The authors would like to send a big thank you to Clemson Broadcast Productions, audio recording by J. Bright, Narration by A. D. McQuiston, Audio sweetening, K. Murphy, videography by G. Spake, Graphic arts by T. Messervy, Video editing by T. Messervy and E. Rodgers. Technical assistance and scientific advice was inspired by the advice of S. Kawada, S. Kubo, J. Hudson, T. Darroudi, D. Mulwee, H. Qian, Y. W. Lam, M. B. Johnstone, C. Campanati, A. C. Lane, and R. Dineshram. This study was funded by three GRF grants from the HKSAR-RGC (Grant Numbers: 705511P, 705112P, and 17304914).

<ol>
	<li>Aizenberg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeleton+of+Euplectella+sp.:+structural+hierarchy+from+the+nanoscale+to+the+macroscale.">Skeleton of Euplectella sp.: structural hierarchy from the nanoscale to the macroscale.</a> Science. 309 (5732), 275-278 (2005).</li><li>de Nooijer, L. J., Toyofuku, T., Oguri, K., Nomaki, H., Kitazato, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pH+distribution+in+foraminifera+determined+by+the+fluorescent+probe+HPTS.">Intracellular pH distribution in foraminifera determined by the fluorescent probe HPTS.</a> Limnol Oceanogr Methods. 6 (11), 610-618 (2008).</li><li>de Nooijer, L. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Settlement+of+the+serpulid+polychaete+Hydroides+elegans+(Haswell)+on+the+arborescent+bryozoan+Bugula+neritina+(L.):+evidence+of+a+chemically+mediated+relationship.">Settlement of the serpulid polychaete Hydroides elegans (Haswell) on the arborescent bryozoan Bugula neritina (L.): evidence of a chemically mediated relationship.</a> J Exp Mar Biol Ecol. 220, 171-190 (1998).</li><li>Chan, V. B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+compositional+and+ultrastructural+shifts+during+the+development+of+calcareous+tubes+in+the+biofouling+tubeworm,+Hydroides+elegans.">Evidence of compositional and ultrastructural shifts during the development of calcareous tubes in the biofouling tubeworm, Hydroides elegans.</a> J. Struct. Biol. 189 (3), 230-237 (2015).</li><li>Dickson, A. G., Goyet, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of methods for the analysis of the various parameters of the carbon dioxide system in sea water. Version 2. , (1994).</li><li>Chan, V. B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+deposition+of+crystalline+aragonite+in+the+controlled+biomineralization+of+the+calcareous+tubeworm.">Direct deposition of crystalline aragonite in the controlled biomineralization of the calcareous tubeworm.</a> Front Mar Sci. 2, 97 (2015).</li><li>Bond, J., Varley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+flow+cytometry+and+SNARF+to+calibrate+and+measure+intracellular+pH+in+NS0+cells.">Use of flow cytometry and SNARF to calibrate and measure intracellular pH in NS0 cells.</a> Cytometry A. 64, 43-50 (2005).</li><li>Lloyd, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+number+and+crystallographic+contrast+images+with+the+SEM:+a+review+of+backscattered+electron+techniques.">Atomic number and crystallographic contrast images with the SEM: a review of backscattered electron techniques.</a> Mineral Mag. 51, 3-19 (1987).</li><li>Perez-Huerta, A., Dauphin, Y., Cuif, J. P., Cusack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+electron+backscatter+diffraction+(EBSD)+data+from+calcite+biominerals+in+recent+gastropod+shells.">High resolution electron backscatter diffraction (EBSD) data from calcite biominerals in recent gastropod shells.</a> Micron. 42 (3), 246-251 (2011).</li><li>Bandli, B. R., Gunter, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles and techniques of electron microscopy: biological applications. , (2000).</li><li>Wirth, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused+Ion+Beam+(FIB)+combined+with+SEM+and+TEM:+Advanced+analytical+tools+for+studies+of+chemical+composition,+microstructure+and+crystal+structure+in+geomaterials+on+a+nanometre+scale.">Focused Ion Beam (FIB) combined with SEM and TEM: Advanced analytical tools for studies of chemical composition, microstructure and crystal structure in geomaterials on a nanometre scale.</a> Chem Geo. 261, 217-229 (2009).</li><li>Volkert, C. A., Minor, A. 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The authors have nothing to disclose.

Live optical imaging is a useful method for observing cellular events in a multicellular organism. Here, internal pH and calcium ion indicators were used to measure the flux of ions at the mineralization sites. In these regions, active ion pumping is required to elevate pH and Ca2+ concentration to enable calcification2,3. When applying fluorescent molecules to study an organism, it is critical to ensure that the concentration used has negligible toxic...

Biomineralization is a complex series of events, which bridges a suite of cellular activities resulting in the production of exquisitely ordered minerals1. The challenge is to characterize both the dynamic cellular process and the sophisticated mineral structures using a combination of optical and electron microscopy methods. An elevation of intracellular pH favors the formation of CaCO3 crystals, hence, identifying the life stage that has an increased pH reveals the time when calcification is likely to be occurring2,3.
The tubeworms from the family Serpulidae are common calcifiers in the ocean4. It is also a popular invertebrate model for marine research, especially in biofouling5,6. In this study, the process of calcification in mineralizing compartments during biomineralization is observed. The rapid process of metamorphosis includes the emergence of calcium carbonate structures7,8.
We demonstrate how internal pH measurements can be performed on the tubeworm, and how life stages and tissues relevant for calcification can be screened. After the life stage of interest is identified, the tissue responsible for calcification can be characterized at a higher resolution using electron microscopy methods. Using fluorescent microscopy, we determine the time required for calcium carbonate to appear after metamorphic induction. A similar stage of life was subsequently visualized with SEM-EDS for elemental composition distribution, and the deposited mineral was analyzed using two different electron microscopy methods, specifically SEM-EBSD and FIB-TEM.

<table><tbody><tr><td>Hexamethyldisilazane&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>16700(EM)</td><td></td></tr><tr><td>Osmium Tetroxide 2% Aqueous Solution</td><td>Electron Microscopy Sciences</td><td>19192</td><td></td></tr><tr><td>IBMX 3-Isobutyl-1-methylxanthine</td><td>ThermoFisher Scientific</td><td>PHZ1124</td><td></td></tr><tr><td>Nigericin, Free Acid</td><td>ThermoFisher Scientific</td><td>N7143-5MG</td><td></td></tr><tr><td>35 mm diameter dish, hole size 27 mm, Glass No.0, Non-coat</td><td>ThermoFisher Scientific</td><td>D110400</td><td></td></tr><tr><td>5-(and-6)-Carboxy SNARF-1, Acetoxymethyl Ester, Acetate</td><td>ThermoFisher Scientific</td><td>C-1271</td><td></td></tr><tr><td>BDH Potassium Chloride, ACS Grade</td><td>VWR</td><td>BDH0258-500G</td><td></td></tr><tr><td>Paraformaldehyde&lt;br/&gt; reagent grade, crystalline</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>1 M Hydrochloric Acid for Volumetric Analysis</td><td>Wako Pure Chemical Industries, Ltd</td><td>083-01095</td><td></td></tr><tr><td>0.05 M Sodium Hydroxide Solution for Volumetric Analysis</td><td>Wako Pure Chemical Industries, Ltd</td><td>199-02185</td><td></td></tr><tr><td>Calcein</td><td>Sigma</td><td>C0875</td><td></td></tr><tr><td>FASW</td><td>Iwaki Co. Ltd.</td><td>Rei-sea Marine</td><td></td></tr><tr><td>Mixed Cellulose Ester Membranes; 47 mm dia, 0.45 &amp;micro;m</td><td>ADVANTEC</td><td>A045A047A</td><td></td></tr><tr><td>ethanol</td><td>Wako Pure Chemical Industries, Ltd</td><td>051-00476</td><td></td></tr><tr><td>Artificial seawater for buffers</td><td></td><td></td><td>by SOP06 of DOE (1994), cdiac.ornl.gov/ftp/cdiac74/sop06.pdf</td></tr><tr><td>Sodium Chloride</td><td>Wako Pure Chemical Industries, Ltd</td><td>191-01665</td><td></td></tr><tr><td>Potassium Chloride</td><td>Wako Pure Chemical Industries, Ltd</td><td>163-03545</td><td></td></tr><tr><td>Magnesium Chloride Hexahydrate</td><td>Wako Pure Chemical Industries, Ltd</td><td>135-00165</td><td></td></tr><tr><td>Calcium Chloride</td><td>Wako Pure Chemical Industries, Ltd</td><td>039-00475</td><td></td></tr><tr><td>Sodium Sulfate</td><td>Wako Pure Chemical Industries, Ltd</td><td>197-03345</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Wako Pure Chemical Industries, Ltd</td><td>089-08415</td><td></td></tr><tr><td>2-amino-2-hydroxymethyl-1,3-propanediol (tris)</td><td>Wako Pure Chemical Industries, Ltd</td><td>207-06275</td><td></td></tr><tr><td>2-aminopyridine</td><td>Wako Pure Chemical Industries, Ltd</td><td>011-02775</td><td></td></tr><tr><td>Orion 5-star Plus pH meter</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>PrpHecT ROSS Micro Combination pH Electrode 8220BNWP</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Axiovision, Version 4.6, Axio Observer Z1</td><td>Zeiss</td><td></td><td></td></tr><tr><td>ImageJ</td><td>NIH, Bethesda, MD, USA</td><td></td><td></td></tr><tr><td>HRTEM H500</td><td>Hitachi</td><td></td><td></td></tr><tr><td>SU6600 VPSEM</td><td>Hitachi</td><td></td><td></td></tr><tr><td>NB5000 Focused Ion and Electron Beam (FIB-SEM) system</td><td>Hitachi&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

characterization of calcification events using live optical and electron microscopy techniques in a marine tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

The following are some observations of the calcification process during the metamorphosis of the tubeworm. Figure 1 shows that the pH values near the collar region is higher than the other tissues after metamorphosis. Figure 2i shows a tubeworm with homogeneous distribution of Ca, suggesting no major calcification events have begun; Figure 2ii shows a tubeworm that has calcified for a longer period, suggesting calcification has gone beyon...

Watch this Scientific Journal Video about Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm at JoVE.com

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

characterization-calcification-events-using-live-optical-electron

Research

JoVE Journal

Biology

7.4K Views.  Clemson University. We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization. 

Video: Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps&#160;and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter&#160;scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,&#160;(2) superior optical sectioning ability,&#160;and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

An integrated suite of imaging techniques has been applied to determine polyp morphology and tissue structure in the Caribbean corals Montastraeaannularis and M. faveolata. Fluorescence, serial block face,&#160;and two-photon confocal laser scanning microscopy have identified lobate structure, polyp walls, and estimated chromatophore and zooxanthellae densities and distributions.

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues ...

Environment

Microfocus X-ray CT (microCT) Imaging of Actinia equina (Cnidaria), Harmothoe sp. (Annelida), and Xenoturbella japonica (Xenacoelomorpha)

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. Non-destructive microfocus X-ray computed tomography (microCT) imaging has become a powerful and emerging protocol in biology, due to technological advancements in sample staining methods and innovations in microCT hardware, processing computers, and data analysis software. However, this protocol is not commonly used, as it is in the medical and industrial fields. One of the reasons for this limited use is the lack of a simple and comprehensible manual that covers all of the necessary steps: sample collection, fixation, staining, mounting, scanning, and data analyses. Another reason is the vast diversity of metazoans, particularly marine invertebrates. Because of marine invertebrates&#8217; diverse sizes, morphologies, and physiologies, it is crucial to adjust experimental conditions and hardware configurations at each step, depending on the sample. Here, microCT imaging methods are explained in detail using three phylogenetically diverse marine invertebrates: Actinia equina (Anthozoa, Cnidaria), Harmothoe sp. (Polychaeta, Annelida), and Xenoturbella japonica (Xenoturbellida, Xenacoelomorpha). Suggestions on performing microCT imaging on various animals are also provided.

Microfocus X-ray CT microCT Imaging of Actinia equina Cnidaria, Harmothoe sp. Annelida, and Xenoturbella japonica Xenacoelomorpha

Here, protocols for performing microfocus X-ray computed tomography (microCT) imaging of three marine invertebrate animals are explained in detail. This study describes steps such as sample fixation, staining, mounting, scanning, image reconstruction, and data analyses. Suggestions on how the protocol can be adjusted for different samples are also provided.

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. ...

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Biochemistry

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. Animals cope with these stimuli by modifying their behavior. Understanding the mechanisms underlying such behavioral modification requires quantification of neural activity during the behavior of interest. Here, we report a method for calcium imaging in freely behaving Caenorhabditis elegans with nonlocalized vibration of specific frequency, displacement, and duration. This method allows the production of well-controlled, nonlocalized vibration using an acoustic transducer and quantification of evoked calcium responses at single-cell resolution. As a proof of principle, the calcium response of a single interneuron, AVA, during the escape response of C. elegans to vibration is demonstrated. This system will facilitate understanding of neural mechanisms underlying behavioral responses to mechanical stimuli.

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Reported here is a system for calcium imaging in freely behaving Caenorhabditis elegans with well-controlled, nonlocalized vibration. This system allows researchers to evoke nonlocalized vibrations with well-controlled properties at nano-scale displacement and to quantify calcium currents during responses of C. elegans to the vibrations.

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. ...

Behavior

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence of calcification inhibitors, that concomitantly lead to a loss of vessel elasticity and function. Vascular calcification is an important contributor to morbidity and mortality in many pathologies, including chronic kidney disease, diabetes mellitus, and atherosclerosis. Current research models to study vascular calcification are limited and are only viable at the late stages of calcification development in vivo. In vitro tools for studying vascular calcification use end-point measurements, increasing the demands on biological material and risking the introduction of variability to research studies. We demonstrate the application of a novel fluorescently labeled probe that binds to in vitro calcification development on human vascular smooth muscle cells and determines the real-time development of in vitro calcification. In this protocol, we describe the application of our newly developed calcification assay, a novel tool in disease modeling that has potential translational applications. We envisage this assay to be relevant in a broader spectrum of mineral deposition research, including applications in bone, cartilage, or dental research.

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Cardiovascular disease is the leading cause of death worldwide. Vascular calcification contributes substantially to the burden of cardiovascular morbidity and mortality. This protocol describes a simple method to quantify vascular smooth muscle cell-mediated calcium precipitation in vitro by fluorescent imaging.

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Neuroscience

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

Bioengineering

Tracking Calcium in the Early Developmental Phases of C. elegans

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 &#176;C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter &#34;CaFE&#34; for Calcium during Fertilization in C. elegans. The CaFE reporter was&#160;engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.

Here, we present protocols and tools for visualizing calcium during fertilization and early embryogenesis using a genetically encoded calcium reporter expressed in the germline of the model nematode C. elegans.

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode ...

Developmental Biology

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf can measure PSII absorption cross-section (&#963;PSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq&#8242;/Fm&#8242;), and non-photochemical quenching (NPQNSV) for various eukaryotic algae and cyanobacteria, almost all FRRf studies to date have focused on phytoplankton. Here, the protocol describes how to measure PSII photophysiology of an epizoic alga Colacium sp.&#160;Ehrenberg 1834 (Euglenophyta), in its attached stage (attached to zooplankton), using cuvette-type FRRf. First, we estimated the effects of substrate zooplankton (Scapholeberis mucronata O.F. M&#252;ller 1776, Cladocera, Daphniidae) on baseline fluorescence and &#963;PSII, Fv/Fm, Fq&#8242;/Fm&#8242;, and NPQNSV of planktonic Colacium sp. To validate this methodology, we recorded photophysiology measurements of attached Colacium sp. on S. mucronata and compared these results with its planktonic stage. Representative results showed how the protocol could determine the effects of calcium (Ca) and manganese (Mn) on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II photophysiology and primary productivity. Here we describe a protocol to measure PSII photophysiology of epizoic alga, Colacium sp. on substrate zooplankton using cuvette-type FRRf.

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

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