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Keywords:

  • blood vessels;
  • confocal laser microscopy;
  • quail;
  • time-lapse imaging;
  • transgenic bird

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References

Avian embryos are important model organism to study higher vertebrate development. Easy accessibility to developing avian embryos enables a variety of experimental applications to understand specific functions of molecules, tissue–tissue interactions, and cell lineages. The whole-mount ex ovo culture technique for avian embryos permits time-lapse imaging analysis for a better understanding of cell behaviors underlying tissue morphogenesis in physiological conditions. To study mechanisms of blood vessel formation and remodeling in developing embryos by using a time-lapse imaging approach, a transgenic quail model, Tg(tie1:H2B-eYFP), was generated. From a cell behavior perspective, Tg(tie1:H2B-eYFP) quail embryos are a suitable model to shed light on how the structure and pattern of blood vessels are established in higher vertebrates. In this manuscript, we give an overview on the biological and technological background of the transgenic quail model and describe procedures for the ex ovo culture of quail embryos and time-lapse imaging analysis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References

Time-lapse microscopy is an important approach to study dynamic morphogenetic events during embryogenesis on the cellular level. Confocal laser microscopy enables the capture of high-resolution images of cells in xyz-dimensions at regular intervals, permitting us to trace and quantify behaviors of the cells by means of computer image analysis (or a really hard working postdoc can do it by hand). Quantitative analysis of cell behavior helps us to assess how a cell's individual behavior influences tissue shape (i.e. when a gene function is disrupted). Time-lapse imaging and analysis of cultured cells are easy to perform because the cells can be labeled efficiently with fluorescent proteins by DNA transfection. In addition, because they are not multi-layered, the number of z-sections is minimized and light scattering is negligible. In contrast, time-lapse imaging of developing amniote embryos is not achieved easily because large body sizes require several xy and numerous z-sections and the opaque tissue causes light scattering. In addition, drifting or “framing out” of embryos frequently occurs since they are growing in all dimensions. These issues notwithstanding, capturing cell behaviors in vivo is vitally important in the field of developmental biology because the tissue shape changes and functions are dynamically governed by physiological circumstances in a body. Time-lapse imaging analysis of developing embryos requires an animal model that enables: (i) fluorescent labeling of desired cells; (ii) undergoes normal development on a microscope stage and is resist to the deleterious effects of excitating light; and (iii) is amenable to experimental manipulations of its molecular and cellular components.

Developing chick and quail embryos are easily observed by creating a small hole in the eggshell; extended observation is possible if the hole is transparently resealed by using a cover slip, saran wrap, Dupont stuff. Researchers have taken advantage of this built-in culture system from hundreds of years ago (see Stern 2004 for a review), using avian embryos as a model for developmental biology. Time-lapse observation of migrating neural crest cells was performed for the first time in chick embryos in ovo by using fluorescent dye, for example, Rhodamine dextran-, DiI and DiO-labeling techniques (Stern et al. 1988; Kulesa & Fraser 2000). The whole-mount ex ovo culture method permitted the observation and manipulation of avian embryos at early stages (New 1955; Chapman et al. 2001). This culture system facilitated time-lapse imaging of ventral tissues, for example, blood vessels, endoderm, and heart primordium, which are located at an inaccessible deep position in an in ovo environment (Rupp et al. 2004; Kimura et al. 2006; Kidokoro et al. 2008). Viral vectors and DNA electroporation techniques enable delivery of exogenous genes into developing embryos (Yamagata et al. 1994; Itasaki & Nakamura 1996; Funahashi et al. 1999). These methods have permitted the labeling of specific cells with fluorescent proteins and the analysis of gene functions. Tet-on/off inducible vector systems have allowed conditional gene manipulation in avian embryos, thereby giving researchers the ability to analyze common genes in lineage-overlapping tissues (Hilgers et al. 2005; Watanabe et al. 2007). Inhibition of target gene expression by shRNA and morpholino-oligos can also be achieved in the avian embryos, as well as other model animals (Katahira & Nakamura 2003; Sheng et al., 2003). In addition, overexpression of dominant negative- and constitutively active forms of proteins are available in the avian embryos by the electroporation or viral infection methods (see Itasaki et al. 1999; Sauka-Spengler & Barembaum 2008 for reviews). By combining with these gene manipulation techniques by co-electroporation, co-infection, co-expression with IRES or 2A linker, time-lapse imaging analyses of fluorescent-labeled cells have helped in the quantitative characterizations of gene functions in the context of cell behaviors (Krull et al. 1997; Kasemeier-Kulesa et al. 2006; Gros et al. 2009; Benazeraf et al. 2010).

Zebrafish embryo is the most popular vertebrate model for time-lapse imaging analysis because it has advantages in small and transparent body, rapid development, and availability of tissue-specific fluorescent reporter transgenic lines. A lot of beautiful time-lapse imaging analyses of zebrafish embryos have revealed mechanisms of a non-amniote embryogenesis; however, from the aspect of anatomical and environmental diversities of vertebrate development, an amniote model suitable for the time-lapse imaging analysis is required. In recent years, genetic engineering in mammalian model (i.e. mouse), has been developed explosively and enabled both fluorescent labeling of cells and studies of gene functions in vivo. However, feasibility of time-lapse imaging analysis of mouse embryos is restricted to certain tissues in whole-mount ex utero culture conditions. For example, the mouse embryo undergoes inside out turning during late gastrulation stage (Jones et al. 2002), which encloses ventral tissues (i.e. endoderm and intra-embryonic blood vessels) and obscures the ability to see and videorecord them. On the other hand, avian embryos remain relatively flat from early to late gastrulation stages, enabling time-lapse observation of both dorsal and ventral tissues by means of the aforementioned whole-mount ex ovo culture technique. Thus, the avian embryo is an exemplary model system for dynamic analysis of how genes trigger changes in cell behaviors that induce specific morphological events.

These advantages have motivated developmental biologists to generate transgenic chick lines carrying fluorescent reporter genes (Table 1) (McGrew et al. 2004; Chapman et al. 2005). Ubiquitously green fluorescent protein (GFP)-expressing transgenic zebra finch has also been generated to facilitate genetic manipulations of songbird, an established vocal learning model in behavioral and neuronal biology (Agate et al. 2009). However, cells in ubiquitously eGFP-expressing transgenic chick embryos cannot be distinguished individually, unless a piece of tissue taken from the eGFP-transgenic chick embryo is transplanted into a wild type host embryo by microsurgery. To perform time-lapse imaging analysis of a specific tissue, the establishment of tissue-specific transgenic lines has been awaited. Quail has been exploited mainly for the generation of transgenic lines that carry tissue-specific promoters upstream of fluorescent reporter genes (Scott & Lois 2005; Sato et al. 2010; Seidl et al. 2012). With these transgenic quail lines, time-lapse imaging has revealed the dynamic nature of cell behaviors during development. This article will provide protocols and describe key considerations for the time-lapse imaging analysis of transgenic quail embryos by using confocal laser microscopy.

Table 1. Transgenic birds expressing fluorescent proteins
Tissue-specific fluorescent reporter gene expression
TransgeneTissueVectorSpeciesReferences
Synapsin1:eGFPNeuronLentivirusQuailScott & Lois (2005)
Tiel:H2B::eYFPEndothelial cellLentivirusQuailSato et al. (2010)
Ovalbumin 2 kb: eGFPOviductLentivirusChickenByun et al. (2011)
Synapsin1:H2B::eGFPNeuronLentivirusQuail Seidl et al. (2012)
Ubiquitous fluorescent reporter gene expression
TransgeneVectorSpeciesReferences
LTR:eGFP::NeorMoloneyvirusChickenMizuarai et al. (2001)
CMV:eGFPLentivirusChickenMcGrew et al. (2004)
PGK:eGFPLentivirusChickenChapman et al. (2005)
β-actin:eGFP::PurorPlasmidChickenVan De Lavoir et al. (2006)
RSV:eGFPMoloneyvirusChickenKoo et al. (2006)
RSV:eGFPLentivirusQuailShin et al. (2008)
hUbiqutinC:eGFPLentivirusFinchAgate et al. (2009)
β-actin:eGFPLentivirusChickenMotono et al. (2010)
CAGGS:eGFP:IRES::PurorTol2 transposonChickenMacDonald et al. (2012)
CAG:eGFP::NeorpiggyBac transposonChickenPark & Han (2012)

Current situation of transgenic chicken and quail

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References

Chicken (Gallus g. domestics) and quail (Coturnix c. japonica) have been exploited mainly for the generation of transgenic avian lines expressing fluorescent proteins (Table 1). Transgenic chickens are generated to study and improve the efficiency of germline integration of exogenous genes. The industrial application of egg white as a bioreactor for pharmaceutical materials (e.g., antibodies, recombinant proteins) is anticipated because it is difficult to obtain large amounts of such materials from biochemical synthesis (Kamihira et al. 2005; Lillico et al. 2007; Kwon et al. 2010; Penno et al. 2010). Importantly, a recent report has demonstrated that horizontal infection with the avian influenza virus can be prevented in a transgenic chicken line carrying a shRNA construct against the influenza A virus polymerase gene, suggesting that transgenic technologies applied to chickens are practical for the poultry industry (Lyall et al. 2011). In contrast to transgenic chickens in applied poultry biology, quail has been exploited for the generation of tissue-specific fluorescent transgenics, which are essential for time-lapse imaging analysis (Table 1) (Scott & Lois 2005; Sato et al. 2010; Seidl et al. 2012). Quail is a highly effective avian species to perform genetic modifications because of shorter incubation and earlier sexual maturation periods compared to chicken (Poynter et al. 2009c). Moreover, the small body size of quail permits maintenance of transgenic strains by means of rodent cages with a small modification in an air-conditioned, pathogen-controlled environment of an animal facility (Huss et al. 2008). Therefore, quail is a compelling alternative avian model to the conventional chicken, particularly to researchers who want to generate transgenic birds efficiently (see also Bower et al. 2011 for a review).

Tissue-specific promoters for fluorescent reporters

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References

Endothelial- and neuronal cell-specific promoters used for transcription of fluorescent reporter genes in Tg(tie1:H2B-eYFP), Tg(syn1:eGFP), and Tg(syn1: H2B-eGFP) quails, are derived from mouse, human, and rat, respectively (De Palma et al. 2003; Dittgen et al. 2004). These mammalian promoters work in the same tissue-specific manner in quail embryos (Scott & Lois 2005; Sato et al. 2010; Seidl et al. 2012). In fact, comparative whole genome analysis revealed that sequences of non-coding regulatory DNA elements, which are involved in embryogenesis, are highly conserved in vertebrates (Uchikawa et al. 2003; Woolfe et al. 2005). To search genes expressed in tissue of interest, the web database, GEISHA (Gallus Expression in Situ Hybridization Analysis, University of Arizona) is very helpful. GEISHA opens expression patterns of genes in chicken embryos, which are compiled from in situ hybridization screening of chicken ESTs (expression sequence tags) and already published images (Bell et al. 2004). Genes in GEISHA are searchable by tissue name. The availability of chick genome sequences facilitated the identification of tissue-specific regulatory regions by homology analysis with other model animal genomes (International Chicken Genome Sequencing Consortium, 2004). With the aid of these databases, we can narrow down candidate genes available for tissue-specific regulation of fluorescent reporter genes. The desired tissue-specific regulatory region can be obtained from the chicken BAC (bacterial artificial chromosome) library (CHORI-261, available in BACKPAC resource center, Children's Hospital Oakland Research Institute). Strategies for the systematic screening of functional enhancers from the chicken BAC library, and in silico analysis of regulatory sequences are described elsewhere (Brown 2008; Kondoh & Uchikawa 2008). At present, labeling of sub-cellular structures with fluorescent proteins (FPs) is feasible due to the characterization of signal sequences targeting organelles and cytoskeletal components (Teddy et al. 2005). For automated detecting and tracking of an individual cell by computing, visualization of the nucleus by labeling with Histone 2B (H2B)-FP is the most effective method (Kanda et al. 1998).

Generation of transgenic quail

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References

Cultured blastoderm cells, similar to mammalian embryonic stem (ES) cells, cannot colonize efficiently in avian germline tissues (see Petitte et al. 2004; Sang 2004 for reviews). This means that exogenous DNA is not easily delivered into primordial germ cells (PGCs) by either DNA injection into oocytes or implantation of transfected ES cells into embryos at the blastoderm stage, which is different from fish, frog, and mammalians. Viral vectors have been preferentially used for the successful delivery of genes in most transgenic birds (Table 1), because automatic infection mechanisms of the viruses do not require toxic manipulations, for example, electroporation or transfection at a critical stage. Obtaining advantage from gene therapy, lentiviral vectors, which are originally designed for in vivo targeting of tumor blood vessels by Naldini's group, were adopted for generation of transgenic quail (De Palma et al. 2003). The lentiviral vectors injected into embryos at blastoderm stage (Fig. 1A, see also Mozdziak & Petitte 2004 for a review). The viral RNA genome carrying the transgene is released from the viral particle, reverse transcribed, and passed into the nucleus where it integrates into the genome of the quail host cell (Fig. 1B). The injected quail embryos are incubated for 16–17 days until hatching, and are raised for about 2 months when they become sexually mature (generation 0 [G0], Fig. 1A). Since the lentiviral vector is replication incompetent, the G0 quails are chimeric for the transgene and must be bred to achieve complete penetrance of the transgene in the germline. The eggs produced by mating G0 and wild type quails are incubated until hatching, and hatchlings are subjected to visual or DNA screening to identify the transgene. The established transgenic quails (G1) are mated with desired quail to provide transgenic embryos (G2) for imaging experiments.

image

Figure 1. Generation of transgenic quail. (A) Transgenic quail are generated by injecting concentrated lentivirus carrying a tissue-specific promoter that expresses a fluorescent reporter gene into blastoderm cells of newly laid stage X eggs. Quail hatchlings (G0) are raised and crossed with wild type mates to acquire germline transmission of the transgene. All of the next generation hatchlings are subjected to screening by polymerase chain reaction (PCR) analysis, visualization with a fluorescent dissecting microscope, or both to identify transgenic quail (G1) and establish stable lines. G2 embryos obtained from G1 and wild type mating are used for experimentation, including time-lapse imaging analysis. (B) Genomic integration mechanism of exogenous genes by using lentivirus vector. (C) Tg (tie1:H2B-eYFP) quail embryos at HH stage 12 (E2, left) and 18 (E3, right). Endothelial cells are individually illuminated by nuclear eYFP signals (Inset, Scale bar, 40 μm). ISV, inter segmental vessel.

Download figure to PowerPoint

For example, three independent G1 Tg(tie1:H2B-eYFP) quail lines were procured from 129 hatchlings (Sato et al. 2010). Because the TIE1 promoter is active in endothelial cells from early embryogenesis through adulthood, Tg(tie1:H2B-eYFP) hatchlings can be identified easily by using a fluorescence dissecting scope to screen for eYFP+ cells in blood vessels of the chorioallantoic membrane (CAM) that remain inside the eggshell. A detailed protocol for the generation of transgenic quails using lentiviral vectors is described elsewhere (Scott & Lois 2006; Poynter et al. 2009a,b,d). The procedures of how to rear and breed Japanese quail are described by Huss (Huss et al. 2008).

Ex ovo culture of quail embryos

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References

Time-lapse imaging can be conducted on avian embryos using an upright or an inverted microscope. If it is an upright microscope, in ovo time-lapse observation of quail embryos is possible if the hole in the eggshell is sealed with a Teflon membrane (Kulesa et al. 2010a), low quality saran wrap, or a glass cover slip to prevent desiccation. Time-lapse observation of ex ovo cultured whole content of quail eggs is possible by using the upright microscope. This method uses a Petri dish with a Teflon membrane-covered window in a lid instead of the eggshell and allows long-term time-lapse imaging of Tg(tie1:H2B-eYFP) embryos on yolk (Al-Roubaie et al. 2012). Typically the embryos preferentially settle opposite the direction of gravity or at the top of the yolk.

To achieve successful time-lapse imaging of early stage embryos by using inverted microscopes, an ex ovo whole-embryo culture method is often used (New 1955; Chapman et al. 2001; Rupp et al. 2003; Canaria & Lansford 2011b). Chapman's ex ovo culture method uses a semi-solid medium made of egg white albumen and agar, which provides a flexible bed for embryos and nutrients that enable long culture, (i.e. Hamburger and Hamilton [HH] stage 4 through 17 for about 40 h) (Hamburger & Hamilton 1992; Chapman et al. 2001). The semi-solid state of the albumen-agar medium also helps to prevent drifting of the embryo and, thereby facilitates time-lapse observation of avian embryos. For successful ex ovo culture, placement of embryos with ventral-side up on the albumen-agar bed is standard. Conversely, ex ovo culture of embryos with the ventral-side down is difficult prior to HH stage 8 (Chapman et al. 2001). From our experiences, even after HH stage 8, quail embryos die occasionally on the albumen-agar beds after flipping to the ventral-side down due to rapid cessation of the blood flow. The albumen-agar bed may not provide enough space for the beating heart, which may cause reduction of circulation, blood clot formation, etc. However, culturing embryos with the ventral-side down is essential for time-lapse imaging of ventral tissues (e.g., endoderm, blood vessels, splanchnic mesoderm, and notochord) by using an inverted microscope. We have succeeded in the ex ovo culture of Tg(tie1:H2B-eYFP) quail embryos with the ventral-side down. Our tips for ex ovo culture are described in the protocol section. Another protocol for the successful ex ovo culture of early chick embryos with the ventral-side down has been described (Ezin & Fraser 2008). This method uses liquid medium and a fibronectin-coated membrane filter instead of the albumen-agar bed and enables time-lapse imaging of neural crest cells by using an upright microscope.

Incubation system for warm-blooded embryo imaging

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References

For time-lapse imaging of quail embryos, the confocal laser microscope essentially has to be attached to a heating system. Different from tissue culture, ex ovo culture of quail embryos on albumen-agar bed does not require CO2 supply. Various types of heating chamber systems became commercially available in recent years. Figure 3 shows a large box-type incubation system and stage-top heater (Fig. 3A,B). The large box-type system can be used to incubate embryos on stage with warm air from the heater unit. This system permits manipulation of embryos on the microscope stage. In contrast, stage-top chamber systems provide the required temperature in a small chamber space by either an electrothermal mechanism or a water jacket. The water jacket-type of stage-top chamber can be used for both heating and cooling. These incubation systems successfully help developing quail embryos on the microscope stage. Before these incubation systems were commercially available, time-lapse imaging analysis of chick embryos has been successfully performed by means of self-made heating systems (Fig. 3C–E) (Kulesa & Fraser 2000, 2002; see also Kulesa & Kasemeier-Kulesa 2007 for a protocol). The incubation chamber can be built with materials obtained at a lower cost, that is, a cardboard covered with thermal insulation (Reflectix , 8 mm thick), a warming heater for professional use (egg-incubator heater, Lyon Electric, #115–20) (Kulesa & Fraser 2002) or a personal mini fan heater (Lakewood, #ELO90), and a temperature controller (thermostat) (Fig. 3C–F). When customizing the incubation system, the chamber design must be considered (i.e., it should not interfere with the moving parts and radiation units of microscope).

Time-lapse imaging of quail embryos by confocal laser microscopy

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References

To achieve successful time-lapse imaging of dynamic cell behaviors in growing embryos, we must consider the following: (i) minimize photo-toxicity, (ii) scan fast at short intervals to keep track of migratory cells, (iii) set sufficient margins in the frame to allow for the embryo's drift. There is a trade-off relationship between optimum conditions for high-quality imaging and fast, less-toxic imaging. Appropriate imaging frequencies can be determined empirically by first identifying the rate of the developmental events of interest and subsequently imaging at a faster rate. For instance, for tracking cell movement and migration, imaging frequencies between 2 and 10 min typically are ideal. The lower limit of imaging frequency is bound by the time needed to acquire a single 3D image, or z-stack. The upper limit is set empirically. If a migrating cell cannot be clearly identified between two sequential frames, then the imaging frequency should be increased.

A smaller pinhole size improves spatial resolution; however, this requires higher number of z-sections, which prolongs image scan time and may increase cell/embryo death. For imaging of 100 μm-thick dorsal aortae in Tg(tie1:H2B-eYFP) embryos, more than 100 z-sections are required if an optimal pinhole size (1 airy unit [AU]) is selected to capture eYFP signals with subcellular resolution. Reduction of the laser power and scan time must be considered to relieve the embryos from photo-toxicity. A larger pinhole size permits bright and fast image acquisition, whereby reducing photo-toxicity, improving time-resolution, and generating sufficient data. The procedure of time-lapse imaging of Tg(tie1:H2B-eYFP) embryos by conventional confocal laser microscopy is described in the protocol section below. Another protocol for time-lapse imaging of Tg(tie1:H2B-eYFP) quail embryos by using two-photon (2P) excitation microscopy and wider image acquisition by applying tiled xyz-scanning has been described (Canaria & Lansford 2011a,b).

Multispectral labeling and imaging strategies

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References

Capturing the complex activities of cells undergoing morphogenesis often requires concurrent imaging of numerous cells in order to effectively distinguish cell–cell and tissue–tissue interactions. Comprehensive analysis of cell interactions and other behaviors during morphogenesis often requires simultaneous visualization of several different tissues or subcellular structures. It is very difficult to distinguish cells labeled with a single fluorophores from one another, especially if the fluorophores have not been subcellularly targeted. It is nearly impossible to confirm where one cell stops and another begins in regions where the cell density is high and contacts are intimate and extensive. Three fluorescent labeling approaches that can be used to ‘reduce’ the observed cell density within tissues include multispectral, mosaic (“salt and pepper”), and photoactivation/photoconversion (reviewed in Bower et al. 2011).

Multispectral techniques offer an ideal approach to distinguish closely opposed cells in dense tissue (Feng et al. 2000; Hadjantonakis et al. 2002; Hutter 2004; Livet et al. 2007; Kulesa et al. 2010b). For multispectral experiments, forethought should be used in choosing fluorescent reporters that have spectrally well separated profiles to avoid cross talk in excitation and emission wavelengths. There is an ever-growing number of spectrally distinct FPs available including CyanFP, GreenFP, YellowFP, RedFP, or FP, to name a few (see Shaner et al. 2005 for a review) that can be excited using epifluorescence, confocal and multiphoton imaging methods. Integrating imaging and computational methods allows the simultaneous resolution of signals from dyes or FPs with overlapping spectra (Lansford et al. 2001) or from cellular autofluorescence.

The subcellular resolution permitted by multispectral fluorescent labeling of nuclei, membranes, or other cellular structures in an optically and genetically accessible amniote opens the door to unprecedented lineage analysis and dynamic developmental studies in living embryos. Targeting spectrally distinct fluorophores to separate subcellular locations, leverages the benefits of multiple colors to elucidate cellular structures while enabling the use of traditional spectral filters to separate the FP signatures (Larue et al. 2003). Different FPs can be subcellularly localized to specific organelles, allowing these structures to be imaged alone or in combination with other markers. The cellular behavior that you are planning to record should dictate where to target your FPs. For instance, nuclear-localized H2B-FP is a good choice if cell migration and cell proliferation are to be recorded (Kanda et al. 1998; Larue et al. 2003; Sato et al. 2010), whereas it would be a poor choice to view cell–cell contacts. That said, labeling the plasma membrane would be a good choice to view cell–cell contacts and cell migration, but might be a poor choice if trying to track cells using monochromatic labeling techniques in cell dense tissues.

It is possible to label the nucleus green and the mitochondria red from a single infection/transfection by inserting between the two genes an internal ribosome entry sites (Bienkowska-Szewczyk & Ehrenfeld 1988; Jang et al. 1988) or the foot-and-mouth disease virus 2A sequence (Ryan & Drew 1994; Donnelly et al. 2001; Fang et al. 2005). The 2A related sites confer nearly stoichiometric co-expression of two or more genes. This multicolor approach can be used to define the entire cell and its various compartments (i.e. nuclear, cytoplasmic, and membrane) with one vector. The 2A-linker system is useful for generation of multispectral transgenic quail by a single lentiviral vector, because it transduces multiple FPs to quail genome as a polycistronic unit in a single viral RNA-genome.

Photoactivatable and Photoconvertible FPs to mark desired cells and tissues

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References

A myriad of photoactivatable and photoconvertible FPs can be used to special mark cells or tissues of interest in a spatiotemporal precise manner within living avian embryos in order to dynamically image cell behaviors using confocal or two-photon (2P) excitation microscopy (see Patterson, 2008, Chudakov et al. 2010 for reviews). Individual cells or groups of cells can be photoactivated or photoconverted to distinguish them from surrounding cells for easier visualization and tracking. Several FPs have been designed that convert from one spectral signature to another upon light-induced conformation changes. One such example is the monomeric Dendra2 protein, which is green until activated with 405 nm light, after which it fluoresces red (Gurskaya et al. 2006; Adam et al. 2009; McKinney et al. 2009). Dendra2 can also be photoconverted to the red fluorescent state in response to intense-blue-light irradiation using a 488 nm Ar laser line. Thus, light of the same wavelength may be required for both visualization and photoconversion of Dendra2, but photoconversion only occurs at high light intensities, whereas Dendra2 green fluorescence can be detected at low light intensities. Photoconversion increases the red-to-green signal intensity, allowing labeled structures to be clearly seen. Other photoactivatable and photoconvertible proteins, such as KikGR, PS-CFP2, Dronpa, have unique photoactivatable features that may be desirable depending on the questions being studied.

The preferred labeling pattern should decide whether confocal or 2P imaging is most suitable for activating or converting the FP. Confocal lasers likely activate FPs at all Z-depths along the path of the laser. However, 2P imaging permits deeper imaging and only provides sufficient excitation energy at the region of focus (Denk et al. 1990; Williams et al. 1994). Thus, using 2P laser illumination likely does not induce out-of-plane photoactivation and is ideal for labeling single cells, groups of cells, and cells deeper within a tissue (Pantazis & Gonzalez-Gaitan 2007; Kulesa et al. 2009). Photoactivatable and photoconvertible FPs expression vectors have been electroporated into developing chick embryos to enabled single-cell labeling (Stark & Kulesa 2007; Kulesa et al. 2009). Transgenic quail that express photoactivatable or photoconvertible FPs will enable tracking and fate mapping of individual and/or clusters of optically-labeled cells.

4D-tracking of cells with computer software

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References

Time-lapse imaging of Tg(tie1:H2B-eYFP) embryos is performed for the quantitative characterization of endothelial cell lineages and evaluation of cellular behaviors under normal and experimental conditions. To that end, tracking of FP+ cells by computer image analysis is indispensable in order to gather quantitative data. To achieve cell-tracking, image data preparation is done through (i) 3- and 4-dimensional registration of images, (ii) segmentation of the target object from the raw image, (iii) identification of the target object as being the same object at subsequent time points, (iv) connection of identical objects across time points to establish tracks, and (v) interpretation of tracking result by graphic and numerical representations (i.e. anatomical maps and graphs). At present, free or commercial image analysis software is available to quantitate cellular behaviors including cell proliferation, movement, differentiation, polarity, and death, to name a few. Fiji/ImageJ is an open-source software program for image processing and analysis (Abramoff et al. 2004; Schindelin et al. 2012). Imaris (Bitplane) and Volocity (PerkinElmer) are commercial image analysis software, which offer various kinds of algorithms required for image processing and analysis with user-friendly interfaces. The commercial software helps inexperienced biologists to work on image processing without the need to write their own programs in Matlab (MathWorks) or the like. Of course, these commercial software programs also allow to plug-in customized programs such as Fiji/ImageJ and Matlab. We will describe an example of image processing using Imaris software in protocol section below.

Perspective

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References

The establishment of a variety of tissue-specific transgenic quail lines is expected to facilitate studies of cellular mechanisms underlying the morphogenesis of amniote embryos by facilitating dynamic tools to study dynamic events. These studies will complement mammalian models that have strong genetic tools by applying time-lapse imaging analysis. Microscopy and fluorescent probes are always evolving. The latest imaging technology of light-sheet microscopy, that is, single plane illumination microscopy (SPIM) and digital scanned laser light-sheet fluorescence microscopy (DSLM) enable even illumination of thick organisms and reduce light scattering artifacts. This advanced technology enabled time-lapse in toto observation of whole zebrafish embryos at cellular level resolution (Keller et al. 2008; Keller & Stelzer 2010), is now being used for time-lapse in toto imaging of transgenic quail embryos. Moreover, adoption of the multi-photon excitation system to light-sheet microscopy further improves time-lapse imaging (Truong et al. 2011) by improving the speed and depth of collection, opening up new regions of development to dynamic analysis such as endoderm derived organs. With the introduction of these advanced microscopy, time-lapse imaging analysis of the transgenic quail embryos uncovers cells lying deep within live tissue and this will have a strong impact on developmental biology.

Protocols

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References

Ex ovo culture of quail embryos in glass-bottomed chambers

Materials
  • Transgenic quail embryos (if not available, FP-electroporated or fluorescent dye-labeled embryos)
  • Fertilized chicken or quail eggs for albumen-agar bed preparation
  • 0.6% Bacto agar, 123 mmol/L NaCl in H2O, autoclaved
  • 10% glucose in H2O, filtered
  • Hanks balanced salt solution (HBSS) with calcium and magnesium (Gibco/Invitrogen 14025), sterile
Equipment
  • Filter papers (Whatman No.1, 1001-185)
  • Hole puncher
  • Disposable Petri dishes, 35 mm, 60 mm, 100 mm
  • Beaker, 100 mL, sterile
  • Glass-bottomed dishes, 35 mm, glass-diameter 27 mm (IWAKI 3960-035) or 2-well chamber slides (Lab-Tek 155380)
  • Plastic container, clear, aprox.15 × 20 cm in size
  • Syringe, 5 mL
  • Needle, 25 gauge
  • Scissors, curved and straight, sterile
  • Forceps, fine tip, sterile
  • Incubator, set at 37°C
  • Epi-fluorescent binocular microscope
  • Confocal laser microscope, inverted
  • Kimwipe

Procedure

Preparations (made in advance)
  1. For the preparation of the albumen-agar bed, incubate fertilized chick or quail eggs for 1–2 days (see note below); six chick or 30 quail eggs yield approximately 60 mL of liquid albumen-agar.
  2. Cut filter paper into 15 mm-width stripes by using office-style scissors. Create single holes or fused holes using a hole puncher. Cut the punched stripes into 15-mm squares by office-style scissors. Store in sterile 100-mm Petri dishes (see note below).
  3. Start incubation of fertilized quail eggs as embryos reach the desired stage at the time of imaging.

Note: For imaging of ventrally located blood vessels in Tg (tie1:H2B-eYFP) by inverted microscopes, embryos should be ex ovo cultured with ventral-side down. To maintain intact circulation throughout the culture, the albumen-agar bed should not cause stress to the developing heart and blood vessels. Probably due to the embryonic metabolism, egg white albumen collected from 1–2 day-incubated fertilized eggs is thinner than that of unincubated eggs, which causes softness of the albumen-agar bed. The punched filter paper (paper ring) is used to hold the embryos under tension (Fig. 2A). Choose a single- or a fused-hole type of paper ring according to the size of the embryo. A box-type window created by cutter is also available (Fig. 2B). The developmental stages of quail embryos have been described previously (Ruffins et al. 2007; Ainsworth et al. 2010).

image

Figure 2. Ex ovo culture of quail embryos. (A) Preparation of quail embryos for ex ovo culture. Place punched filter paper (paper ring) on embryo and allow it to attach (i). Cut excess edges of the paper ring with underneath vitelline membrane (ii), and isolate embryo with the paper ring from yolk (iii). (B) Diagram shows paper ring preparation. The paper ring should not overlap with the embryo body. (C) Embryo in 35-mm glass-bottomed dish. (D) Embryo in 2-well chamber slide. Empty well can be used for humidification. (E) Cross-section view of glass-bottomed vessel for time-lapse imaging of ventrally locating blood vessels in a quail embryo by using an inverted microscope.

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Albumen-agar bed
  1. Collect 30 mL egg white from the incubated chick or quail eggs in a beaker using a syringe. About 5 mL and 1 mL of thin egg albumen per egg are obtained from chick and quail eggs, respectively.
  2. Add 900 μL of 10% glucose to 30 mL of egg white, then mix. Place the beaker in a 37°C incubator until the liquid agar is ready.
  3. Dissolve 0.6% agar, 123 mmol/L NaCl in H2O by using either autoclave or microwave. Then, allow the liquid gel to cool to approximately 50°C on the bench.
  4. While the liquid gel is cooling, prepare 35-mm glass-bottomed dishes or 2-well chamber slides and 35-mm Petri dishes (see note below).
  5. Add 30 mL of liquid agar solution into the beaker that contains 30 mL egg white, then mix gently by swirling the beaker.
  6. Next, aliquot the liquid albumen-agar mixture quickly in glass-bottomed vessels and 35-mm Petri dishes. For 35-mm glass-bottomed dishes (27 mm glass-diameter) and 2-well chamber slides, 200 μL and 120 μL of albumen-agar are the optimum volume, respectively (see note below). For 35-mm petri dishes, 1 mL albumen-agar is sufficient.
  7. Allow the liquid albumen-agar to solidify on the bench.
  8. To keep moist, store the albumen-agar gel dishes in a plastic container lined with humidified paper towels until use.

Note: 35-mm Petri dishes with albumen-agar beds are prepared for temporal culture of embryos before time-lapse observation. As shown in Figure 2C,D, 35-mm glass-bottomed dishes and 2-well chamber slides are prepared for time-lapse observation by inverted microscopes. The type of the imaging chamber is selected according to the shape of the sample holder on the microscope stage. For example, a 35-mm glass-bottomed dish fits in commercial stage-top incubators with single- or 6-circular wells, and a 2-well chamber slide fits in conventional slide holder. If the microscope is equipped with a universal sample holder, both types of chambers can be used. Objective lenses cannot focus into z-positions beyond their working distances. For example, the working distance of Plan-Apochromat 20×, numerical aperture (NA) 0.8 (Zeiss), is 0.55 mm (information on working distances are available on the website of the microscope manufacture). Considering a short working distance of the objective lens, the lining of the albumen-agar bed in glass-bottomed dishes must be as thin as possible. The albumen-agar beds should be prepared on the day of time-lapse imaging. This is important because the humidity and softness of the beds diminish due to water evaporation during storage, which results in significantly lower survival rates of embryos.

Embryo preparation
  1. Cut off the pointed end of the quail egg using curved scissors and gently pour the contents of the egg into a 60-mm Petri dish (Fig. 2A).
  2. (Optional) Thick albumen occasionally interferes with the attachment of early embryos to the paper rings. If the embryo is younger than HH stage 8, remove thick albumen, which is covering the vitelline membrane surface, by using Kimwipe (see Chapman et al. 2001 for details).
  3. Place the paper ring on the embryo using forceps and wait for a few seconds to allow the paper ring to attach to the vitelline membrane (Fig. 2A).
  4. Simultaneously cut off the vitelline membrane and excess edges of the paper ring using straight scissors. Do not disturb the area vasculosa. (Fig. 2A and B).
  5. Hold the paper ring with forceps and carefully transfer it to a 60-mm Petri dish containing HBSS. Remove residual yolk from the embryo by swirling the paper ring gently in HBSS.
  6. Transfer the embryo with the ventral-side up to the albumen-agar bed in a 35-mm Petri dish. Keep the embryo in a humidified plastic container at 37°C.
  7. Screen transgenic embryos or fluorescent-labeled embryos with an epi-fluorescence binocular microscope.
  8. (Optional) If a negligible amount of yolk remains on the ventral surface, gently add a drop of HBSS to the yolk, tilt the dish, and remove the yolk by draining.
  9. Transfer the embryo with the ventral-side down onto albumen-agar bed in the glass-bottomed vessel. (Optional) Prior to the transfer, drop 10 μL of HBSS on the center of the albumen-agar bed. HBSS buffers the bed and embryo, which improves embryo viability.
  10. Incubate the embryo at least for 1 h before starting time-lapse imaging (see note below).

Note: Because the quail embryo moves downward by its own weight, its position is not stable immediately after the transfer to albumen-agar bed. Allow the embryo to settle down on the albumen-agar bed for about 1–2 h prior to starting time-lapse imaging. We usually prepare three to four embryos in glass-bottomed vessels as candidates, and after 1–2 h pre-incubation, choose one embryo that appears healthiest in terms of a vigorous heart beat and smooth blood flow in the vascular plexus. Our method permits time-lapse observation of dorsal aorta formation in Tg(tie1:H2B-eYFP) embryos by using inverted confocal laser microscope for a maximum period of 12 h.

Time-lapse imaging by using a confocal laser microscope

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References

Materials

  • Quail embryos prepared in glass-bottomed vessels (see above section)
  • Water for humidity

Equipment

  • Confocal laser microscope, LSM 510 META NLO, inverted (Zeiss) with a programmable stage (see note below)
  • Incubation system
  • Remote temperature sensor with cable
  • Hard drive

Note: A motorized stage allows for micrometer steps in acquiring static 3D images and tiled images for larger fields-of-view. Acquisition of z-stacks and tiled images is typically controlled by microscope operating software. Reconstruction of z-stacks and stitching of tiled images can be automated using specialized and/or open-source computer programs. An incubating chamber aids in optimizing atmosphere and temperature conditions, as needed for developing tissues or embryos.

Procedure

  1. Warm up an equipped incubator to 37°C during pre-incubation of quail embryos. Place the probe of a remote temperature sensor on the microscope stage to check the temperature around the embryo (see note below).
  2. Set the glass-bottomed vessel in the sample holder on the microscope stage. Find an embryo by using 5× or 10× objective lens.
  3. Set the parameters for time-lapse acquisition. Place an objective lens, Plan-Apochromat 20×, numerical aperture (NA) 0.8, and open the pinhole to the maximum. An argon laser (488 nm wavelength) at a power of 6.0–8.0% is used for excitation of eYFP. Set the long-path filter at 505 nm (LP505) to obtain eYFP signals. Each xy-plane is scanned without averaging at a speed of 1.58 μsec per pixel, 512 × 512 image size. Total of 40–60 z-slices are obtained at 3-μm intervals from bottom to top (z-stack size is 120–180 μm). The image acquisition cycle is configured depending on the speed of the event to be imaged and the magnification, often in the range of 2.0–7.5 min intervals for 12 h (see note below).
  4. Start time-lapse imaging.
  5. Check monitor at 1–2 h intervals as possible. If the embryo is rapidly drifting, stop acquisition at an interval, and save the images. Re-adjust embryo's xyz-position, and re-start at appropriate intervals (see note below).
  6. Save image file on the hard drive and then backup to a desired storage save to free up the microscope computer for additional image data. Original image files should be saved without any modification whatsoever, so that the original data is available to inquiry. Duplicate the image file for additional post-collection image processing, analysis, and visualization.

Note: The humidity inside the glass-bottomed vessels must be maintained as much as possible. If a 35-mm glass-bottomed dish is selected as the culture vessel, place additional 35-mm Petri dishes filled with water in the blank space of the stage-top chamber. In case of a 2-well chamber slide, fill the empty well with water (Fig. 2D). If the space in the incubation chamber permits, put a cup of water in front of the stream of warm air that comes from the heater and is entering the thermal box (Fig. 3F). A larger pinhole size is preferred to reduce scan time and minimize photo-toxicity. If the embryo is not labeled with multicolor fluorophores, a long-path (LP) filter is available to detect almost the full range of the spectrum of emitted light. Using the LP filter helps to obtain brighter signals compared with the band-path (BP) filter. In consideration of the embryo's likely upward or downward drift, set sufficient z-margins by oversampling above and below the planes of interest. Drifting of the embryo can be summarized afterward by registration programs (details are provided in subsequent protocol).

image

Figure 3. Incubation system for warm-blooded animal imaging. (A) Incubation box with heating unit. (B) Stage-top incubator equipped with electrothermal heating system. (C) Self-made incubator covering an inverted confocal laser microscope. (D) Components of self-made incubation system. (E) Another example of a self-made incubator. (F) Schematic diagram of a self-made incubation system.

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Tracking of endothelial cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References

Equipment

  • Image processing and analysis software, Imaris (requires Imaris Track, Imaris Measurement Pro, and Imaris XT modules, after ver. 7) (Bitplane).
  • High-end computer, 3.0–3.4 GHz CPU, 16 Gb DDR RAM, Large SATA hard drive.

Procedure

  1. Load saved image data from the hard drive. Imaris software can convert numerous image file formats in order to analyze with its own format. Loaded image files are automatically reconstructed into xyz plus t and color.
  2. Crop the ROI and time as needed and save the file in Imaris format. This permits faster analysis and may permit the analysis to be carried out using a typical laptop.
  3. (Optional) If the raw images are dirty with speckles, noise reduction using median or Gaussian filters will reduce the speckled artifacts within the images, which will help in the subsequent segmentation processes. We find a median 3 × 3 × 1 filter is very useful to clear up the image data.
  4. To correct the embryo's likely drift, set standard points in each time frame and connect them. Anchored objects other than the target objects are preferred as the standard points. Drift correction is automatically done on the basis of averaging the selected standard points (see note below).
  5. Segment objects by applying spot detection algorithm.
  6. Objects can be tracked by using the Imaris Track algorithm. This connects previous and subsequent objects across time points by using the Brownian motion or Auto-regressive motion model. Depending on how the cells of interest have been labeled, cell tracking may permit cell behaviors such as migration speeds and paths, space and time of mitosis and differentiation to be quantitatively analyzed.
  7. Check the results of the automated tracking. Spots and tracks can be manually added, deleted, merged, and connected as needed (see note below).
  8. Process and analyze the tracking results. Imaris MeasurementPro helps statistical data collection and visual presentation.
  9. The best approach is to collect your own image data for analysis or obtain already published raw image data to work out your own image analysis protocols based on your interests.

Note: Drift correction is an indispensable process to allow detection of the true movement of the objects of interest. The Imaris Track module can correct the embryo's drift by using a landmark-based registration approach. This methodology requires setting of standard points that are connected as identical xyz-position in each time frame. For instance, immotile tissue labeled with DiI or red FP can be used as standard point (Fig. 4; case exploits stochastically distributed H2B-mCherry signals in somites). Instead of the landmark-based registration, voxel value-based registration by using TurboReg (ImageJ plug-in) (Thévenaz et al. 1998) or PoorMan 3D Reg (Michael Liebling lab, University of California, Santa Barbara, USA) (Liebling et al. 2005) can be used.

image

Figure 4. Processing of time-lapse images of objects in quail embryos. (A) Time-lapse imaging of a Tg(tie1:H2B-eYFP) embryo at HH stage 12 by using a inverted confocal laser microscope, LSM 510 (Zeiss). Individual endothelial cells in forming dorsal aortae are visualized by nuclear eYFP signals (upper panels). For the purpose of drift correction, few somitic cells in the Tg(tie1:H2B-eYFP) embryo are labeled with H2B-mCherry by electroporation technique. During image acquisition, the embryo drifted toward lower left as indicated by the drastic change of the marked H2B-mCherry positions (white tracks in lower panels). Scale bar, 100 μm. (B) mCherry signals in the somites are used for standard points by Imaris registration program (i.e. landmark-based registration). After correcting the embryo's drift, positions of mCherry are aligned so that the true motion of eYFP+ endothelial cells can be analyzed. Scale bar, 100 μm. (C) Automatic detection of H2B-eYFP+ endothelial cells signals by Imaris. (D) Color-coded representation of the tracking result. (E) Time-lapse imaging of a mitotic endothelial cell in Tg(tie1:H2B-eYFP) embryo. Higher time-resolution imaging enables analysis of endothelial cell proliferation in vivo. Scale bar, 8 μm.

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The manual correction of tracks requires much effort and time. Higher time-resolution images are effective for bypassing or reducing the need for manual correction. However, the Imaris Track algorithm cannot process dividing cells, for example, as branched tracks, and thus, one of the daughter cells is not properly connected to the mother cell. To track dividing cells accurately, manual correction is needed.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Current situation of transgenic chicken and quail
  5. Tissue-specific promoters for fluorescent reporters
  6. Generation of transgenic quail
  7. Ex ovo culture of quail embryos
  8. Incubation system for warm-blooded embryo imaging
  9. Time-lapse imaging of quail embryos by confocal laser microscopy
  10. Multispectral labeling and imaging strategies
  11. Photoactivatable and Photoconvertible FPs to mark desired cells and tissues
  12. 4D-tracking of cells with computer software
  13. Perspective
  14. Protocols
  15. Time-lapse imaging by using a confocal laser microscope
  16. Tracking of endothelial cells
  17. Acknowledgments
  18. References
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