This work was supported by the U.S. National Institutes of Health Grant NS-024572 (to R.S.W.).

1. Stain the Preparation with Supravital Dyes
<ol>
	<li>For the garter snake dissection protocol see Stewart et al.5 and Teng et al.6 Reptilian tissue remains physiological for longer times, and with less bacterial contamination, when kept at low temperatures (see below). Mammalian tissue is usually maintained at room temperature or higher.</li>
	<li>For lysosomal vital dye staining, dissolve the dye in cold reptilian Ringers solution 1:5,000 (0.2 &#956;M). Incubate at 4 &#176;C for 15 min. Wash several times with cold Ringers. Image as soon as possible.</li>
	<li>For FM1-43 staining using KCl stimulation, dilute the dye in high-KCl Ringers 1:500 (7 &#956;M). Incubate at 4 &#176;C for 30-60 sec maximum. Wash quickly three times with cold Ringers, ~1 min/rinse. Image as soon as possible.</li>
	<li>For FM1-43 staining using hypertonic (sucrose) stimulation, dilute the dye in 0.5 M sucrose Ringers as above. Incubate at 4 &#176;C for 2-5 min. Wash as in step 1.3 above with cold Ringers. Image as soon as possible.</li>
	<li>For FM1-43 staining via electrical stimulation, see Teng et al.6 Briefly, the preparation is placed a dish containing physiological saline solution and the dye. The nerve is stimulated with a preprogrammed train of 200 &#956;sec rectangular pulses ( ~7 V, 30 Hz, 18 &#956;sec). Wash as in step 1.3 above with cold Ringers. Image as soon as possible.</li>
</ol>
2. Configure the Preparation for Imaging
<ol>
	<li>Orient the preparation so that structures of interest are as close as possible to the microscope objective.</li>
	<li>If an inverted microscope is used, utilize a chamber whose bottom contains a thin round cover slip. Typically, the imaging chamber should have a thin stainless steel bottom with a 25 mm diameter #1 thickness glass cover slip at the center. If an upright microscope is used, a bottom coverslip is not required but is useful to permit imaging with transmitted light (e.g. differential interference contrast, DIC) to orient the specimen and locate objects of interest.</li>
	<li>Choose an appropriate objective to obtain the highest possible 3D resolution. There are tradeoffs among numerical aperture (n.a.), working distance, magnification, and type of lens (dry, water- and oil-immersion). For thin preparations like tissue cultures, oil objectives are best. Using an upright microscope, float a cover slip on the aqueous bath, and use immersion oil between the top of the cover slip and the objective.</li>
	<li>If deconvolution software is used, the vendor might provide predetermined point spread functions (PSFs) for certain objectives. If no such information is provided, or if an unsupported objective is chosen, determine the PSF by imaging fluorescent microdots or similar diffraction-limited objects7,8.</li>
</ol>
3. Establish the Desired Depth of Field and Number of Images Desired per Time-lapse Frame
<ol>
	<li>Select the total depth of field so that moving or interacting structures of interest do not disappear or go out of focus as they move in the z-direction. The z-field should slightly exceed the vertical dimension of the cell or cell process being imaged. For vertebrate motor terminals, 15-20 &#181;m is typical.</li>
	<li>Calculate the z-axis step needed for each increment of focus. Resolution along the z-axis varies depending upon properties of the objective; it is about one-fourth of the x-y plane resolution. If either confocal imaging or deconvolution software is used, z-axis resolution is improved. Improve final resolution by deliberate oversampling in the z-direction 5, but see also step 3.3 below. A typical step interval is in the range from 400-1,500 nm for 40-100X objectives. Light should be shuttered off between each z-step image.</li>
	<li>Select the number of images per z-stack, namely the desired depth of field divided by the step interval. Time to complete a typical z-stack is 1-10 sec. Fast-moving objects (100 nm/sec) can appear blurred in a 3D image stack because each image plane corresponds to a slightly different time point. Also, each additional image contributes to photobleaching and possible phototoxicity (see Discussion). If either situation pertains, choose a larger z-step to collect fewer images per stack. Compensate later using image interpolation software (step 6.3 below).</li>
</ol>
4. Select the Time-lapse Frame Rate
Choose by experimentation a rate that is just adequate to smoothly resolve changes with time such as movement, interactions or fusion events9. Under sampling can produce motion artifacts (sampling errors), while oversampling unnecessarily increases exposure to light. Collect either a single long sequence or several repeated sequences from the same preparation, each with a relatively small total time interval (e.g. 10 time points x 30 sec interval = 5 min). If possible, view the preparation with continuous epifluorescence illumination to roughly estimate movement velocities, including drift of the entire preparation if present.
5. Complete All Live Imaging for a Particular Preparation
Reptilian preparations typically remain robust for ~1-2 hr, longer if cooled.
6. Analyze Data According to Desired Use
<ol>
	<li>Retain all raw data files. While digital storage is usually not a problem, processing time is significant even with fast personal computers or workstations. For this reason, crop images into a region of interest [ROI]. Assure that the entire region of interest is in the cropped window during the entire time course.</li>
	<li>Deconvolve image stacks using appropriate software and the correct point spread function. Confirm that resolution is improved and that no artifact has been generated by the deconvolution algorithm. Figure 3 shows example images.</li>
	<li>Use an interpolation algorithm to expand z-axis apparent resolution. A 6X expansion from an actual 1.5 &#956;m image plane separation to an apparent 0.25 &#956;m separation is suggested. Alternatively, use some combination of oversampling (step 3.2) and interpolation.</li>
	<li>Perform contrast, brightness, noise filtering, photobleaching and other typical image processing adjustments if desired. Image manipulation standards dictate that for most scientific work, it is appropriate that the same corrections be applied to all images, both within a stack and at different time points. The consequence of a particular adjustment should be assessed among all images 9.</li>
	<li>Select a particular display mode for export of data. The most straightforward display is stereo video using either red-green or red-blue anaglyphs (examples in Figure 3), stereo pairs, or alternating left/right eye frames with shutter glasses. Anaglyphs are limited to single-color. Enhance apparent image depth if desired to improve z-axis visual resolution.</li>
	<li>Experiment with various filtering and image enhancement techniques. For example, Laplacian filtering is useful to increase contrast of small structures above background. The brightness of pixels at the center of a running window is enhanced while brightness of surrounding areas is reduced. Note that some filtering methods do not work well in combination.</li>
	<li>Apply drift correction if there is substantial movement of the preparation over time. Such movement is common in living muscle, even with drugs added to suppress action potentials and synaptic potentials. A pixel registration algorithm (e.g. IMARIS, Adobe AfterEffects, TurboReg plugin for ImageJ) aligns time-lapse images and can reduce, but usually not completely eliminate, such motion.</li>
</ol>

<ol>
	<li>Frigault, M. M., Lacoste, J., Swift, J. L., Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+microscopy-tips+and+tools.">Live-cell microscopy-tips and tools.</a> J. Cell Sci. 122 (6), 753-767 (2009).</li><li>Tinevez, J. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+method+for+measuring+phototoxicity+of+a+live+cell+imaging+microscope.">A quantitative method for measuring phototoxicity of a live cell imaging microscope.</a> Meth. Enzymol. 506, 291-309 (2012).</li><li>Dunn, K. W., Kamocka, M. M., McDonald, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+evaluating+colocalization+in+biological+microscopy.">A practical guide to evaluating colocalization in biological microscopy.</a> Am. J. Physiol. Cell Physiol. 300, (2011).</li><li>Snapp, E. L., Hegde, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+and+evaluation+of+FRETexperiments+to+measure+proteinproximities+in+cells.">Rational design and evaluation of FRETexperiments to measure proteinproximities in cells.</a> Curr. Protoc. Cell Biol. 17, (2006).</li><li>Stewart, R. S., Teng, H., Wilkinson, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late"+macroendosomes+and+acidic+endosomes+in+vertebrate+motor+nerve+terminals.">Late" macroendosomes and acidic endosomes in vertebrate motor nerve terminals.</a> J. Comp. Neurol. 520, 4275-4293 (2012).</li><li>Teng, H., Lin, M. Y., Wilkinson, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroendocytosis+and+endosome+processing+in+snake+motor+boutons.">Macroendocytosis and endosome processing in snake motor boutons.</a> J. Physiol. 582, 243-262 (2007).</li><li>McNally, J. G., Karpova, T., Cooper, J., Conchello, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+by+deconvolution+microscopy.">Three-dimensional imaging by deconvolution microscopy.</a> Methods. 19, 373-385 (1999).</li><li>Swedlow, J. R., Platani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+imaging+using+wide-field+microscopy+and+deconvolution.">Live cell imaging using wide-field microscopy and deconvolution.</a> Cell Struct. Funct. 27, 335-341 (2002).</li><li>Cromey, D. W., Taatjes, D. J., Roth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+images+are+data:+and+should+be+treated+as+such.">Digital images are data: and should be treated as such.</a> Methods Mol. Biol. 931, 1-27 (2013).</li><li>Teng, H., Wilkinson, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin-mediated+endocytosis+near+active+zones+in+snake+motor+terminals.">Clathrin-mediated endocytosis near active zones in snake motor terminals.</a> J. Neurosci. 20 (21), 7986-7993 (2000).</li><li>Teng, H., Cole, J. C., Roberts, R. L., Wilkinson, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytic+active+zones:+hot+spots+for+endocytosis+in+vertebrate+nerve+terminals.">Endocytic active zones: hot spots for endocytosis in vertebrate nerve terminals.</a> J. Neurosci. 19 (12), 4855-4866 (1999).</li><li>Tan, T. T. T., Khaw, C., Ng, M. M. L., Mendez-Vilas, A., Diaz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+recent+advances+in+live+cell+bioimaging.">Challenges and recent advances in live cell bioimaging.</a> Microscopy: Science, Technology, Applications and Education. , 1495-1505 (2010).</li><li>Verbrugghe, K. J. C., Chan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+C.+elegans+embryos+using+an+epifluorescent+microscope+and+open+source+software.">Imaging C. elegans embryos using an epifluorescent microscope and open source software.</a> J. Vis. Exp. (49), (2011).</li></ol>

The authors declare no competing financial interests.

The most critical aspect of 4D imaging is management of the duration and intensity of light exposure. Photobleaching decreases image signal-to-noise ratio and can be problematic or not depending on various factors, including choice of fluorophores. Nonspecific damage to living tissue (phototoxicity) is related to photobleaching, and can sometimes be identified using fluorescent probes designed for the purpose2,12 or by examination of morphology with suitable brightfield optics, such as differential interferenc...

Time-lapse imaging of living tissue provides visual access to dynamical structure-function relations that cannot be appreciated in fixed or living preparations imaged at a single point in time. Often, however, the tradeoff for access to temporal information is a decrease in optical resolution. High numerical aperture oil-immersion objectives are impractical in living tissue because of their narrow range of focus, leaving water immersion or dry objectives as the only alternatives. Moreover, the increased resolution afforded by confocal optics cannot be utilized in some living preparations due to phototoxicity from the relatively high levels of illumination required1,2. Lastly, while several real-time or time-lapse optical techniques are available that offer enhanced resolution, their applicability is limited to preparations where structures of interest can be positioned within a few hundred nanometers of the objective1. The method described makes use of relatively low-cost equipment, is versatile, yet offers improved resolution compared to more commonly-used time-lapse techniques. It is intended for use in individual laboratories as well as imaging facilities.
The method utilizes conventional epifluorescence microscopy, combined with a sensitive digital camera and with hardware designed to rapidly acquire sets of images at slightly different focal planes (z-stacks). Each z-stack is digitally deconvolved to increase resolution. One feature of 3D time-lapse (4D) imaging is precise tracking of moving organelles or other structures. When properly set up, imaged structures do not go out of focus, and movement in all three directions can be observed and quantified. Thus it is impossible for a stained structure to disappear over one or more time-lapse frames merely by drifting above or below a narrow focal plane. The method also serves as a sensitive tool for assessing the interactions and possible fusion of small structures. Conventional epifluorescence or confocal images of structures near the diffraction limit (a few hundred nm) do not confirm fusion even if merged images show overlap of their respective labels3. Fusion is suggested, but it remains possible that the objects are separated horizontally or vertically by a distance that is below the diffraction limit. Three- or four-dimensional imaging, in contrast, permits viewing the objects in each of three orthogonal directions. The appearance of fusion in all three views increases the level of certainty. And, in some living preparations, directed or Brownian movement of putatively fused objects provides further proof when both labels move together in time. Of course, when near the diffraction limit the level of certainty in discerning structures from background, or showing that they contain two dyes (fusion), is not absolute. If applicable, specialized techniques, such as fluorescence resonance energy transfer (FRET)4, are more appropriate.

<table border="0" cellpadding="0" cellspacing="0" style="width:720px;" width="720">
	<colgroup>
		<col span="4" />
	</colgroup>
	<tbody>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">Name of Material/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:180px;">Catalog Number</td>
			<td style="width:180px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Reagents:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SGC5</td>
			<td>Biotium [Hayward, CA]</td>
			<td>70057</td>
			<td>Final conc:10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FM1-43FX</td>
			<td>Invitrogen [Carlsbad, CA]</td>
			<td>F35335</td>
			<td>Final conc:7 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LysoTracker Red</td>
			<td>Invitrogen [Carlsbad, CA]</td>
			<td>L7528</td>
			<td>Final conc:0.2 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Solutions:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reptilian Ringers pH 7.2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>145 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>2.5&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">CaCl2</td>
			<td>3.6&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">MgSO4</td>
			<td>1.8&#160; mM</td>
			<td style="width:180px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">KH2PO4 (Dibasic)</td>
			<td>1.0&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>5.0&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:21px;">High KCL Reptilian Ringers pH 7.2</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>&#160;86&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>&#160;60&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">CaCl2</td>
			<td>3.6&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">MgSO4</td>
			<td>1.8&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">KH2PO4 (Dibasic)</td>
			<td>1.0&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>5.0&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:21px;">High Sucrose Ringers pH 7.2</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>145 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>2. 5 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">CaCl2</td>
			<td>3.6&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">MgSO4</td>
			<td>1.8&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">KH2PO4 (Dibasic)</td>
			<td>1.0&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>5.0&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>0.5 M (17.1 gm/50 mL)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Comments</td>
			<td>Comments(website)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Axioplan 200 inverted microscope</td>
			<td colspan="2">Carl Zeiss [Thornwood, NY]</td>
			<td><a href="http://www.zeiss.com/">www.zeiss.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">N-Achroplan 63X water objective; n.a.=0.9; Working distance=2.4mm&#160;</td>
			<td colspan="2">Carl Zeiss [Thornwood, NY]</td>
			<td><a href="http://www.zeiss.com/">www.zeiss.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">DG4 combination light source/excitation filterwheel switcher</td>
			<td>Sutter instruments [Novato, CA]</td>
			<td>175W Xenon arc lamp</td>
			<td><a href="http://www.sutter.com/">www.sutter.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Lambda 10-2&#160; emission filterwheel switcher</td>
			<td colspan="2">Sutter instruments [Novato, CA]</td>
			<td><a href="http://www.sutter.com/">www.sutter.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sensicam CCD camera</td>
			<td colspan="2">Cooke Instruments [Tonawanda, NY]</td>
			<td><a href="http://www.cookecorp.com/">www.cookecorp.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cascade 512 CCD camera</td>
			<td colspan="2">Photometrics [Tucson, AZ]</td>
			<td><a href="http://www.photometrics.com/">www.photometrics.com</a></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;">Imaging dishes- made in-house-11cm dia.; 25 mm dia. #1 coverslip embedded; magnetic pins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Comments</td>
			<td>Comments(website)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slidebook 5.0</td>
			<td>Intelligent Imaging Innovations [Denver, CO]</td>
			<td>Deconvolution; Drift correction;3D and 4D data presentation</td>
			<td><a href="http://www.intelligent-imaging.com/">www.intelligent-imaging.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IMARIS 7.5.2</td>
			<td>Bitplane [South Windsor, CT]</td>
			<td>Drift correction; 3D and 4D data presentation</td>
			<td><a href="http://www.bitplane.com/">www.bitplane.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AfterEffects CS6</td>
			<td>Adobe [San Jose, CA]</td>
			<td>Drift correction</td>
			<td><a href="http://www.adobe.com/">www.adobe.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ 1.46</td>
			<td>National Institutes of Health [Bethesda, MD]</td>
			<td>Multiple plugins available;Stereo pair construction</td>
			<td><a href="http://rsbweb.nih.gov/ij">http://rsbweb.nih.gov/ij</a></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Zeiss LSM</td>
			<td>Carl Zeiss [Thornwood, NY]</td>
			<td>Stereo pair construction</td>
			<td><a href="http://www.zeiss.com/">www.zeiss.com</a></td>
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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.

Data shown are from snake neuromuscular terminals (see low and high magnification views in Figure 3; the endocytic dye (FM1-43) uptake creates a haze that fills each bouton) and, in particular, macroendosomes (MEs) and acidic endosomes (AEs) within these terminals5. MEs are created by bulk endocytosis during neural activity10 and their number declines exponentially with time after activity has ceased6. Use of 4D live imaging was to determine if MEs move towards the plasma...

Watch this Scientific Journal Video about Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging at JoVE.com

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

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 ...

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Research

JoVE Journal

Neuroscience

8.9K Views.  Washington University School of Medicine. 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.

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

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4. Rod photoreceptors are responsible for vision in dim light, cones in bright light. Phototransduction takes place in the outer segment of the photoreceptor cell, a specialized compartment that contains a high concentration of visual pigment, the primary light detector. The visual pigment is composed of a chromophore, 11-cis retinal, attached to a protein, opsin. A photon absorbed by the visual pigment isomerizes the chromophore from 11-cis to all-trans. This photoisomerization brings about a conformational change in the visual pigment that 
initiates a cascade of reactions culminating in a change in membrane potential, and bringing about the transduction of the light stimulus to an electrical signal. The 
recovery of the cell from light stimulation involves the deactivation of the intermediates activated by light, and the reestablishment of the membrane potential. Ca2+ modulates the activity of several of the enzymes involved in phototransduction, and its concentration is reduced upon light stimulation. In this way, Ca2+ plays an important role in the recovery of the cell from light stimulation and its adaptation to background light.
Another essential part of the recovery process is the regeneration of the visual pigment that has been destroyed during light-detection by the 
photoisomerization of its 11-cis chromophore to all-trans5-7. This regeneration begins with the release of all-trans retinal by 
the photoactivated pigment, leaving behind the apo-protein opsin. The released all-trans retinal is rapidly reduced in a reaction utilizing NADPH to all-
trans retinol, and opsin combines with fresh 11-cis retinal brought into the outer segment to reform the visual pigment. All-trans retinol is 
then transferred out of the outer segment and into neighboring cells by the specialized carrier Interphotoreceptor Retinoid Binding Protein (IRBP).
Fluorescence imaging of single photoreceptor cells can be used to study their physiology and cell biology. Ca2+-sensitive fluorescent dyes can be used to examine in detail the interplay between outer 
segment Ca2+ changes and response to light8-12 as well as the role of inner segment Ca2+ stores in Ca2+ homeostasis13,14. 
Fluorescent dyes can also be used for measuring Mg2+ concentration15, pH, and as tracers of aqueous and membrane compartments16. 
Finally, the intrinsic fluorescence of all-trans retinol (vitamin A) can be used to monitor the kinetics of its formation and removal in single 
photoreceptor cells17-19.

A method is described for the preparation of single living photoreceptor cells from different vertebrate species for fluorescence imaging. The method can be used to image the fluorescence of endogenous fluorophores, such as NADH or vitamin A, or that of exogenously added fluorescent dyes sensitive to Ca2+ or other factors.

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Visualization of Amyloid &amp;#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...