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, ...

<ol>
	<li>Sudhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synaptic+vesicle+cycle.">The synaptic vesicle cycle.</a> Annu. Rev. Neurosci. 27, 509-547 (1146).</li><li>Denk, W., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photon+upmanship:+why+multiphoton+imaging+is+more+than+a+gimmick.">Photon upmanship: why multiphoton imaging is more than a gimmick.</a> Neuron. 18 (3), 351-357 (1997).</li><li>Helmchen, F., Svoboda, K., Denk, W., Tank, D. 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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.

<table border="0" cellpadding="0" cellspacing="0" width="1414">
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			<td height="21" style="height:21px;width:259px;">Equipment</td>
			<td style="width:199px;"></td>
			<td style="width:119px;"></td>
			<td style="width:371px;"></td>
			<td style="width:468px;"></td>
		</tr>
		<tr height="21">
			<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 
			<a href="http://www.zeiss.com/microscopy/en_de/products/light-microscopes/axio-observer-for-biology.html#introduction">http://www.zeiss.com/microscopy/en_de/products/light-microscopes/axio-observer-for-biology.html#introduction</a></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:259px;">Laser TIRF slider</td>
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
	</tbody>
</table>

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 ...

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10.7K 조회수.  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.