Near Infrared Microspectroscopy, Fluorescence Microspectroscopy, Infrared Chemical Imaging and High-Resolution Nuclear Magnetic Resonance Analysis of Soybean Seeds, Embryos and Single Cells

Chemical analysis of soybean seeds, somatic embryos and single cells were carried out by Fourier Transform Infrared (FT-IR), Fourier Transform Near Infrared (FT-NIR) Microspectroscopy, Fluorescence and High-Resolution NMR (HR-NMR). The first FT-NIR chemical images of biological systems approaching 1 micron (1μ) resolution are presented here. Chemical images obtained by FT-NIR and FT-IR Microspectroscopy are presented for oil in soybean seeds and somatic embryos under physiological conditions. FT-NIR spectra of oil and proteins were obtained for volumes as small as 2μ3. Related, HR-NMR analyses of oil contents in somatic embryos are also presented here with nanoliter precision. Such 400 MHz 1H NMR analyses allowed the selection of mutagenized embryos with higher oil content (e.g. ~20%) compared to non-mutagenized control embryos. Moreover, developmental changes in single soybean seeds and/or somatic embryos may be monitored by FT-NIR with a precision approaching the picogram level. Indeed, detailed chemical analyses of oils and phytochemicals are now becoming possible by FT-NIR Chemical Imaging/ Microspectroscopy of single cells. The cost, speed and analytical requirements of plant breeding and genetic selection programs are fully satisfied by FT-NIR spectroscopy and Microspectroscopy for soybeans and soybean embryos. FT-NIR Microspectroscopy and Chemical Imaging are also shown to be potentially important in functional Genomics and Proteomics research through the rapid and accurate detection of high-content microarrays (HCMA). Multi-photon (MP), pulsed femtosecond laser NIR Fluorescence Excitation techniques were shown to be capable of Single Molecule Detection (SMD). Therefore, such powerful techniques allow for the most sensitive and reliable quantitative analyses to be carried out both in vitro and in vivo. Thus, MP NIR excitation for Fluorescence Correlation Spectroscopy (FCS) allows not only single molecule detection, but also molecular dynamics and high resolution, submicron imaging of femtoliter volumes inside living cells and tissues. These novel, ultra-sensitive and rapid NIR/FCS analyses have numerous applications in important research areas, such as: agricultural biotechnology, food safety, pharmacology, medical research and clinical diagnosis of viral diseases and cancers

Methods for Spatio-Temporal Analysis of Embryo Cleavage In Vitro

Automated or semiautomated time-lapse analysis of early stage embryo images during the cleavage stage can give insight into the timing of mitosis, regularity of both division timing and pattern, as well as cell lineage. Simultaneous monitoring of molecular processes enables the study of connections between genetic expression and cell physiology and development. The study of live embryos poses not only new requirements on the hardware and embryo-holding equipment but also indirectly on analytical software and data analysis as four-dimensional video sequencing of embryos easily creates high quantities of data. The ability to continuously film and automatically analyze growing embryos gives new insights into temporal embryo development by studying morphokinetics as well as morphology. Until recently, this was not possible unless by a tedious manual process. In recent years, several methods have been developed that enable this dynamic monitoring of live embryos. Here we describe three methods with variations in hardware and software analysis and give examples of the outcomes. Together, these methods open a window to new information in developmental embryology, as embryo division pattern and lineage are studied in vivo

Automating assessment of human embryo images and time-lapse sequences for IVF treatment

As the number of couples using In Vitro Fertilization (IVF) treatment to give birth increases, so too does the need for robust tools to assist embryologists in selecting the highest quality embryos for implantation. Quality scores assigned to embryonic structures are critical markers for predicting implantation potential of human blastocyst-stage embryos. Timing at which embryos reach certain cell and development stages in vitro also provides valuable information about their development progress and potential to become a positive pregnancy. The current workflow of grading blastocysts by visual assessment is susceptible to subjectivity between embryologists. Visually verifying when embryo cell stage increases is tedious and confirming onset of later development stages is also prone to subjective assessment. This thesis proposes methods to automate embryo image and time-lapse sequence assessment to provide objective evaluation of blastocyst structure quality, cell counting, and timing of development stages

Green Fluorescent Protein in the sea urchin: new experimental approaches to transcriptional regulatory analysis in embryos and larvae

The use of Green Fluorescent Protein (GFP) as a reporter for expression transgenes opens the way to several new experimental strategies for the study of gene regulation in sea urchin development. A GFP coding sequence was associated with three different previously studied cis-regulatory systems, viz those of the SM50 gene, expressed in skeletogenic mesenchyme, the CyIIa gene, expressed in archenteron, skeletogenic and secondary mesenchyme, and the Endo16 gene, expressed in vegetal plate, archenteron and midgut. We demonstrate that the sensitivity with which expression can be detected is equal to or greater than that of whole-mount in situ hybridization applied to detection of CAT mRNA synthesized under the control of the same cis-regulatory systems. However, in addition to the important feature that it can be visualized nondestructively in living embryos, GFP has other advantages. First, it freely diffuses even within fine cytoplasmic cables, and thus reveals connections between cells, which in sea urchin embryos is particularly useful for observations on regulatory systems that operate in the syncytial skeletogenic mesenchyme. Second, GFP expression can be dramatically visualized in postembryonic larval tissues. This brings postembryonic larval developmental processes for the first time within the easy range of gene transfer analyses. Third, GFP permits identification and segregation of embryos in which the clonal incorporation of injected DNA has occurred in any particular desired region of the embryo. Thus, we show explicitly that, as expected, GFP transgenes are incorporated in the same nuclei together with other transgenes with which they are co-injected

Computational efficient segmentation of cell nuclei in 2D and 3D fluorescent micrographs

This paper proposes a new segmentation technique developed for the segmentation of cell nuclei in both 2D and 3D fluorescent micrographs. The proposed method can deal with both blurred edges as with touching nuclei. Using a dual scan line algorithm its both memory as computational efficient, making it interesting for the analysis of images coming from high throughput systems or the analysis of 3D microscopic images. Experiments show good results, i.e. recall of over 0.98

Dynamic in vivo imaging and cell tracking using a histone fluorescent protein fusion in mice

BACKGROUND: Advances in optical imaging modalities and the continued evolution of genetically-encoded fluorescent proteins are coming together to facilitate the study of cell behavior at high resolution in living organisms. As a result, imaging using autofluorescent protein reporters is gaining popularity in mouse transgenic and targeted mutagenesis applications. RESULTS: We have used embryonic stem cell-mediated transgenesis to label cells at sub-cellular resolution in vivo, and to evaluate fusion of a human histone protein to green fluorescent protein for ubiquitous fluorescent labeling of nucleosomes in mice. To this end we have generated embryonic stem cells and a corresponding strain of mice that is viable and fertile and exhibits widespread chromatin-localized reporter expression. High levels of transgene expression are maintained in a constitutive manner. Viability and fertility of homozygous transgenic animals demonstrates that this reporter is developmentally neutral and does not interfere with mitosis or meiosis. CONCLUSIONS: Using various optical imaging modalities including wide-field, spinning disc confocal, and laser scanning confocal and multiphoton excitation microscopy, we can identify cells in various stages of the cell cycle. We can identify cells in interphase, cells undergoing mitosis or cell death. We demonstrate that this histone fusion reporter allows the direct visualization of active chromatin in situ. Since this reporter segments three-dimensional space, it permits the visualization of individual cells within a population, and so facilitates tracking cell position over time. It is therefore attractive for use in multidimensional studies of in vivo cell behavior and cell fate

Quantitative analysis of the mechanical environment in the embryonic heart with respect to its relationship in cardiac development

Includes bibliographical references.2015 Fall.In order to understand the causes of congenital heart defects, which afflict at least 4 infants per 1,000 live births, research has implemented the use of animal models to study embryonic heart development. Zebrafish (Danio rerio) have become one of the more prominent of these animal models due to the fact that their heart morphology at the earliest stages of development is remarkably similar to humans, and because embryos lack pigmentation, rendering them transparent. This transparency allows for high-speed images of blood flow to be acquired in the developing heart so that the mechanotransductive relationship between the intracardiac flow environment and myocardial progenitor cell differentiation can be understood. One particular aspect of the flow environment, a cyclic retrograde flow at the junction of the forming atrium and ventricle, has been shown to be necessary for valve formation, though the mechanisms causing it to occur had previously been unknown. By comparing the results of two-dimensional spatiotemporal analysis applied to embryos both with normal retrograde flow and inhibited retrograde flow, this study shows that a particular range of pressures associated with the pumping mechanics of the heart as well as resistance due to systolic contractile closure must exist in order to maintain adequate retrograde flow to induce valve formation. The use of two-dimensional spatiotemporal analysis was sufficient to acquire these results, however when applied to analysis of other aspects of the intracardiac flow environment, this computational method is subject to critical limitations. Therefore, this study includes the development of methodology to integrate the results of spatiotemporal analysis on multiple focal planes bisecting the heart into a more accurate, three-dimensional result. The results of this study not only increase our understanding of the mechanics behind an important factor in embryonic development, but also enable future experiments pertaining to the measurement of embryonic intracardiac blood flow to be performed with increased certainty

Integrated holographic system for all-optical manipulation of developing embryos

We demonstrate a system for the combined optical injection and trapping of developing embryos. A Ti:sapphire femtosecond laser in tandem with a spatial light modulator, is used to perform fast and accurate beam-steering and multiplexing. We show successful intracellular delivery of a range of impermeable molecules into individual blastomeres of the annelid Pomatoceros lamarckii embryo by optoinjection, even when the embryo is still enclosed in a chorion. We also demonstrate the ability of the femtosecond laser optoinjection to deliver materials into inner layers of cells in a well-developed embryo. By switching to the continuous wave mode of the Ti:sapphire laser, the same system can be employed to optically trap and orient the 60 μm sized P. lamarckii embryo whilst maintaining its viability. Hence, a complete all-optical manipulation platform is demonstrated paving the way towards single-cell genetic modification and cell lineage mapping in emerging developmental biology model species