This JoVE Method Collection issue was funded by NIH 1K99DC01883001. I thank my postdoc mentor Liqun Luo, my collaborators Eric Betzig and Tian-Ming Fu for the strong support to develop my interest in time-lapse imaging during my postdoc project. I also thank the School of Brain Science and Brain Medicine in Zhejiang University for recruiting me and supporting me to continue time-lapse imaging study in my own lab.

<ol>
	<li>Rieger, S., Wang, F., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+imaging+of+neural+development:+zebrafish+lead+the+way+into+the+fourth+dimension.">Time-lapse imaging of neural development: zebrafish lead the way into the fourth dimension.</a> Genesis. 49 (7), 534-545 (2011).</li><li>Wong, C., Chen, A. A., Behr, B., Shen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+microscopy+and+image+analysis+in+basic+and+clinical+embryo+development+research.">Time-lapse microscopy and image analysis in basic and clinical embryo development research.</a> Reproductive Biomedicine Online. 26 (2), 120-129 (2013).</li><li>Ezin, M., Fraser, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+imaging+of+the+early+avian+embryo.">Time-lapse imaging of the early avian embryo.</a> Methods in Cell Biology. 87, 211-236 (2008).</li><li>Poggi, L., Zolessi, F. R., Harris, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+analysis+of+retinal+differentiation.">Time-lapse analysis of retinal differentiation.</a> Current Opinion in Cell Biology. 17 (6), 676-681 (2005).</li><li>Svensson, C. M., Medyukhina, A., Belyaev, I., Al-Zaben, N., Figge, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untangling+cell+tracks:+Quantifying+cell+migration+by+time+lapse+image+data+analysis.">Untangling cell tracks: Quantifying cell migration by time lapse image data analysis.</a> Cytometry. Part A. 93 (3), 357-370 (2018).</li><li>Christen, P., Muller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+visualisation+and+quantification+of+bone+resorption+and+bone+formation+from+time-lapse+imaging.">In vivo visualisation and quantification of bone resorption and bone formation from time-lapse imaging.</a> Current Osteoporosis Reports. 15 (4), 311-317 (2017).</li><li>Januschke, J., Loyer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+immobilization+of+Drosophila+tissues+with+fibrin+clots+for+live+imaging.">Applications of immobilization of Drosophila tissues with fibrin clots for live imaging.</a> Journal of Visualized Experiments. (166), e61954 (2020).</li><li>Li, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+explant+system+for+time-lapse+imaging+studies+of+olfactory+circuit+assembly+in+Drosophila.">An explant system for time-lapse imaging studies of olfactory circuit assembly in Drosophila.</a> Journal of Visualized Experiments. (176), e62983 (2021).</li><li>Schramm, P., Hetsch, F., Meier, J. C., Koster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+fully+active+brain+tissue+in+awake+Zebrafish+larvae+and+juveniles+by+skull+and+skin+removal.">In vivo imaging of fully active brain tissue in awake Zebrafish larvae and juveniles by skull and skin removal.</a> Journal of Visualized Experiments. (168), e62166 (2021).</li><li>Ratke, J., Kramer, F., Strobl, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+live+imaging+of+multiple+insect+embryos+in+sample+chamber-based+light+sheet+fluorescence+microscopes.">Simultaneous live imaging of multiple insect embryos in sample chamber-based light sheet fluorescence microscopes.</a> Journal of Visualized Experiments. (163), e61713 (2020).</li><li>Terzi, A., Alam, S. M. S., Suter, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS+live+cell+imaging+during+neuronal+development.">ROS live cell imaging during neuronal development.</a> Journal of Visualized Experiments. (168), e62165 (2021).</li><li>Mutlu, A. S., Chen, T., Deng, D., Wang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+imaging+of+lipid+storage+dynamics+in+Caenorhabditis+elegans+using+stimulated+Raman+scattering+microscopy.">Label-free imaging of lipid storage dynamics in Caenorhabditis elegans using stimulated Raman scattering microscopy.</a> Journal of Visualized Experiments. (171), e61870 (2021).</li><li>Boutillon, A., Escot, S., David, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+and+spatially+controlled+volume+ablations+using+a+two-photon+microscope+in+the+Zebrafish+gastrula.">Deep and spatially controlled volume ablations using a two-photon microscope in the Zebrafish gastrula.</a> Journal of Visualized Experiments. (173), e62815 (2021).</li></ol>

The author has nothing to disclose.

Development is characterized by the dynamic change of structures on the levels of organelles, cells, and organs. The ability of time-lapse imaging to collect four-dimensional information from the same structure in its physiological condition makes it an ideal technique to study the dynamic process of development1,2,3,4,5,6. The vast amount of data collected through live imaging also enables the quantitative analysis of development. Although time-lapse imaging has been more widely used for single-layered cell culture, imaging of three-dimensional live tissues or animals is much more challenging due to technical difficulties, such as light scattering of live tissues, developing an appropriate mounting strategy and labeling method, maintaining normal physiological condition during an extensive imaging period, accessibility to a suitable microscope, etc. These challenges restrict the usage of live imaging in many labs. In this method collection, seven papers reported new methods of live sample preparation, labeling, and laser-based manipulation. All of these methods facilitate the live imaging of developmental processes from different angles.
Direct imaging of deep regions is technically challenging due to the strong scattering of live tissues. This scattering can be minimized by the exposure of internal organs through dissection,&#160;thus improving&#160;the spatial resolution of images. In this collection, Januschke and Loyer7 described a protocol for dissecting Drosophila larval brains and a mounting method using Fibrin clots. This mounting strategy allowed time-lapse imaging using inverted microscopes of multiple samples on the same dish as it only required the&#160;addition of fibrinogen and thrombin. Li and Luo8 presented a protocol for the dissection of an antennae-brain explant from fly pupae and an ex vivo culture condition that maintained normal development of the olfactory circuit for 24 h. Their system enabled visualizing axonal and dendritic targeting in the assembly of a fly olfactory circuit from its physiological condition. This explant system is suitable for two-photon microscope and lattice lightsheet microscope with adaptive optics-based imaging, allowing both long-term continuous imaging and fast imaging with high spatiotemporal resolution in a short time period. Schramm et al.9 developed a minimally invasive surgical procedure to remove the skin and soft skull cartilage of zebrafish through micro-peeling. This allowed them to observe some key events in later developmental stages, including neuronal differentiation, maturation, plasticity, and Ca2+ transient up to 30 days post-fertilization. It also allowed them&#160;to perform live imaging after pigmentation of the zebrafish larval skins. In addition to dissection, appropriate mounting of samples is also crucial for live imaging. Ratke et al.10 presented an experimental framework for simultaneous live imaging of multiple fly embryos using light sheet microscopy. This protocol increased the overall throughput and is ideal for comparative study.
In addition to sample preparation, new labeling methods facilitate visualizing specific structures in live tissues. Terzi et al.11 reported a genetically encoded H2O2-specific biosensor, roGFP2-Orp1, for imaging intracellular H2O2 levels from cultured zebrafish neurons and whole larvae during development. One important advantage of this genetically encoded H2O2 biosensor was the improvement of the temporal and spatial resolution of H2O2 detection. Another study from Mutlu et al.12 described stimulated Raman scattering (SRS) microscopy, a label-free chemical imaging method for rapid and quantitative detection of lipids in live cells with a subcellular resolution. Both H2O2 and lipids are easily lost during sample fixation. The development of labeling or imaging methods in live tissue not only circumvented this challenge, but also retained spatial and temporal information of these structures in biological processes.
Finally, light can also be used for cell ablation. Boutillon et al.13 showed a method to ablate deep and spatially well-defined volumes in zebrafish using a two-photon microscope. Compared with ultraviolet laser-based cell ablations, the two-photon laser improved axial resolution and tissue penetration. This protocol provided a perturbation strategy to study cell-cell interaction during development.
The advancement of microscopic technologies allowed us to understand many developmental processes in four dimensions. Protocols in this collection will help more scientists to overcome the technical barrier of live imaging in different biological systems. We anticipate that more methods that facilitate live imaging will be developed in the future.

methods and protocols for live imaging in development

ARTICLES DISCUSSED:
Januschke, J., Loyer, N. Applications of immobilization of Drosophila tissues with fibrin clots for live imaging. Journal of Visualized Experiments. (166), 10.3791/61954 (2020).
Li, T., Luo, L. An explant system for time-lapse imaging studies of olfactory circuit assembly in Drosophila. Journal of Visualized Experiments. (176), 10.3791/62983 (2021).
Schramm, P., Hetsch, F., Meier, J. C., Koster, R. W. In vivo imaging of fully active brain tissue in awake zebrafish larvae and juveniles by skull and skin removal. Journal of Visualized Experiments. (168), 10.3791/62166 (2021).
Ratke, J., Kramer, F., Strobl, F. Simultaneous live imaging of multiple insect embryos in sample chamber-based light sheet fluorescence microscopes. Journal of Visualized Experiments. (163), 10.3791/61713 (2020).
Terzi, A., Alam, S. M. S., Suter, D. M. ROS live cell imaging during neuronal development. Journal of Visualized Experiments. (168), 10.3791/62165 (2021).
Mutlu, A. S., Chen, T., Deng, D., Wang, M. C. Label-Free imaging of lipid storage dynamics in Caenorhabditis elegans using stimulated Raman scattering microscopy. Journal of Visualized Experiments. (171), 10.3791/61870 (2021).
Boutillon, A., Escot, S., David, N. B. Deep and spatially controlled volume ablations using a two-photon microscope in the zebrafish gastrula. Journal of Visualized Experiments. (173), 10.3791/62815 (2021).

Read this Scientific Article Protocol about Methods And Protocols For Live Imaging In Development at JoVE.com