visualization of vesicle motilities in neurons using fluorescence imaging

Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging

In this procedure, mix 4 micrograms of plasmid with 200 microliters of serum-free medium. In a separate tube, dilute eight microliters of the transfection reagent in 200 microliters of serum-free medium. Then, incubate the solution for 5 minutes at room temperature. After five minutes, mix the plasmid solution and the transfection reagent solution, and incubate the mixture for 20 minutes at room temperature. Following that, add the mixture to the coated glass bottom dishes.
Incubate the neurons at 37 degrees Celsius for 30 minutes. Afterward, replace the medium with 2 milliliters of cerebral cortical culture medium, and incubate the neurons at 37 degrees Celsius for one to two days.
For imaging, use a fluorescence microscope equipped with a CCD camera with the 40x objective lenses. Keep the temperature at 37 degrees Celsius using an incubation system. Acquire neuron images at 1 frame every 5 seconds over a 100-second period controlled by the image acquisition software.
To analyze the images, open all the sequential images in ImageJ software with manual tracking. To combine the sequential images, choose Image, then Stacks followed by Images to Stack, and then click OK. After that, choose Plugins, then Manual Tracking. A tracking window will pop up. Click on the checkbox of Show Parameters, and define the tracking parameters in the parameters section.
Then, click Add Track to start a new track. After determining the vesicles of interest, click on the center of the signals in the sequential images to record the XY coordinates. Repeat this procedure to collect the XY coordinates of the vesicles from all sequential images. Each image automatically advances to the successive image after the reference point is selected.

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1. Imaging Vesicle Motility
<ol>
	<li>Prepare the plasmids that will label each type of vesicle. For instance, use EGFP-Rab5 for early endosomes, EGFP-Rab7 for late endosomes, LAMP-EGFP for lysosomes, and EGFP-LC3 for autophagosomes. 
	NOTE: These plasmids are available on request from tsuruta.fuminori.fn@u.tsukuba.ac.jp. Typically, 3 -5 days in vitro (Division or DIV) neurons can be transfected with these plasmids using transfection reagents.</li>
	<li>Mix 4.0 &#181;g of plasmids with 200 &#181;L of serum-free medium by pipetting. In a separate tube, dilute 8 &#181;L of transfection reagent (see Table of Materials) in 200 &#181;L of serum-free medium. Incubate for 5 minutes at room temperature (RT).</li>
	<li>Mix the plasmid solution and transfection regent solution in step 1.2 by pipetting. Incubate for 20 min at RT.</li>
	<li>Add serum-free medium and 400 &#181;L of the plasmid solution from step 1.3 to each coated glass-bottom dish containing cortical neurons cerebral cortical culture medium. Incubate the neurons at 37 &#176;C in a 5% CO2 incubator for 30 min. Replace the medium with 2 mL of cerebral cortical culture medium. Incubate the neurons at 37 &#176;C in a 5% CO2 incubator for 1-2 days. 
	NOTE: Prepare a cerebral cortical culture medium consisting of neuron basal medium supplemented with 1x B-27 supplements, 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin.</li>
	<li>After 1 - 2 days of transfection, select the transfected cells for image acquisition. 
	NOTE: Premature neurons less than 7 DIV have a tendency to exhibit dynamic vesicle motility. Select neurons that moderately express the fluorescence probe because clear images cannot be obtained when a highly expressing neuron is selected. Also, select a typical neuronal shape, such as pyramidal neurons, which have long axons and intricate dendrites, for ease of identification of the axon and dendrites (see Figure 1A).</li>
	<li>Image acquisition using a time-lapse imaging system: For imaging, use a fluorescence microscope equipped with a Charge-Coupled Device (CCD) camera with 40X Plan Apo 0.95NA or 100X Plan Apo VC NA1.4 objective lenses. Control the temperature at 37 &#176;C using an incubation system. Acquire neuron images at one frame/5 s over a 100 s period controlled by the image acquisition software. Alternatively, acquire images at shorter intervals and for longer time periods, such as at one frame per 2 s over a 300-s period. 
	NOTE: A variety of applications are available for taking sequential images, many of which would be suitable for this method. Therefore, no particular application is recommended; instead, each researcher should use whatever application is available.</li>
</ol>
2. Image Analysis
<ol>
	<li>Open all sequential images in ImageJ software with Manual Tracking. 
	NOTE: To date, Manual Tracking has been widely used in tracking experiments. Manual Tracking was developed by Dr. Fabrice Cordeli&#232;res. Detailed written instructions are available online. For the analysis, the quality of the imaging data is improved with a sufficient number of images. It is recommended to use more than 20 images.</li>
	<li>To combine the sequential images, choose&#8594; &#8594; . Click .</li>
	<li>Choose &#8594; ; a tracking window will pop up.</li>
	<li>Click on the checkbox of and define the tracking parameters in the parameters section. 
	NOTE: Several parameters can be set, including time interval, x/y calibration, z calibration, search square size for centering, dot size, line width, and font size. In this simplified assay, both time interval and x/y calibration were defined. Under other experimental circumstances, it may be necessary to define other parameters as well. The parameters used here are as follows: the is 5 s, and the is either 0.26642 &#181;m for a 40X Plan Apo 0.95NA objective lens or 0.10657 &#181;m for a 100X Plan Apo VC NA1.4 objective lens.</li>
	<li>After the parameters are defined, click on to start a new track.</li>
	<li>After determining the vesicles of interest (e.g., the vesicles indicated by blue and red arrowheads in Figure 1A (right panel)), click on the center of the signals in the sequential images to record the xy coordinates. The results of the recorded xy coordinates, distance, and velocity show up in a new window. 
	NOTE: In the case of lysosomes, large bright fluorescent signals (e.g., Figure 1A (right panel), orange arrowhead) are often observed in neurons. These signals occasionally exhibit either accumulated vesicles or pathological varicosities, so these signals should be avoided for quantifying motility.</li>
	<li>Repeat step 2.6 to collect the xy coordinates of vesicles from all sequential images; after the reference point is selected, each image automatically advances to the successive image.</li>
	<li>Export these data to a new table (e.g., a spreadsheet). Use appropriate software to create a suitable graph (see Table of Materials). 
	NOTE: To analyze the data, select the column, sum up all values of this column, and show a motility distance as a bar graph, such as in Figure 1B. Additionally, repeat this step, calculate the average of the total motility distance, and show as a bar graph, as in Figure 1C. The software to create a bar graph and waveform data is shown in the Table of Materials.</li>
</ol>

Source: Tsuruta, F., et al. <a href="https://app.jove.com/v/55488">Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes.</a> J. Vis. Exp. (2017).
This video demonstrates how neuronal vesicles are labeled with specific fluorescent proteins to visualize their movement. First, a plasmid coding for a fluorescent protein is incubated with a transfection agent and then introduced into a culture of neurons. Subsequently, images of the neurons are acquired and analyzed to track the vesicles&#39; movement.

<img alt="Figure 1" src="/files/ftp_upload/22989/22989_Figure1.jpg" /> 
Figure 1: Lysosome Motility in Neuronal Dendrites. (A) Mouse cortical neurons (4 DIV) were transfected with LAMP-EGFP plasmid for 2 days. This image shows the motility of LAMP-EGFP-containing vesicles in the dendrites. The inset shows the time-lapse image of the LAMP-EGFP-positive vesicles. The red arrowhead indicates a moving vesicle, and the blue arrowhead indicates a resting vesicle. The orang...

1. Imaging Vesicle Motility

	Prepare the plasmids that will label each type of vesicle. For instance, use EGFP-Rab5 for early endosomes, EGFP-Rab7 for ...

Take the desired plasmid in serum-free media. This plasmid encodes vesicle-specific proteins with fluorescent tags.
&#160;In another tube, take the transfection reagent diluted in serum-free media.
Mix the two solutions and incubate.
The transfection reagent combines with the plasmid, forming a complex.
Take a well containing adherent neurons in the media with serum. Add the complexes and incubate. The complex facilitates the plasmid&#39;s entry into the cell.
Discard the media to remove non-internalized complexes.
Add fresh media and then incubate.
The plasmid enters the nucleus to be transcribed and is then translated into vesicle-specific fluorescent proteins that label the vesicles in the neurons.
Using a fluorescence microscope under controlled temperature, acquire images at specified time intervals.
To analyze the images, define tracking parameters.
Identify the vesicle of interest and record its coordinates in each of the images.
Compile the data to visualize the vesicle&#39;s movement.

9 Views. Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging

Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging

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