Tomoko Watanabe

Image-based adaptive optics for two-photon microscopy

We demonstrate wavefront sensorless aberration correction in a two-photon excited fluorescence microscope. Using analysis of the image-formation process, we have developed an optimized correction scheme permitting image-quality improvement with minimal additional exposure of the sample. We show that, as a result, our correction process induces little photobleaching and significantly improves the quality of images of biological samples. In particular, increased visibility of small structures is demonstrated. Finally, we illustrate the use of this technique on various fresh and fixed biological tissues.

Multi-Cellular Rosettes in the Mouse Visceral Endoderm Facilitate the Ordered Migration of Anterior Visceral Endoderm Cells

Modeling and experimental results suggest a role for Planar Cell Polarity-dependent multi-cellular rosette structures in ensuring correct epithelial cell migration in the mouse visceral endoderm.

Limited predictive value of blastomere angle of division in trophectoderm and inner cell mass specification

The formation of trophectoderm (TE) and pluripotent inner cell mass (ICM) is one of the earliest events during mammalian embryogenesis. It is believed that the orientation of division of polarised blastomeres in the 8- and 16-cell stage embryo determines the fate of daughter cells, based on how asymmetrically distributed lineage determinants are segregated. To investigate the relationship between angle of division and subsequent fate in unperturbed embryos, we constructed cellular resolution digital representations of the development of mouse embryos from the morula to early blastocyst stage, based on 4D confocal image volumes. We find that at the 16-cell stage, very few inside cells are initially produced as a result of cell division, but that the number increases due to cell movement. Contrary to expectations, outside cells at the 16-cell stage represent a heterogeneous population, with some fated to contributing exclusively to the TE and others capable of contributing to both the TE and ICM. Our data support the view that factors other than the angle of division, such as the position of a blastomere, play a major role in the specification of TE and ICM.

Adaptive harmonic generation microscopy of mammalian embryos

Adaptive optics is implemented in a harmonic generation microscope using a wavefront sensorless correction scheme. Both the second- and third-harmonic intensity signals are used as the optimization metric. Aberration correction is performed to compensate both system- and specimen-induced aberrations by using an efficient optimization routine based upon Zernike polynomial modes. Images of live mouse embryos show an improved signal level and resolution.

Characterisation of the dynamic behaviour of lipid droplets in the early mouse embryo using adaptive harmonic generation microscopy.

BackgroundLipid droplets (LD) are organelles with an important role in normal metabolism and disease. The lipid content of embryos has a major impact on viability and development. LD in Drosophila embryos and cultured cell lines have been shown to move and fuse in a microtubule dependent manner. Due to limitations in current imaging technology, little is known about the behaviour of LD in the mammalian embryo. Harmonic generation microscopy (HGM) allows one to image LD without the use of exogenous labels. Adaptive optics can be used to correct aberrations that would otherwise degrade the quality and information content of images.ResultsWe have built a harmonic generation microscope with adaptive optics to characterise early mouse embryogenesis. At fertilization, LD are small and uniformly distributed, but in the implanting blastocyst, LD are larger and enriched in the invading giant cells of the trophectoderm. Time-lapse studies reveal that LD move continuously and collide but do not fuse, instead forming aggregates that subsequently behave as single units. Using specific inhibitors, we show that the velocity and dynamic behaviour of LD is dependent not only on microtubules as in other systems, but also on microfilaments. We explore the limits within which HGM can be used to study living embryos without compromising viability and make the counterintuitive finding that 16 J of energy delivered continuously over a period of minutes can be less deleterious than an order of magnitude lower energy delivered dis-continuously over a period of hours.ConclusionsLD in pre-implantation mouse embryos show a previously unappreciated complexity of behaviour that is dependent not only on microtubules, but also microfilaments. Unlike LD in other systems, LD in the mouse embryo do not fuse but form aggregates. This study establishes HGM with adaptive optics as a powerful tool for the study of LD biology and provides insights into the photo-toxic effects of imaging embryos.

Long-term imaging of mouse embryos using adaptive harmonic generation microscopy.

We present a detailed description of an adaptive harmonic generation (HG) microscope and culture techniques that permit long-term, three-dimensional imaging of mouse embryos. HG signal from both pre- and postimplantation stage (0.5-5.5 day-old) mouse embryos are fully characterized. The second HG images reveal central spindles during cytokinesis whereas third HG images show several features, such as lipid droplets, nucleoli, and plasma membranes. The embryos are found to develop normally during one-day-long discontinuous HG imaging, permitting the observation of several dynamic events, such as morula compaction and blastocyst formation.

Dissection of Embryonic Mouse Kidney, Culture In Vitro, and Imaging of the Developing Organ

INTRODUCTIONnIn this article, we outline procedures for the dissection of intact kidneys and the isolation of the ureteric bud (UB) and the metanephric mesenchyme (MM) from mouse embryos. The apparatus required for the culture of these tissues in vitro is described in detail as well as the equipment necessary for performing time-lapse imaging studies of the developing kidney.

Imaging kidney development

INTRODUCTIONnDevelopment of the kidney involves interactions between several cell lineages and complex morphogenetic processes, such as branching of the ureteric bud (UB) to form the collecting duct system and condensation and differentiation of the mesenchymal progenitors to form the nephron epithelia. One of the advantages of the mouse kidney as an experimental system is that it can develop in culture, from the stage of initial branching of the UB (E11.5) for up to a week (although it achieves the size and degree of development of only an E13.5-E14.5 kidney in vivo). The availability of fluorescent proteins (FPs) has provided powerful tools for visualizing the morphogenesis of specific renal structures in organ cultures. Two categories of genetically modified mice that express FPs are useful for visualizing different cell lineages and developmental processes in these organ cultures: (1) transgenic mice that express a fluorescent reporter in the pattern of a specific gene; and (2) Cre reporter mice, which turn on an FP in cells with Cre recombinase activity (and their daughter cells), used in conjunction with cell type-specific Cre transgenic mice. Here, we describe some of the currently available Cre and FP transgenic lines that are useful for the study of kidney development.

Towards understanding the roles of position and geometry on cell fate decisions during preimplantation development.

The first lineage segregation event in mouse embryos produces two separate cell populations: inner cell mass and trophectoderm. This is understood to be brought about by cells sensing their position within the embryo and differentiating accordingly. The cellular and molecular underpinnings of this process remain under investigation and have variously been considered to be completely stochastic or alternately, subject to some predisposition set up at fertilisation or before. Here, we consider these views in light of recent publications, discuss the possible role of cell geometry and mechanical forces in this process and describe how modelling could contribute in addressing this issue.

Adaptive multiphoton and harmonic generation microscopy for developmental biology

Specimen-induced aberrations often affect microscopes, particularly when high numerical aperture lenses are used to image deep into biological specimens. These aberrations cause a reduction in resolution and focal intensity. This is particularly problematic in multiphoton microscopy, where the non-linear nature of the signal generation process means that the signal level is strongly affected by changes in the focal spot intensity. We have applied the techniques of adaptive optics to correct aberrations in two-photon fluorescence and harmonic generation microscopes, restoring image quality. In particular we have used these microscopes for studies in developmental biology and for the imaging of mammalian embryos.