Name: 개발 Neuroepithelium의 세포 문제의 고해상도 라이브 영상 (동영상) | JoVE Journal
Uploaded: 2012-04-12T13:00:00.000+00:00
Duration: 10 min 59 s
Description: 학습하세요 발달 중인 신경상피의 세포 행동을 고해상도 라이브 영상으로 관찰하는 방법을. 형광 단백질을 이용한 병아리 배아 신경관 절편 준비와 장시간 현미경 촬영 기술을 익혀보세요.

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1. Dish Preparation
<ol>
 <li>Dishes used for slice culture are glass-bottomed (with a coverslip as the base) (WillCo dishes). These are placed on lens tissue in a 6 cm tissue culture dish to keep the glass bottom clean.</li>
 <li>On the day before the experiment, add 2 ml 0.1% poly-L-lysine solution to the glass bottomed dish and incubate for 5min at room temperature to allow the poly-L-lysine to coat the glass bottom.</li>
 <li>Remove poly-L-lysine solution and rinse three times in deionised water, and then once in 70% ethanol.</li>
 <li>Leave to dry overnight. Dishes can also be dried by gently heating in a microwave at low power for 30-45 seconds.</li>
</ol>
2. Embryo Electroporation
<ol>
 <li>Incubate eggs at 37 °C to Hamburger-Hamilton (HH) stage 10 (~36 hours) (or other desired stage).</li>
 <li>Before beginning prepare glass needles (we use a Flaming/Brown model p87 microcapillary puller) and break off the tip of the needle using a fine forceps under a dissecting microscope. The end of the needle should be sharp enough to pierce the embryo while not being so narrow that it impedes injection of the DNA solution.</li>
 <li>Window eggs and place electrodes (5mm apart) on either side of the embryo.</li>
 <li>Inject DNA (~0.025 - 0.5 μg/μl in deionised water colored with a small amount of fast green) into the neural tube.</li>
 <li>Apply current - 12-17 V three times, 50ms pulse length with 950 ms between pulses.</li>
 <li>We use low concentrations of DNA and low electroporation voltages to achieve mosaic expression so that we can follow individual cells.</li>
 <li>Cover window in the eggshell with cellotape and make sure it is sealed.</li>
 <li>Allow embryos to recover for 3-4 hours or overnight at 37 °C.</li>
</ol>
3. Collagen and Slice Culture Medium Preparation
<ol>
 <li>Prepare collagen mix and slice culture medium about an hour before slicing.</li>
 <li>To 300 μl type 1 collagen add 100 μl 0.1% acetic acid solution and 100 μl 5 x L15 medium. Vortex thoroughly after each addition. The solution should turn yellow.</li>
 <li>Now add 15-20 μl 7.5% sodium bicarbonate and thoroughly vortex. The solution should become slightly pink, and the volume of sodium bicarbonate required for this may differ. Keep on ice and make up fresh each time.</li>
 <li>To 10 ml neurobasal medium add B-27 supplement and Glutamax, to a 1 x final concentration and 10 μl Gentamicin solution.</li>
 <li>Place medium in a 37 °C incubator buffered with 5% CO2. Leave the top of the container loose to allow it to equilibrate with the CO2.</li>
</ol>
4. Slice Culture
<ol>
 <li>Remove embryos from egg and wash in L15 medium.</li>
 <li>Place in a tissue culture dish with a layer of sylgard at the bottom and pin them out through the surrounding extra-embryonic membranes so that they are stretched taut.</li>
 <li>Using a microknife, slice embryo as straight as possible through region of interest. For spinal cord, slices should be between 1-2 somites thick. Leave slices attached to embryo while you slice other embryos so you don't lose them.</li>
 <li>Prepare a 200 μl micropipette tip by cutting off ~1 mm of the tip and attaching this to a p2 or p10. This tip will be used to transfer the spinal cord slices to the glass bottom dish. A 10 μl tip is not broad enough to pick up slices.</li>
 <li>Coat the inside of the tip with collagen by pipetting 1 μl collagen mix prepared earlier. Leave for 1-2 mins and then rinse with L15 medium. This will prevent the tissue from sticking to the inside of the tip.</li>
 <li>Detach slice from embryo using a microknife and remove from the dish using a p2 or p10 fitted with a 200 μl tip and set to 1 μl. Try to take up as little medium as possible.</li>
 <li>Vortex the collagen mix and drop 5-8 μl of this on to the poly-l-lysine coated dish.</li>
 <li>Immediately put embryo slice into collagen and position into place with a pair of fine forceps. Slices should be positioned so that the side to be imaged is flush with the coverslip. The tissue should adhere to the poly-l-lysine coating on the coverslip.</li>
 <li>Repeat this until you have several slices on the coverslip. We can usually place 6-9 slices on one dish.</li>
 <li>Once all the slices are in place, cover the dish and allow the collagen to set for 20 minutes. Some of the earliest placed collagen may already have started to dry out. To prevent this add a small amount of L15 to these before covering.</li>
 <li>Once set, carefully add 2ml slice culture medium that has been equilibrated in 5% CO2 at 37 °C for at least an hour. Care must be taken to not dislodge the collagen from the coverslip.</li>
 <li>Place in a 37 °/ 5% CO2 incubator and allow slices to recover for at least 3 hours before imaging. If there is no humidity, place dish in a perspex box with a bit of damp tissue paper in one corner.</li>
</ol>
5. Imaging Embryo Slices
<ol>
 <li>We use a DeltaVision Core wide-field microscope fitted with a WeatherStation environmental chamber to image slices. The chamber is constantly maintained at 37 °C, with a CO2 perfusion apparatus to maintain the microscope stage at 5% CO2 / 95% air.</li>
 <li>Imaging is normally carried out using a 40 x / 1.30 NA oil immersion lens and images are captured with a CoolSnap HQ2 cooled CCD camera.</li>
 <li>Z-sections are captured every 1.5 μm through 45 μm tissue. Exposure time should be kept as low as possible. We normally expose for 5-50 ms for each Z-section. Images are captured every 7 minutes and up to 9 slices can be visited using the DeltaVision system's precise point visiting function.</li>
</ol>
6. Representative Results
An example time-lapse sequence of a spinal cord progenitor cell is shown in Fig. 2a and corresponding Movie 1. Imaging was started on a spinal cord slice from a two-day-old (HH stage 12) embryo. This cell was transfected with a construct expressing GFP-αTubulin. During this early stage, neural progenitor cells undergo predominantly progenitor-progenitor mode divisions during which the cells divide to generate two further cycling progenitor cells. Fig. 2b and corresponding Movie 2 shows a cell transfected with GFP-GPI (GPI anchored GFP), marking the cell membrane. This cell undergoes a division during which the basal process splits into two and is equally inherited by the daughter cells.
<img alt="Neural tube DNA injection, slicing, mounting, imaging; Deltavision widefield microscopy setup." src="/files/ftp_upload/3920/3920fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Slice culture protocol for time-lapse imaging. 
<img alt="Time-lapse microscopy of GFP-Tubulin and GFP-GPI in cells; fluorescence analysis over time." src="/files/ftp_upload/3920/3920fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Cell behavior during normal spinal cord development. (a) Selected frames from Movie 1. Cell expressing GFP-tubulin divides with a cleavage plane that is perpendicular to the apical surface (2 h 20 min), generating two daughter cells that divide once again (24 h 23 min and 25 h 54 min). (b) Selected frames from Movie 2. Cell transfected with GFP-GPI undergoes cell division during which the basal process is split (white arrows) and equally inherited by the daughter cells.
Movie 1. <a href="https://www.jove.com/files/ftp_upload/3920/3920movie1.mov" target="_blank">Click here to view movie</a>.
Movie 2. <a href="https://www.jove.com/files/ftp_upload/3920/3920movie1.mov" target="_blank">Click here to view movie</a>.

<ol><li>Ulloa, F., Briscoe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphogens+and+the+Control+of+Cell+Proliferation+and+Patterning+in+the+Spinal+Cord%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">Morphogens and the Control of Cell Proliferation and Patterning in the Spinal Cord.</a> Cell Cycle. 6, 2640-2649 (2007).</li>
<li>Briscoe, J., Novitch, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulatory+pathways+linking+progenitor+patterning%2C+cell+fates+and+neurogenesis+in+the+ventral+neural+tube%5BTitle%5D)+AND+%22Philosophical+Transactions+of+the+Royal+Society+B%3A+Biological+Sciences%22%5BJournal%5D)">Regulatory pathways linking progenitor patterning, cell fates and neurogenesis in the ventral neural tube.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 363, 57-70 (2008).</li>
<li>Shiau, C., Das, R., Storey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+effective+assay+for+high+cellular+resolution+time-lapse+imaging+of+sensory+placode+formation+and+morphogenesis%5BTitle%5D)+AND+%22BMC+Neuroscience%22%5BJournal%5D)">An effective assay for high cellular resolution time-lapse imaging of sensory placode formation and morphogenesis.</a> BMC Neuroscience. 12, 37(2011).</li>
<li>Wilcock, A. C., Swedlow, J. R., Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitotic+spindle+orientation+distinguishes+stem+cell+and+terminal+modes+of+neuron+production+in+the+early+spinal+cord%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Mitotic spindle orientation distinguishes stem cell and terminal modes of neuron production in the early spinal cord.</a> Development. 134, 1943-1954 (2007).</li>
<li>Vilas-Boas, F., Fior, R., Swedlow, J. R., Storey, K. G., Henrique, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+reporter+of+notch+signalling+indicates+regulated+and+random+notch+activation+during+Vertebrate+neurogenesis%5BTitle%5D)+AND+%22BMC+Biology%22%5BJournal%5D)">A novel reporter of notch signalling indicates regulated and random notch activation during Vertebrate neurogenesis.</a> BMC Biology. 9, 58(2011).</li>
<li>Herman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+microscopy%5BTitle%5D)+AND+%22Bios+Scientific+Publishers%22%5BJournal%5D)">Fluorescence microscopy.</a> Bios Scientific Publishers. , (1998).</li>
<li>Conchello, J. -A., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+sectioning+microscopy%5BTitle%5D)+AND+%22Nat.+Meth%22%5BJournal%5D)">Optical sectioning microscopy.</a> Nat. Meth. 2, 920-931 (2005).</li>
<li>Pawley, J. B. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPawley%2C+JB+%22Fundamental+Limits+in+Confocal+Microscopy+Handbook+Of+Biological+Confocal%22">Fundamental Limits in Confocal Microscopy Handbook Of Biological Confocal</a>. Pawley, J. B. , Springer US. (2006).</li>
<li>Swedlow, J. R., Hu, K., Andrews, P. D., Roos, D. S., Murray, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measuring+tubulin+content+in+Toxoplasma+gondii%3A+A+comparison+of+laser-scanning+confocal+and+wide-field+fluorescence+microscopy%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Measuring tubulin content in Toxoplasma gondii: A comparison of laser-scanning confocal and wide-field fluorescence microscopy.</a> Proceedings of the National Academy of Sciences. 99, 2014-2019 (2002).</li>
<li>Murray, J. M., Appleton, P. L., Swedlow, J. R., Waters, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluating+performance+in+three-dimensional+fluorescence+microscopy%5BTitle%5D)+AND+%22Journal+of+Microscopy%22%5BJournal%5D)">Evaluating performance in three-dimensional fluorescence microscopy.</a> Journal of Microscopy. 228, 390-405 (2007).</li>
<li>Biggs, D. S. C. 3D Deconvolution Microscopy. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBiggs%2C+DSC+%22Current+Protocols+in+Cytometry%22">Current Protocols in Cytometry</a>. , John Wiley & Sons, Inc. (2001).</li>
<li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W., Prasher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Green+fluorescent+protein+as+a+marker+for+gene+expression%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Green fluorescent protein as a marker for gene expression.</a> Science. 263, 802-805 (1994).</li>
<li>Wiedenmann, J., Oswald, F., Nienhaus, G. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+proteins+for+live+cell+imaging%3A+Opportunities%2C+limitations%2C+and+challenges%5BTitle%5D)+AND+%22IUBMB+Life%22%5BJournal%5D)">Fluorescent proteins for live cell imaging: Opportunities, limitations, and challenges.</a> IUBMB Life. 61, 1029-1042 (2009).</li>
</ol>

No conflicts of interest declared.

We present here a novel time-lapse imaging assay to monitor cell behavior in chicken embryonic slice culture. This assay enables high resolution imaging of living tissue for up to 70 hours, although time frames between 24-48 hours are easier to capture. The use of a high NA oil objective and relatively short intervals between time-points enables image acquisition at high resolution, as well as allowing us to monitor cell behavior that often occurs rapidly, and can be easily missed during conventional time-lapse imaging using confocal microscopy.
The major advantage of this assay is the ability to image cells over long periods of time. The use of wide-field6 instead of confocal laser scanning7 microscopy is critical for this. Confocal microscopy has traditionally offered several advantages over wide-field microscopy, including the ability to take optical sections and eliminate out of focus information directly7, but the use of a pinhole leads to a loss of light to the detector8, excluding a significant amount of information, and necessitating longer exposure times. Wide-field microscopy, on the other hand, uses full field illumination and all the light passing through the objective is sent to the detector. This coupled with a high quantum efficiency cooled coupled device (CCD) camera ensures imaging with a high signal to noise ratio compared to confocal microscopy9, with very fast exposure times. In our application, we must image for long periods, record 3D stacks and ultimately resolve small&#x2014;near-diffraction limited structures. Although confocal microscopy prevents out-of-focus light from ever reaching the detector and thus significantly reduces background in thick, densely-labeled samples7, 8, it only achieves a usable signal-to-noise ratio with much larger light input than wide-field microscopy10. For very light-sensitive sparsely-labeled samples like our electroporated spinal cord slices, therefore, wide-field microscopy performs better than confocal microscopy. When combined with image restoration by deconvolution, which removes out of focus information and improves contrast, wide-field is then particularly good for detection of small, dim objects11. While not appropriate for every tissue imaging experiment, this approach has been very effective for our live cell imaging application.
The choice of fluorescent protein is an important factor that can influence cell survival. We find that the best results are obtained from constructs that use green fluorescent protein (GFP)12 as a marker. We are currently assessing a range of red fluorescent proteins that can be effectively used in combination with GFP for dual channel time-lapses. There are a variety of such proteins available13. Although many proteins are stable as fusions with a fluorescent protein, some proteins may be rendered unstable by fusion, resulting in reduced cell viability. In such cases, the use of constructs containing an internal ribosome entry site (IRES) is useful to separate the protein of interest from the fluorescent protein. 
When imaging spinal cord slices, care must be taken to minimize exposure to fluorescent light. The microscope eyepieces must be used only with transmitted light (brightfield) to locate and position slices. Fluorescent images should always be acquired using the microscope software using minimal exposure times. These should be kept in the range of 5-50 ms and the use of neutral density filters should be experimented with. Although longer exposure times may initially produce clearer images, the phototoxic effects of fluorescent light exposure may lead to cell death. The first 5-10 &mu;m of tissue that is closest to the coverslip should not be imaged as this region contains tissue that may have been damaged during slicing. 
Although the examples presented here are of embryos electroporated at HH stage 10 and imaged 6-7 hours later, this method can be used for embryos up to HH stage 18. At later stages, the spinal cord tissue is much larger and so is difficult to slice by hand. In these cases, embedding the embryos in low melting point agarose and sectioning with a vibratome may yield better results. Even though we present this assay as a method for imaging cells in the developing spinal cord, this approach has also been modified to image other embryonic tissues, including the sensory placodes3.

high-resolution live imaging of cell behavior in the developing neuroepithelium

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

The overall goal of this procedure is to image cell behavior within the embryonic neural tube at high resolution for long periods of time. This is accomplished by first transecting the early neural tube with a construct driving expression of a fluorescent protein of choice to mark single cells. The next step is to make slices of the early embryo and mount them onto glass bottom dishes.After recovery in an incubator, the embryo slices are imaged on a wide field microscope at regular intervals. Ultimately, this technique can be used to study cell behavior within the developing neuro epithelium over long periods of time with high spatial and temporal resolution. The main advantage of this technique over existing methods for imaging living tissue is that it allows us to observe individual cell behavior over long periods of time at high spatial and temporal resolution.This method allows you to address key questions in the field of, of developmental neurobiology, such as the role of mitotic spindle orientation in sulfate choice, or how signaling dynamics might be regulating cell behavior Prior to electroporation of plasmids into the neural tube. Glass needles are prepared using a micro capillary puller under a dissecting microscope. Use fine forceps to break off the tip of the needle.The end of the needle should be sharp enough to pierce the neural tube while not being so narrow that it impedes injection of the DNA solution. The X for this experiment have been incubated at 37 degrees Celsius for about 36 hours to hamburger Hamilton. Stage 10.To begin this procedure, window and egg and place electrodes five millimeters apart on either side of the embryo, inject DNA into the neural tube. The DNA is colored with a small amount of fast green for visualization. Apply a current of 12 to 17 volts and 50 millisecond pulse length three times with 950 milliseconds between pulses.Low concentrations of DNA and low electroporation voltages are used to achieve mosaic expression so that individual cells can be followed. Finally, cover the window in the eggshell with cellar tape, making sure it is sealed. Allow the embryos to recover for three to four hours or overnight at 37 degrees Celsius.The collagen mixture and slice culture medium should be prepared about an hour before slicing the embryos. To prepare the collagen mixture at 100 microliters of 0.1%acetic acid solution to 300 microliters of type one collagen and vortex thoroughly, add 100 microliters of five times L 15, medium and vortex thoroughly. Again, the solution should turn yellow.Next, add 15 to 20 microliters of 7.5%sodium bicarbonate, and vortex thoroughly. The solution should become slightly pink. Keep on ice.This collagen mix should be prepared fresh each time. To prepare the slice culture medium at the following to 10 milliliters of neuro basal medium, B 27 supplement glut max and Gentamycin solution, place the medium in a 37 degree Celsius incubator, buffered with 5%carbon dioxide. Leave the top of the container loose to allow it to equilibrate with the carbon dioxide for at least an hour.To begin this procedure, use a pair of small scissors to cut out the embryo. Remove the embryo from the egg with tweezers washing L 15 medium. Place the embryo in a tissue culture dish with a layer of syl guard at the bottom.Pin the embryo out through the surrounding extra embryonic membranes so that the membranes are stretched taut. Using a micro knife, slice the embryo as straight as possible through the region of interest. For spinal cord slices should be between one to two somites thick leaf slices attached to the lateral tissue of the embryo so they are not lost while other embryos are being sliced.A customized micro perpet tip is needed for transferring the spinal cord slices to the glass bottom dish. To prepare this, attach a 200 microliter tip to a P two or P 10 perman and cut off approximately one millimeter from the end of the tip. Code the inside of the tip with collagen by preparing one microliter of the collagen mix that was prepared earlier.Leave for one to two minutes and then rinse with L 15 medium. This will prevent the tissue from sticking to the inside of the tip. Now detach a slice from the embryo using a micro knife and remove the slice from the dish using the P two or P 10 fitted With the 200 microliter tip.Try to take up as little medium as possible. Vortex the collagen mix and drop five to eight microliters of it onto a polyol lysine coated glass bottom dish with a cover slip as the base, immediately put the embryo slice into the collagen and position it with a pair of fine forceps. LICs should be positioned so that the side to be imaged is flush with the cover slip.The tissue should adhere to the polyol lysine coating on the cover slip. Repeat this until you have several slices on the cover slip. Usually six to nine slices are placed on one dish.When all the slices are in place, add two to three microliters of L 15, medium to the earliest place collagen and cover the dish. Allow the collagen to set for 20 minutes. Once the collagen is set carefully add two milliliters of slice culture medium that has been equilibrated for at least an hour in 5%carbon dioxide and 37 degrees Celsius.Care must be taken not to dislodge the collagen from the cover slip place in a 37 degree Celsius 5%carbon dioxide incubator and allow slices to recover for at least three hours before imaging. Imaging cell behavior within tissue for long periods of time at high temporal and spatial resolution is challenging. A combination of optimal culture conditions and the use of white field microscopy is essential for obtaining good results.A delta vision core wide field microscope fitted with a weather station environmental chamber is used to image the slices. The chamber is constantly maintained at 37 degrees Celsius with a carbon dioxide perfusion apparatus to maintain the microscope stage at 5%carbon dioxide and 95%Air imaging is normally carried out using a 40 times 1.30 NA oil immersion lens and images are captured with a core snap HQ two called CCD camera Z Sections are captured every 1.5 microns through 45 microns of tissue exposure. Time should be kept as low as possible, for example, five to 50 milliseconds.For each said section images are captured every seven minutes. Up to nine slices can be visited using the Delta vision systems. Precise point visiting function shown here is an example time-lapse sequence of a spinal cord progenitor cell transfected with a construct expressing GFP alpha Tubulin imaging was started on a spinal cord slice from a two day old HH stage 12 embryo.During this early stage, neural progenitor cells undergo predominantly progenitor progenitor mode divisions during which the cells divide to generate two further cycling progenitor cells. This figure shows selected frames from the time-lapse sequence just shown. At two hours and 20 minutes, the cell divides with a cleavage plane that is perpendicular to the apical surface generating two daughter cells.These two cells divide once again at 24 hours and 23 minutes and 25 hours and 54 minutes. This next time-lapse sequence is of a cell transfected with G-F-P-G-P-I marking the cell membrane. This cell undergoes a division during which the basal process splits in two indicated by the white arrows and is equally inherited by the daughter cells.Selected frames from this time-lapse sequence show that the cell division occurs at zero hours and 49 minutes. After watching this video, you should have a good idea of how to prepare and image spinal cord slices. Remember, if you are new to this technique, you may struggle initially as there are a number of technically challenging steps that all need to come together for this to work.

high-resolution-live-imaging-cell-behavior-developing

Research

JoVE Journal

Neuroscience

This protocol describes imaging of individual  neurons or neural crest cells in living zebrafish embryos.  This method is used to examine cellular behaviors and actin localization using fluorescence  confocal time-lapse microscopy.

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Live Imaging of Drosophila Larval Neuroblasts

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

학습하세요 발달 중인 신경상피의 세포 행동을 고해상도 라이브 영상으로 관찰하는 방법을. 형광 단백질을 이용한 병아리 배아 신경관 절편 준비와 장시간 현미경 촬영 기술을 익혀보세요.

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

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