The authors would like to acknowledge the Universit&#224; degli Studi di Milano for support to Eliana Di Cairano (Post-doctoral fellowship) and Stefania Moretti (Ph.D. fellowship). This work was supported by the University Research Program PUR to C.P.
We would like to thank Prof. Jeremy M. Henley, School of Biochemistry, University of Bristol, United Kingdom, for the pHluorin construct and Dr. Dotti Francesco for assistance in data analysis, and Silvia Marsicano for technical assistance.

1. Cell Culture and Transfection
<ol>
	<li>SH-SY5Y cell culture 
	NOTE: The experiments have been performed using the human neuroblastoma SH- SY5Y (ATCC# CRL-2266)15. SH-SY5Y cells grow as a mixture of floating clusters and adherent cells. Follow the instructions reported in the protocol (cell density, splitting ratio, etc.) to have cells that grow firmly attached to glass cover, which is crucial for TIRFM.
	<ol>
		<li>Before starting, under the laminar flow biosafety cabinet, make the opportune volume of sterile phosphate buffer saline solution (PBS) and culture medium.
		<ol>
			<li>Make 50 ml of PBS with concentrations of 150 mM NaCl, 24 mM phosphate buffer, pH 7.4. Filter the solution.</li>
			<li>Make 50 ml of cell medium from Dulbecco&#8217;s modified Eagle medium (DMEM) with high glucose, 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 &#181;g/ml), L-glutamine (2 mM), and sodium pyruvate (1 mM). Filter the solution.</li>
		</ol>
		</li>
		<li>Remove complete growth medium and wash the cells with 3 ml of PBS.</li>
		<li>Incubate cells with 2 ml of 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) (for 6 cm Petri dish) for 5 min at 37 &#176;C, 5% CO2 and detach cells using pipette.</li>
		<li>Inactivate trypsin by adding 2 ml of DMEM, and collect cells by centrifugation at 300 x g for 5 min.</li>
		<li>Remove the supernatant, add 1 ml of DMEM to the pellet and pipette the solution up and down sufficiently to disperse cells into a single cell suspension.</li>
		<li>Split them 1:4 in a new 6 cm diameter Petri dish containing 3 ml of complete medium. Maintain cells in culture in 6 cm diameter Petri dishes, at 37 &#176;C in a 5% CO2 incubator. Sub-culture once a week or when they have covered 80 - 90% of the surface area.</li>
	</ol>
	</li>
	<li>SH-SY5Y cell plating for imaging
	<ol>
		<li>For TIRFM experiments, plate cells onto glass covers. Employ glass covers with 0.17 &#177; 0.005 mm thickness and a 1.5255 &#177; 0.00015 refractive index. Before starting, prepare the glass coverslips as follow:
		<ol>
			<li>Clean glass covers with 90% ethanol, O/N.</li>
			<li>Rinse them thoroughly in distilled water (three changes of distilled water). Dry glass covers in a drying oven.</li>
			<li>Place covers in glass Petri dishes and sterilize in a preheated oven at 200 &#176;C for 3 hr.</li>
		</ol>
		</li>
		<li>The day before transfection, place each coverslip in a 3.5 cm Petri dish, add 1 ml of culture medium and incubate at 37 &#176;C in a 5% CO2 incubator.</li>
		<li>Trypsinize cells as described in steps 1.1.3 - 1.1.5, suspend the cell pellet in 1 ml of complete medium and count. Calculate the correct volume of cell suspension to add to each Petri dish to yield 3 x 105 cells/well. This density is required for optimal cell growth and efficient transfection. Incubate at 37 &#176;C in a 5% CO2 incubator O/N.</li>
	</ol>
	</li>
	<li>SH-SY5Ytransfection by polyethylenimine (PEI) 
	NOTE: To visualize synaptic vesicles dynamics, pCB6 vector containing synapto-pHluorin has been used. The synapto-pHluorin has been generated by in frame fusion of a pH-sensitive variant of the green fluorescent protein (GFP)16 and the vesicular membrane protein synaptobrevin 2. The construct has been extensively employed to investigate synaptic vesicle properties within neurons9.
	<ol>
		<li>Before starting transfection, make 10 ml of the following solutions. Keep the solutions as maximal as 1 month.
		<ol>
			<li>Make a 150 mM NaCl solution. Adjust to pH 5.5 with 0.01 N HCl.</li>
			<li>Make a PEI solution at 10% polyethylenimine (PEI; 25 kDa linear) in 150 mM NaCl solution. The pH of solution rises to 8.8. Adjust pH to 7.8 with 0.01 N HCl.</li>
		</ol>
		</li>
		<li>24 hr after plating, remove the medium and refresh with 1.5 ml of complete medium. Keep the cells at 37 &#176;C, in a 5% CO2 incubator.</li>
		<li>Under the laminar flow biosafety cabinet, in a 1.5 ml microfuge tube, add 3 &#181;g of plasmid DNA to 25 &#956;l of 150 mM NaCl solution and 100 &#956;l of PEI solution per 3.5 cm Petri dish.</li>
		<li>Vortex for 10 sec, then incubate the DNA/PEI mixture for 30 min at RT.</li>
		<li>Carefully add the DNA/PEI mixture to the Petri dish containing coverslips with cells and gently shake to equally distribute the reagent in the Petri dish.</li>
		<li>After 4 hr change the medium and incubate the cells O/N at 37 &#176;C in a 5% CO2 incubator. Perform imaging experiments 24 - 48 hr after transfection.</li>
	</ol>
	</li>
</ol>
2. Cell Imaging by Total Internal Reflection Fluorescence Microscopy (TIRFM)
<ol>
	<li>Imaging set-up
	<ol>
		<li>Perform TIRF imaging with the set-up described in Figure 1. It comprises a motorized inverted microscope (Figure 1, inset A), the laser source (Figure 1, inset B) and the TIRF-slider (Figure 1, inset C). Reach TIRFM illumination through a high numerical aperture (NA 1.45 Alpha Plan-Fluar) 100X oil, immersion objective.</li>
		<li>For TIRFM illumination, employ a multi-line (458/488/514 nm) 100 mW argon-ion laser. Using a mono mode fiber, introduce the linearly polarized laser light into the beam path, via the TIRF slider. Insert the TIRF slider into the luminous field diaphragm plane of the reflected-light beam path.
		<ol>
			<li>For wide field illumination, connect the microscope to a conventional mercury short-arc lamp HBO white light. A polarization-maintaining double prism in the slider ensures the simultaneous combination of TIRF illumination and white light.</li>
		</ol>
		</li>
		<li>Filter the laser light with an excitation filter (band width 488/10 nm) mounted on a filter wheel, introduced into the laser path. Employ a high speed, software-controlled, shutter to allow fast control of laser illumination. For pHluorin analysis, mount a band pass 525/50 nm emission filter. Capture digital images (512 x 512 pixels) on a cooled Fast CCD camera with the Image ProPlus software.</li>
	</ol>
	</li>
	<li>Achieving TIRF illumination (Figure 2)
	<ol>
		<li>Turn on the lasers, the computer, the camera, the filter wheel, and the shutter controllers; then, wait 20 min before starting the experiment as the lasers need to warm up and stabilize.</li>
		<li>Before imaging, make the opportune volume of the following solutions.
		<ol>
			<li>Make 50 ml of Krebs (KRH) solution at 125 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) (buffered to pH 7.4), 2 mM CaCl2, and 6 mM glucose.</li>
			<li>Make 10 ml of KCl-KRH solution (pH 7.4) at 80 mM NaCl, 50 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM HEPES (buffered to pH 7.4), 2 mM CaCl2, and 6 mM glucose.</li>
		</ol>
		</li>
		<li>Remove the glass cover with transfected cells and insert it in the appropriate imaging chamber. Assemble the chamber and add 500 &#181;l of KRH solution in the center of the glass.</li>
		<li>Add oil over the objective. Place the imaging chamber on the stage of the microscope and position the objective under the glass coverslip. Position the safe cover over the sample.</li>
		<li>In epifluorescence mode, focus on the coverslip (upper surface) and choose transfected cells placed in the chamber center. Select cells whose fluorescent signal can be clearly recorded using an exposure time below 80 msec.</li>
		<li>Under software control, switch to TIRF illumination in live mode.</li>
		<li>To set the TIRF configuration, check the position of the beam that emerges out of the objective, on the sample cover (Figure 2B). When the beam is positioned in the center of the objective lens (Figure 2A, left), a spot is visible in the center of the TIRF sample cover (Figure 2B, left) and the cell is imaged in epifluorescence mode (several focus planes, high background fluorescence; Figure 2C, left).</li>
		<li>To reach the critical angle, move the focused spot in the Y direction (forward or backward;&#160;Figure 2B, center) using the angle adjustment screw on the TIRF slider (Figure 1C). When the beam converges on the sample plane at an angle larger than the critical angle (Figure 2A, right), the spot disappears and a straight, thin, focused line is evident in the middle of the sample cover (Figure 2B, right).</li>
		<li>To fine-tune the TIRF angle use the cell sample (Figure 2C). Watch the fluorescence image on the video, at this stage, an epifluorescence-like image is still visible. Gently, move the screw until TIRF condition is achieved: only one optical plane of the cell is in focus (i.e., the plasma membrane in contact with the cover-slip), this results in a flat image with high contrast (Figure 2C, right).</li>
	</ol>
	</li>
	<li>Sample imaging
	<ol>
		<li>Set the single-channel time-lapse experiment. To minimize photobleaching, capture the image using low exposure time and high gain. Appropriate exposure times are between 40 - 80 msec. Acquire images at 1 - 2 Hz sampling frequency. Vesicle kinetics may be better appreciated sampling at higher frequency (10 Hz). The regular time of observation is usually 2 min.</li>
		<li>Add 500 &#181;l of KRH solution and record cells in TIRFM mode. This is the resting condition. Save the time sequential images.</li>
		<li>Focus on the same cell and record under the same conditions of resting (laser power, time exposure, frame number). After five frames, add 500 &#181;l of KCl-KRH solution and keep KCl in the chamber. This is the stimulated condition; save the time sequential images.</li>
	</ol>
	</li>
</ol>
3. Image Analysis and Data Processing
NOTE: To analyze images, macros have been developed in the lab, based on existing functions of the image analysis software; similar macros are available online (URL provided in Table of Materials and Equipment).
<ol>
	<li>Fluorescence intensity quantification
	<ol>
		<li>Use a &#8220;Sequence fluorescence intensity&#8221; macro for fluorescence intensity quantification in a region of interest (ROI) of the image, over the course of the movie.</li>
		<li>Open the time-sequential images. Go to the macro menu and select &#39;Sequence fluorescence intensity&#39;. In the &#39;Analysis&#39; window appears &#8220;select the ROI&#8221;.</li>
		<li>Choose one of the selection tools in the menu to create the ROI. Place 3 ROIs in regions of the cell membrane without spots (background ROI). Employ this &#8220;background ROI&#8221; to evaluate the photobleaching and to set the threshold for fusion event analysis (Figure 3A).</li>
	</ol>
	</li>
	<li>Photobleaching correction and threshold determination (Figure 3B)
	<ol>
		<li>To evaluate the photobleaching, open the fluorescence intensity rows &#8220;background ROIs&#8221;, (Figure 3Ba). Normalize the fluorescence intensity values in each frame to the initial intensity value (F0) (F/F0) (Figure 3Bb). Average the values.</li>
		<li>Highlight the average data and create a line plot using the chart menu options.</li>
		<li>From the data analysis menu, select &#8220;trendline&#8221; to open the plot analysis dialog. Select the type of regression. Set &#8220;exponential&#8221; regression. Then select &#8220;display equation on chart&#8221;. In the graph window, the exponential equation appears and the parameter values are automatically assigned, (Figure 3Bc).</li>
		<li>Apply the exponential correction to the intensity values in each frame as follow: 
		Fn (corrected) = Fn / exp(-n * a) 
		Fn = experimental fluorescence intensity measured at frame n; n = number of frames; a = bleaching factor (constant expressing&#160;the rate of intensity loss due to photobleaching;&#160;Figure 3Bd).</li>
		<li>To set the threshold, open a normalized and corrected &#8220;background ROI&#8221;, calculate the average fluorescence signal and its standard deviation (SD). The average value plus 3 SD represents the threshold (Figure 3Be). Use this threshold for data analysis.</li>
	</ol>
	</li>
	<li>Selection of fusion events using a semiautomatic procedure
	<ol>
		<li>Open the time-sequential images with image analysis software. Apply a Gaussian filter to the active image sequence.</li>
		<li>Analyze images using the tool &#8220;count objects&#8221; or a macro which allows the selection of an object whose pixels have average fluorescence intensity within a defined range. Set the intensity range manually, using the threshold function (go to the bar menu, set measure &#8594; threshold to highlight the area of interest). An adequate threshold is 30% over the local fluorescent background signal.</li>
		<li>Apply a macro &#8220;Filters objects&#8221; to select only objects meeting the following criteria:
		<ol>
			<li>Apply ranges option (min and max inclusive) for aspect. Aspect reports the ratio between the major axis and the minor axis of the ellipse equivalent to the object. Aspect is always &#8805;1. Adequate values are min = 1, max = 3.</li>
			<li>Apply ranges for diameter. Diameter reports the average length of the diameters measured at two degree intervals joining two outline points and passing through the centroid of the object. Set the range in pixels (or in &#181;m, if using a calibrated system).</li>
			<li>Define the optimal range in preliminary experiments: select manually the spots of interest and then measure their diameter using the plot profile function.</li>
		</ol>
		</li>
		<li>Select &#8220;display objects&#8221;: selected objects will appear superimposed to the TIRFM image (Figure 4B).</li>
		<li>Include in the analysis only those spots that show a short (one to three frames) transient increase in fluorescence intensity, immediately followed by a marked loss of signal (transient spots). Employ the circular selection to create a ROI approximately one-spot diameter radially around the selected vesicle/spots (experimental ROIs). Perform this step manually.</li>
		<li>With the ROIs selected, calculate the average fluorescence intensity of each ROI over the course of the movie.</li>
	</ol>
	</li>
	<li>Data analysis (Figure 3C-D)
	<ol>
		<li>Export the time-course of the fluorescence changes measured in each &#8220;experimental ROI&#8221; to a spreadsheet; (Figure 3Da). Normalize the intensity value in each frame to the initial fluorescence intensity (F/F0), (Figure 3Db).</li>
		<li>Apply the exponential correction to the intensity values in each frame as reported in step 3.2.4 (Figure 3Dc).</li>
		<li>To calculate the total number of fusion events (peak number), the time each fusion occurs (peak width) and the amplitude of fluorescent peak (peak height and AUC) apply logical functions using spreadsheet or math packages. An example of fusion event analysis using logical formulas is reported in Figure 3Dd and 3De.</li>
		<li>Assume the increase of fluorescence intensity exceeding the threshold (average background fluorescence intensity &#177; 3SD) as vesicle fusion to the plasma membrane and the resulting peak as a fusion event.</li>
		<li>Calculate the peak width as difference between the last and the first x value of each peak. Multiply this value for 1 / (sampling frequency). Consider this value as the time of vesicle fusion and adhesion at the plasma membrane before vesicle re-acidification and recycling (Figure 3Dd).</li>
		<li>Calculate the whole-cell AUC as a sum of values over threshold. Consider this value as net fluorescent change during the recording time due to the spontaneous (resting) or evoked (stimulated) synaptic activity.</li>
		<li>Calculate the peak height as the difference between the maximal y value of each peak and the threshold. Consider this value as indicative of the fusion type (single vs. simultaneous/sequential fusion or transient vs. full fusion).</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

This paper presents a protocol to image and analyze vesicles dynamics in secreting cells, using fluorescent cDNA-encoded vectors and TIRFM. Key elements of successful imaging by TIRFM are the selection of the cellular model and cell transfection with genetically-encoded optical indicators of vesicle release and recycling.
TIRFM is ideally suited for cells growing adherent to a glass cover and sufficiently flat to allow stable visualization of membranes and fusion events. Vesicles should ideall...

Chemical synaptic transmission between neurons is a major mechanism of communication in the nervous system. It relies on the release of neurotransmitters through a dynamic cycle of vesicle fusion and retrieval at the presynaptic site. Many of the proteins involved in vesicle dynamics have been identified; however, their specific contribution to the phenomenon remains to be clarified1.
Our understanding is partly limited by the fact that the most widely used assays for exo/endocytosis are not always the most appropriate. Several studies related to vesicle fusion and dynamics rely on electrophysiological techniques. This technique provides an optimal temporal resolution and is excellent for investigating the initial fusion of vesicles to the plasma membrane but is unable to detect many of the underlying molecular events that support presynaptic function. Electron microscopy, on the other side, provides the finest morphological description of each singular step, but the dynamic aspect of the event cannot be captured, because samples must be fixed in order to be analyzed.
The advent of new optical recording techniques2,3, in combination with advances in fluorescent molecular probes development4-6, enables the visualization of exocytic processes in live cells, thus providing new levels of information about the synaptic structure and function.
Initial studies exploited activity-dependent styryl dyes (FM1&#8211;43 and related organic dyes)7,8. State-of-the-art imaging techniques employ pH-sensitive variants of the Green Fluorescent Protein (GFP) (pHluorin) tethered to luminal vesicles proteins9. These probes are normally switched off when present in the vesicles because of the low luminal pH. After fusion with the plasma membrane, the vesicle interior is exposed to the neutral extracellular space, the pH abruptly increases, relieves the proton-dependent quenching of pHluorin and the fluorescent signal rapidly appears. As the change in pHluorin is faster than the fusion event, by monitoring fluorescence increases, vesicle fusion with the membrane can be measured and analyzed. Because surface pHluorin-tagged molecules are endocytosed, the fluorescence signal subsequently returns to basal level, therefore the same construct may be used also to monitor vesicle recycling9.
While the vesicle-tagged pH-sensor ensures the visualization only of those vesicles that really fuse with the plasma membrane, imaging at high spatial and temporal resolution is required to describe in details the steps involved in the exo/endocytic&#160;processes. The optical technique that provides the necessary spatio-temporal resolution is total internal reflection fluorescence microscopy (TIRFM), an application of fluorescence microscopy10.
Total internal reflection occurs at the interface between the glass cover-slip and the sample. When the light path reaches the glass cover-slip with an incident angle larger than the critical angle, the excitation light is not transmitted into the sample but is completely reflected back. Under these conditions, an evanescent light wave forms at the interface and propagates in the medium with less optical density (the sample). As the intensity of the evanescent field decays exponentially with distance from the interface (with a penetration depth of about 100 nm) only the fluorophores in closest proximity to the cover-slip can be excited while those further away from the boundary are not. In cells transfected with GFP-constructs, this depth corresponds to proteins expressed on the plasma membrane or in vesicular structures approaching it. As fluorophores in the cell interior cannot be excited, the background fluorescence is minimized, and an image with a very high signal/background ratio is formed11.
Several characteristics make TIRFM the technique of choice for monitoring vesicles dynamics. The perfect contrast and the high signal-to-noise-ratio allow the detection of very low signals deriving from single vesicles. Chip-based image acquisition in each frame provides the temporal resolution necessary to detect highly dynamic processes. Finally, the minimal exposure of cells to light at any other plane in the sample strongly reduces phototoxicity and enables long lasting time-lapse recording12.
Data analysis remains the most challenging and crucial aspect of this technique. The simplest way to monitor vesicle fusion is to measure the accumulation of reporter fluorescent proteins at the cell surface, over time13. As fusion increases, net fluorescence signal increases as well. However, this method may underestimate the process, particularly in large cells and in resting conditions, because endocytosis and photobleaching processes offset the increase in fluorescence intensity due to vesicle exocytosis. An alternative method is to follow each single fusion event14. This latter method is very sensitive and can reveal important details about the fusion mechanisms. However, it requires the manual selection of single events, because completely automated procedures to follow vesicles and to register the fluctuation of their fluorescent signals are not always available. Observation of vesicle dynamics requires sampling cells at high frequency. This generates a large amount of data that can hardly be analyzed manually.
The proposal of this paper is to optimize the TIRFM imaging technique for monitoring the basal and stimulated neurotransmitter release in the SH-5YSY neuroblastoma cell line, and to describe, step-by-step, a procedure developed in the laboratory to analyze data, both at whole-cell and single-vesicle levels.

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			<td height="21" style="height:21px;width:259px;">Equipment</td>
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			<td style="width:371px;"></td>
			<td style="width:468px;"></td>
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			<td height="21" style="height:21px;width:259px;">Axio Observer Z1</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:119px;">491912-9850-000</td>
			<td>inverted microscope 
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			<td height="21" style="height:21px;width:259px;">Multiline Argon Laser Lasos 77</td>
			<td style="width:199px;">Lasos</td>
			<td style="width:119px;">00000-1312-752</td>
			<td>multi-line (458/488/514 nm), 100mW argon-ion laser 
			<a href="http://www.lasos.com/products/argon-ion-laser">http://www.lasos.com/products/argon-ion-laser</a></td>
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			<td style="width:199px;">Zeiss</td>
			<td style="width:119px;">423681-9901-000</td>
			<td><a href="http://www.zeiss.com/microscopy/en_de/products/imaging-systems/single-molecular-imaging-laser-tirf-3.html">http://www.zeiss.com/microscopy/en_de/products/imaging-systems/single-molecular-imaging-laser-tirf-3.html</a></td>
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			<td height="21" style="height:21px;width:259px;">100x Objective</td>
			<td style="width:199px;">Zeiss</td>
			<td>421190-9900-000</td>
			<td>Oil, NA 1.45 Alpha-Plan 
			<a href="https://www.micro-shop.zeiss.com/?l=en&amp;p=us&amp;f=o&amp;a=v&amp;m=a&amp;id=421190-9900-000&amp;ss=1">https://www.micro-shop.zeiss.com/?l=en&#38;p=us&#38;f=o&#38;a=v&#38;m=a&#38;id=421 
			190-9900-000&#38;ss=1</a></td>
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			<td height="21" style="height:21px;width:259px;">CCD Camera RetigaSRV Fast 1394&#160;</td>
			<td style="width:199px;">QImaging</td>
			<td style="width:119px;"></td>
			<td><a href="http://www.qimaging.com/products/datasheets/Retiga-SRV.pdf">http://www.qimaging.com/products/datasheets/Retiga-SRV.pdf</a></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:259px;">LAMBDA 10-3 optical filter changer with SmartShutter</td>
			<td style="width:199px;">Sutter Instrument Company</td>
			<td style="width:119px;"></td>
			<td><a href="http://www.sutter.com/IMAGING/lambda103.html">http://www.sutter.com/IMAGING/lambda103.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;"></td>
			<td style="width:199px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Software</td>
			<td style="width:199px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image ProPlus 6.3 Software</td>
			<td style="width:199px;">Media Cybernetics</td>
			<td style="width:119px;"></td>
			<td>spot selection, ROI selection, fluorescence intensity determination 
			<a href="http://www.mediacy.com/index.aspx?page=IPP">http://www.mediacy.com/index.aspx?page=IPP</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Excel</td>
			<td style="width:199px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td>photobleaching correction, whole-cell and single-vesicle analyses 
			<a href="http://office.microsoft.com/it-it/excel/">http://office.microsoft.com/it-it/excel/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GraphPad Prism 4.00&#160;</td>
			<td>GraphPad Software, Inc.</td>
			<td style="width:119px;"></td>
			<td>statistical analysis 
			<a href="http://www.graphpad.com/scientific-software/prism/">http://www.graphpad.com/scientific-software/prism/</a></td>
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tirfm and ph-sensitive gfp-probes to evaluate neurotransmitter vesicle dynamics in sh-sy5y neuroblastoma cells: cell imaging and data analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

The TIRF imaging and data analysis procedures described are designed to study vesicles dynamics in cellular systems. This technique can be used to determine the effects of signaling molecules and drugs on fusion events and neurotransmitter vesicle dynamics17. Using GFP-tagged plasma membrane proteins, the TIRFM analysis has been employed to characterize the constitutive trafficking of GFP-tagged glutamate transporters in glial and epithelial cells18,19.
To validate the im...

Watch this Scientific Journal Video about TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis at JoVE.com

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

tirfm-ph-sensitive-gfp-probes-to-evaluate-neurotransmitter-vesicle

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Neuroscience

10.7K visualizações.  Università degli Studi di Milano. This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

Vídeo: TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized cells use this process to execute vital body functions such as insulin secretion from &#946; cells and neurotransmitter release from chemical synapses. Owing to its physiological significance, exo-/endocytosis has been one of the most studied topics in cell biology. Many tools have been developed to study this process at the gene and protein level, because of which much is known about the protein machinery participating in this process. However, very few methods have been developed to measure membrane lipid turnover, which is the physical basis of exo-/endocytosis.
This paper introduces a class of new fluorescent lipid analogs exhibiting pH-dependent fluorescence and demonstrates their use to trace lipid recycling between the plasma membrane and the secretory vesicles. Aided by simple pH manipulations, those analogs also allow the quantification of lipid distribution across the surface and the intracellular membrane compartments, as well as the measurement of lipid turnover rate during exo-/endocytosis. These novel lipid reporters will be of great interest to various biological research fields such as cell biology and neuroscience.

This protocol presents a fluorescence imaging method that uses a class of pH-sensitive lipid fluorophores to monitor lipid membrane trafficking during cell exocytosis and the endocytosis cycle.

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...