This work was supported by National Institutes of Health grants R01-HD093827 and R03-HD090304 (A.J.T).

All animal procedures were performed under the guidelines and approved by the Institutional Animal Care and Use Committee at Cornell University (#2002-0095). 8-10 weeks old AcroSensE mice2 were used for the present study. Requests for information on the availability of the AcroSensE mice can be submitted to the corresponding author.
1. Sperm collection and washing
<ol>
	<li>Collect cauda epididymal sperm (more details on this procedure are provided in11) from AcroSensE mice. Allow 15 min swim-out procedure in a 3.5 cm tissue-culture dish containing 0.5 mL of Modified Whitten&#39;s medium (MW) at 37 &#176;C. Dish should be covered to reduce evaporation of the MW, and should be tilted so that the medium has enough depth to cover the cauda epididymides. 
	NOTE: Modified Whitten&#39;s medium (MW) includes 22 mM HEPES, 1.2 mM MgCl2, 100 mM NaCl, 4.7 mM KCl, 1 mM pyruvic acid, 4.8 mM lactic acid hemi-Ca2+ salt, pH 7.35. Glucose (5.5 mM), NaHCO3 (10 mM), and 2-hydroxypropyl--cyclodextrin (CD; 3 mM) are supplemented (see Table of Materials) as needed for induction of capacitation, and concentrations can be modified for specific experiments. In addition, alternative sterol acceptors could be used instead of CD (e.g., bovine serum albumin or high density lipoproteins).</li>
	<li>Using fine forceps, remove any epididymal tissue from the swim-out collection dish. Transfer the medium containing the sperm into a 15 mL conical tube, and centrifuge the suspension at 100 x g for 1 min in a swinging bucket rotor at room temperature. Using a plastic pipette, remove the MW supernatant containing the sperm from any gross tissue debris that will have pelleted at this step.</li>
	<li>Bring the MW supernatant containing the sperm to a total volume of 3 mL by adding MW at 37 &#176;C. Centrifuge at 400 x g for 8 min (at room temperature) in a round-bottomed tube. Slowly remove the supernatant of MW from the loosely-pelleted sperm.</li>
	<li>If viable, the sperm should appear as a &#34;fluffy&#34;-looking pellet. Gently resuspend the sperm via trituration with a large-bore transfer pipette or via gentle manual taps on the tube (&#34;finger flicking&#34;). Once resuspended evenly, transfer them into a new round-bottom tube. Use 10-20 &#181;L of the sperm suspension to count and assess motility. Dilute sperm in MW for use. 
	NOTE: For most experiments involving mouse sperm capacitation, a final concentration of 5-10 million sperm/mL MW is utilized. The imaging protocol described here does not require the capacitation of sperm, although it is necessary for sperm to acquire the ability to undergo physiologically-relevant acrosome exocytosis. One could study pathological exocytosis or exocytosis induced through reagents such as a calcium ionophore without capacitating the sperm. Additionally, one could explore potential changes in acrosomal calcium in non-capacitated cells in response to various stimuli.</li>
	<li>Use the sperm within 3-5 h from collection, keeping the time as short as possible and consistent among experimental trials.</li>
	<li>Perform all steps of collection and washing at 37 &#176;C using MW medium, employing methods to minimize membrane damage. This includes the use of large-bore plastic transfer pipettes or large-orifice pipette tips, which are recommended for use during all stages of the procedure.</li>
</ol>
2. Poly-D-lysine-coating of coverslip dishes for imaging
NOTE: Poly-D-lysine (PDL) dishes should be prepared fresh before each experiment.
<ol>
	<li>Dispense a 0.5 &#956;L droplet of PDL (0.5 mg/mL, see Table of Materials) at the center of a coverslip dish.</li>
	<li>Using a 10 &#956;L graduated tip, smear the PDL droplet across the coverslip (crisscross pattern as shown in Figure 1B).</li>
	<li>Allow to dry at 37 &#730;C for 10 min. Keep PDL-coated dishes covered and at 37 &#730;C until use.</li>
</ol>
3. Capillary pulling, loading for puffing
<ol>
	<li>Pull borosilicate glass capillaries (O.D, 2 mm, I.D, 1.56 mm, see Table of Materials) using settings as follows- Heat: 330, Pull: 250, Velocity: 250, and Time: 70. This step should be optimized for the specific puller in use.</li>
	<li>Before each experiment, load a single capillary with the stimulating solution containing 3-5x the concentration of the stimulant (to account for the rapid diffusion of the solution upon puffing). Before filling, use fine tweezers to break open the capillary tip at approximately 1-2 mm&#160;from the end. This should produce a &#8764;5 &#181;m opening diameter. Heat polishing is not required.</li>
	<li>Using a thin plastic transfer pipette, slowly inject the stimulating solution into the capillary until three-fourths of its length is full.</li>
	<li>Remove any air bubbles formed during filling by gentle flicking of the capillary.</li>
	<li>Mount filled capillary on the micromanipulator (see Table of Materials).</li>
</ol>
4. Preparation of the delivery of stimulation for puffing
NOTE: To minimize the risk of user injury, safety goggles should be worn during the operation of the puffing procedure.
<ol>
	<li>Set up the single-cell delivery system settings to provide a 10 s pulse at 5 psi.</li>
	<li>Connect the borosilicate capillary to the single-cell delivery system pipette holder. Ensure seals are tight.</li>
	<li>While observing the capillary tip, apply a short puff of 2 s at 20 psi to ensure that the solution is indeed dispensed through the tip&#39;s end (a small drop should be obvious at the end of the tip).</li>
</ol>
5. Microscopy and image acquisition
NOTE: Multiple brands of microscopes are available and can be used; however, a minimum frame rate of 3 frames/s is desirable. Regarding temperature and environmental control, please note that the actual temperature of the medium in the dish should be confirmed, for example, using a non-contact infrared thermometer.
<ol>
	<li>Add 80 &#956;L of diluted sperm to the center of a poly-D-lysine-coated 35 mm coverslip dish. For most experiments involving mouse sperm capacitation, a final concentration of 5-10 million sperm/mL is utilized.</li>
	<li>Slowly add to the sperm 3 mL of warm (37 &#730;C) MW base media supplemented with 10 mM CaCl2. 
	NOTE: One could perform experiments at room temperature to slow cellular processes, but it&#39;s important to note that because capacitation is mediated by lipid exchanges and the fluidity of micro- and macro-domains, one must keep in mind that physiologically-relevant capacitation and acrosome exocytosis are temperature-dependent processes.</li>
	<li>Mount the dish with the sperm on the microscope (pre-heated to 37 &#730;C).</li>
	<li>Excite the GCaMP3 using a 488 nm laser line and view with a 505-550 nm bandpass (BP) filter. Excite the mCherry using a 555 nm laser line and view with a 575-615 nm BP filter.</li>
	<li>Using a micromanipulator (it is recommended to attach the manipulator to the air-table on which the microscope was set up.), position the tip of the capillary approximately 100 &#956;m to the side and 5-10 &#956;m above the plane of the cell of interest. 
	NOTE: The stimulating solutions are made at 3-5x the normal concentration to compensate for the rapid diffusion occurring in the volume between the capillary and the cell.</li>
	<li>Start the imaging sequence on the microscope.</li>
	<li>Image sperm cells at a high frame rate, preferably &#62;10 frames/s. Here, a resonant scanner was used that enables imaging at 33 ms/frame.</li>
	<li>Ten seconds after starting the image acquisition on the microscope, activate the single-cell delivery system to initiate the 10 s puff of stimulant.</li>
	<li>Image cells for a duration of 10-15 min.</li>
	<li>Using the single-cell delivery system, image multiple cells from a single dish according to the locations provided in Figure 1B. 
	&#8203;NOTE: Untreated/non-stimulated sperm under the microscope should appear mostly red, with a low intensity of the green signal. Sperm heads should be attached to the coverslip, and live sperm can be identified by their moving tails. In sperm that undergo AE, the GCaMP3 green signal will initially increase, and both the mCherry red signal and GCaMP3 green signal will disappear if AE is complete, as illustrated in Figure 2. The reduction of mCherry fluorescence provides a more specific indicator of the time when AE begins because changes in Ca2+ concentrations will continually cause changes in the GCaMP3 fluorescence intensity.</li>
</ol>
6. Image and data analysis
NOTE: Offline image analysis is conducted using ImageJ (see Table of Materials). Previously, several intermediate steps were reported in the process leading to AE, including acrosomal calcium rise (ACR), and full membrane fusion. The latter leads to the loss of mCherry fluorescence and therefore represents full AE. In some cells, signals akin to pre-spike foot events (PSF) are observed when using amperometric approaches in studies of exocytosis (for more details, please see2). Several optional methods are available for analyzing the AcroSensE raw data to quantify ACR, AE, and its intermediate steps. The following describes some of the basic analytical methods.
<ol>
	<li>Depending on the specific microscope system file extension, use the split channel tool (Image &#62; Colors &#62; Split Channels) to generate individual windows for the GCaMP3 and mCherry channels.</li>
	<li>Synchronize the individual windows using the &#34;Synchronize Window&#34; tool (Analyze &#62; Tools &#62; Synchronize Windows).</li>
	<li>Select a region of interest (ROI) around the sperm head in the red channel to include the acrosome region (Figure 3A,B). The synchronize tool allows the same area selection to be applied automatically on the green channel, in which the sperm are still too dim to be visible.</li>
	<li>Choose &#34;Z Profiler&#34; plugin (Plugins &#62; Stacks &#62; Z Profiler) or &#34;Time Series Analyzer&#34; (Plugins &#62; Stacks &#62; Time Series Analyzer) to plot the change in fluorescence intensity as a function of frames/time for each of the two channels.</li>
	<li>Copy data to a spreadsheet software (e.g., Microsoft Excel) for plotting and further analysis. 
	NOTE: Once the data are copied to a spreadsheet, multiple parameters can be extracted for analysis, including the following, as illustrated in Figure 4.
	<ol>
		<li>Fusion Pore (FP) start time: measure the time from timepoint 0 to the timepoint where the GCaMP3 signal starts to rise. This parameter indicates the delay between the stimulation and the sperm ACR response.</li>
		<li>AE start time: measure the time from timepoint 0 to the timepoint where the mCherry signal starts to decay. This parameter indicates the delay between the stimulation and the sperm AE response. This parameter could also be used to calculate the delay between FP formation and AE response.</li>
		<li>Peak FP amplitude: measure the fluorescence signal from the base to the peak of the GCaMP3 signal rise. This parameter indicates the total increase in ACR response.</li>
		<li>Rate of loss of mCherry: determine this parameter by a linear or an exponential fit on the section of the mCherry signal loss (as indicated by &#34;slope&#34; in Figure 4B). 
		NOTE: Alternatively, determine the decay rate by the residual (R) signal method at various time points (e.g., R60: duration time in seconds to a residual signal of 60% from the baseline of the mCherry signal). This parameter can be used to quantify the rate at which the acrosomal content is dispersed upon AE.</li>
		<li>Signal decrease for mCherry loss: determine the difference in amplitude between the baseline mCherry fluorescence intensity and the signal following AE. This parameter indicates the total amount of acrosomal content dispersed upon AE.</li>
		<li>Plot the changes in fluorescence (F) for both the red and green channels as raw data, or normalize them by the initial fluorescence (F0) and express them as &#916;F/F0. The latter provides better visualization of the changes in both red and green on a single plot (e.g., see Figure 3D and Figure 3H). 
		NOTE: Finally, the different measured parameters can be compared between different conditions to determine the relationship between changes in acrosomal calcium concentration and AE. For examples of various experimental condition comparisons, see reference2.</li>
	</ol>
	</li>
</ol>

Combining Imaging with a Stimulant Delivery System to Study Live Mouse Sperm

<ol>
	<li>Austin, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+penetration+of+the+sperm+in+the+mammalian+egg.">Observations on the penetration of the sperm in the mammalian egg.</a> Aust J Sci Res B. 4 (4), 581-596 (1951).</li><li>Cohen, R., 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+genetically+targeted+sensor+reveals+spatial+and+temporal+dynamics+of+acrosomal+calcium+and+sperm+acrosome+exocytosis.">A genetically targeted sensor reveals spatial and temporal dynamics of acrosomal calcium and sperm acrosome exocytosis.</a> J Biol Chem. 298 (5), 101868 (2022).</li><li>Cohen, R., Mukai, C., Travis, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+regulation+of+acrosome+exocytosis.">Lipid regulation of acrosome exocytosis.</a> Adv Anat Embryol Cell Biol. 220, 107-127 (2016).</li><li>Harper, C. V., Cummerson, J. A., White, M. R., Publicover, S. J., Johnson, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+resolution+of+acrosomal+exocytosis+in+human+sperm.">Dynamic resolution of acrosomal exocytosis in human sperm.</a> J Cell Sci. 121, 2130-2135 (2008).</li><li>Sosa, C. M., 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=Kinetics+of+human+sperm+acrosomal+exocytosis).">Kinetics of human sperm acrosomal exocytosis).</a> Mol Hum Reprod. 21 (3), 244-254 (2015).</li><li>Balestrini, P. A., 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=Seeing+is+believing:+Current+methods+to+observe+sperm+acrosomal+exocytosis+in+real+time.">Seeing is believing: Current methods to observe sperm acrosomal exocytosis in real time.</a> Mol Reprod Dev. 87 (12), 1188-1198 (2020).</li><li>Nakanishi, T., 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=Real-time+observation+of+acrosomal+dispersal+from+mouse+sperm+using+GFP+as+a+marker+protein.">Real-time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein.</a> FEBS Lett. 449 (2-3), 277-283 (1999).</li><li>Kim, K. S., Gerton, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+release+of+soluble+and+matrix+components:+evidence+for+intermediate+states+of+secretion+during+spontaneous+acrosomal+exocytosis+in+mouse+sperm.">Differential release of soluble and matrix components: evidence for intermediate states of secretion during spontaneous acrosomal exocytosis in mouse sperm.</a> Dev Biol. 264 (1), 141-152 (2003).</li><li>Hasuwa, H., 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=Transgenic+mouse+sperm+that+have+green+acrosome+and+red+mitochondria+allow+visualization+of+sperm+and+their+acrosome+reaction+in+vivo.">Transgenic mouse sperm that have green acrosome and red mitochondria allow visualization of sperm and their acrosome reaction in vivo.</a> Experimental animals / Japanese Association for Laboratory Animal Science. 59 (1), 105-107 (2010).</li><li>Henderson, M. J., 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+Low-Affinity+GCaMP3+Variant+(GCaMPer)+for+imaging+the+endoplasmic+reticulum+calcium+store.">A Low-Affinity GCaMP3 Variant (GCaMPer) for imaging the endoplasmic reticulum calcium store.</a> PloS one. 10 (10), 0139273 (2015).</li><li>Travis, A. J., 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=Functional+relationships+between+capacitation-dependent+cell+signaling+and+compartmentalized+metabolic+pathways+in+murine+spermatozoa.">Functional relationships between capacitation-dependent cell signaling and compartmentalized metabolic pathways in murine spermatozoa.</a> J Biol Chem. 276 (10), 7630-7636 (2001).</li><li>Cohen, R., 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=Lipid+modulation+of+calcium+flux+through+CaV2.3+regulates+acrosome+exocytosis+and+fertilization.">Lipid modulation of calcium flux through CaV2.3 regulates acrosome exocytosis and fertilization.</a> Dev Cell. 28 (3), 310-321 (2014).</li><li>Sanchez-Cardenas, C., 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=Acrosome+reaction+and+Ca(2)(+)+imaging+in+single+human+spermatozoa:+new+regulatory+roles+of+[Ca(2)(+)]i.">Acrosome reaction and Ca(2)(+) imaging in single human spermatozoa: new regulatory roles of [Ca(2)(+)]i.</a> Biol Reprod. 91 (2), 67 (2014).</li></ol>

No authors have conflicts of interest to report related to this work.

Here, a microscopy-based method is described to utilize the newly generated AcroSensE mouse model for real-time, single-cell monitoring and analysis of the interplay between acrosomal calcium dynamics and intermediate steps leading to AE. Together with readily available genetic approaches, such as crossbreeding with other mouse genetic models or gene editing, this model and method provide a powerful system to study the role of various components and pathways that take part in sperm signaling pathways related to capacitat...

Sperm acquire the ability to fertilize during a process called capacitation1. One endpoint of this process is that the sperm acquire the ability to undergo AE. Over two decades of data support the presence of a complex, multi-step model of AE in mammalian sperm (summarized in2,3). However, studying AE in live sperm is challenging, and currently available methods to monitor this process with adequate resolution are cumbersome and require multiple preparation steps4, are limited to the detection of the final step of AE (e.g., using PNA5), are limited to measurements of changes in cytosolic calcium (in contrast to acrosomal calcium dynamics), or are limited to measurements of either cytosolic calcium dynamics or AE6.
To overcome some of the key limitations of real-time AE studies under physiological conditions and to enable investigation of the interplay between calcium dynamics and AE, a unique mouse model was generated. In this mouse model, a fusion protein composed of the genetically encoded Ca2+-sensor (GCaMP3) and mCherry is expressed and targeted to the acrosome using an acrosin promoter and signaling peptide2. The targeted dual GCaMP3-mCherry sensor enables simultaneous real-time measurements of calcium concentrations and the status of the acrosomal contents in live sperm under physiological conditions using microscopy and a single-cell stimulant delivery system (Figure 1). As a component of the acrosomal matrix, membrane fusion, and AE would result in the loss of the photostable and pH-insensitive mCherry fluorescence from the sperm, as this protein diffuses out of the acrosome vesicle. In this regard, the model&#39;s ability to reflect the timing and occurrence of AE is akin to the benefits of the acrosome-targeted GFP mouse line7,8,9.
The GCaMP3 variant used in this transgenic mouse line has an approximate KD of 400 &#181;M and a dynamic range for Ca2+ of 10-4-10-3 M10, which is suitable for this vesicle. We showed that this combination of features of GCaMP3 could reveal fusion pore formation between the plasma membrane and the outer acrosomal membrane (OAM)2. The fusion pore detection is a result of the pore size being too small to allow the AcroSensE protein to disperse out of the acrosome (via loss of acrosome content) while providing a membrane &#34;channel&#34; that enables the influx of Ca2+ ions into the acrosome lumen, leading to an increase in fluorescence intensity of the GCaMP3.
The bright, monomeric, non-calcium-sensitive fluorescent protein mCherry supports visualization of the acrosome while the GCaMP3 signal is faint (e.g., prior to Ca2+ binding, Figure 2), and importantly, it also allows for the identification of acrosome-intact sperm cells suitable for imaging.
The following protocol describes the utilization of the unique AcroSensE mouse model and the methods for microscopy used experimentally to study AE and sperm calcium dynamics with high spatial and temporal resolution.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100x oil objective&#160;</td>
			<td>Olympus Japan</td>
			<td>UPlanApo,</td>
			<td></td>
		</tr>
		<tr>
			<td>2-hydroxypropyl-b-cyclodextrin&#160;</td>
			<td>Sigma</td>
			<td>C0926</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm coverslip dish, 1.5 thickness</td>
			<td>MatTek Corp.&#160;</td>
			<td>P35G-1.5-20-C&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL round-bottomed tube</td>
			<td>Falcon</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capilarries</td>
			<td>Sutter Instrument Co. CA USA</td>
			<td>B200-156-10</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus Japan</td>
			<td>Olympus FluoView&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose&#160;</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated tip&#160;</td>
			<td>TipOne, USA Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H7006</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ&#160;</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/plugins/index.html</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactic acid</td>
			<td>Sigma</td>
			<td>G5889</td>
			<td></td>
		</tr>
		<tr>
			<td>Live-Cell Microscope Incubation Systems&#160;</td>
			<td>TOKAI HIT Shizuoka, Japan</td>
			<td>Model STX</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller&#160;</td>
			<td>Sutter Instrument Co. CA USA</td>
			<td>Model P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6297</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic transfer pipette&#160;</td>
			<td>FisherBrand&#160;</td>
			<td>13-711-6M</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine&#160;</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvic acid</td>
			<td>Sigma</td>
			<td>107360</td>
			<td></td>
		</tr>
		<tr>
			<td>Single cell delivery system</td>
			<td>Parker, Hauppauge, NY</td>
			<td>Picospritzer III</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time imaging of acrosomal calcium dynamics and exocytosis in live mouse sperm

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process essential for fertilization. However, our understanding of how calcium signaling regulates AE is still incomplete. In particular, the interplay between intra-acrosomal calcium dynamics and the intermediate steps leading to AE is not well-defined. Here, we describe a method that provides spatial and temporal insights into acrosomal calcium dynamics and their relationship to membrane fusion and subsequent exocytosis of the acrosome vesicle. The method utilizes a novel transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE). The sensor combines a genetically encoded calcium indicator (GCaMP) fused with mCherry. This fusion protein was specifically designed to enable the concurrent observation of acrosomal calcium dynamics and membrane fusion events. Real-time monitoring of acrosomal calcium dynamics and AE in live AcroSensE sperm is achieved using a combination of high frame-rate imaging and a stimulant delivery system that can target single sperm. This protocol also provides several examples of basic methods to quantify and analyze the raw data. Because the AcroSensE model is genetically encoded, its scientific significance can be augmented by using readily available genetic tools, such as crossbreeding with other mouse genetic models or gene-editing (CRISPR) based methods. With this strategy, the roles of additional signaling pathways in sperm capacitation and fertilization can be resolved. In summary, the method described here provides a convenient and effective tool to study calcium dynamics in a specific subcellular compartment-the sperm acrosome-and how those dynamics regulate the intermediate steps leading to membrane fusion and acrosome exocytosis.

Figure 2 provides a simplified illustration showing the sequence of fluorescence changes expected following the successful stimulation of sperm. The top panel of Figure 2 illustrates the changes in GCaMP3 fluorescence intensity, where the signal is initially dim (baseline acrosomal calcium concentrations are lower than GCaMP3 KD), and upon the entry of calcium ions via fusion pores, the fluorescence increases in brightness. Finally, upon AE, there is a ...

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Author Spotlight: A Live Cell Imaging Technique to Study Calcium Signaling and Acrosome Exocytosis in Mouse Sperm 

The AcroSensE mouse model and live cell imaging methods described here provide a new approach to studying calcium dynamics in the subcellular compartment of the sperm acrosome and how they regulate intermediate steps leading to membrane fusion and acrosome exocytosis.

Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process ...

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912 Views.  Cornell University. The AcroSensE mouse model and live cell imaging methods described here provide a new approach to studying calcium dynamics in the subcellular compartment of the sperm acrosome and how they regulate intermediate steps leading to membrane fusion and acrosome exocytosis.

Video: Author Spotlight: A Live Cell Imaging Technique to Study Calcium Signaling and Acrosome Exocytosis in Mouse Sperm 

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. 
This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...