques, the photophysical principles of this method can have a harmful effect on the sample. Current advances in light sheet microscopy have created a novel imaging toolbox that allows for rapid acquisition of high-resolution fluorescent images with minimal perturbation of the processes of interest. Each unique design has its own advantages and limitations. In this review, we describe several cutting edge light sheet microscopes and their optimal applications.

Two-photon light-sheet nanoscopy by fluorescence fluctuation correlation analysis

Advances in light-sheet microscopy have enabled the fast three-dimensional (3D) imaging of live cells and bulk specimens with low photodamage and phototoxicity. Combining light-sheet illumination with super-resolution imaging is expected to resolve subcellular structures. Actually, such kind of super-resolution light-sheet microscopy was recently demonstrated using a single-molecule localization algorithm. However, the imaging depth and temporal resolution of this method are limited owing to the requirements of precise single molecule localization and reconstruction. In this work, we present two-photon super-resolution light-sheet imaging via stochastic optical fluctuation imaging (2PLS-SOFI), which acquires high spatiotemporal resolution and excellent optical sectioning ability. 2PLS-SOFI is based on non-linear excitation of fluctuation/blinking probes using our recently developed fast two-photon three-axis digital scanned light-sheet microscope (2P3A-DSLM), which enables both deep penetration and thin sheet of light. Overall, 2PLS-SOFI demonstrates up to 3-fold spatial resolution enhancement compared with conventional two-photon light-sheet (2PLS) microscopy and about 40-fold temporal resolution enhancement compared with individual molecule localization-selective plane illumination microscopy (IML-SPIM). Therefore, 2PLS-SOFI is promising for 3D long-term, deep-tissue imaging with high spatiotemporal resolution.

Let's push things forward: disruptive technologies and the mechanics of tissue assembly

Although many of the molecular mechanisms that regulate tissue assembly in the embryo have been delineated, the physical forces that couple these mechanisms to actual changes in tissue form remain unclear. Qualitative studies suggest that mechanical loads play a regulatory role in development, but clear quantitative evidence has been lacking. This is partly owing to the complex nature of these problems - embryonic tissues typically undergo large deformations and exhibit evolving, highly viscoelastic material properties. Still, despite these challenges, new disruptive technologies are enabling study of the mechanics of tissue assembly in unprecedented detail. Here, we present novel experimental techniques that enable the study of each component of these physical problems: kinematics, forces, and constitutive properties. Specifically, we detail advances in light sheet microscopy, optical coherence tomography, traction force microscopy, fluorescence force spectroscopy, microrheology and micropatterning. Taken together, these technologies are helping elucidate a more quantitative understanding of the mechanics of tissue assembly.