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><tbody><tr><td>100x oil objective&amp;nbsp;</td><td>Olympus Japan</td><td>UPlanApo,</td><td></td></tr><tr><td>2-hydroxypropyl-b-cyclodextrin&amp;nbsp;</td><td>Sigma</td><td>C0926</td><td></td></tr><tr><td>35 mm coverslip dish, 1.5 thickness</td><td>MatTek Corp.&amp;nbsp;</td><td>P35G-1.5-20-C&amp;nbsp;</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>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C4901</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus Japan</td><td>Olympus FluoView&amp;nbsp;</td><td></td></tr><tr><td>Glucose&amp;nbsp;</td><td>Sigma</td><td>G7528</td><td></td></tr><tr><td>Graduated tip&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>TOKAI HIT Shizuoka, Japan</td><td>Model STX</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>Micropipette Puller&amp;nbsp;</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>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S6297</td><td></td></tr><tr><td>Plastic transfer pipette&amp;nbsp;</td><td>FisherBrand&amp;nbsp;</td><td>13-711-6M</td><td></td></tr><tr><td>Poly-D-lysine&amp;nbsp;</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 ...

Watch this Scientific Journal Video about A Live Cell Imaging Technique to Study Calcium Signaling and Acrosome Exocytosis in Mouse Sperm at JoVE.com

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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Developmental Biology

950 Views.  Cornell University. Ons onderzoeksdoel is om de moleculaire mechanismen die de spermafunctie reguleren beter te begrijpen, inclusief de processen zoals capacitatie en bevruchting. In het bijzonder onderzoeken we hoe membraanlipiden calciumsignalering en acrosoomexocytose activeren. Dit protocol pakt de lacune aan in ons begrip van de wisselwerking tussen capacitatie, sperma-activering, calciumsignalering en acrosoom-exocytose.De voordelen van dit protocol zijn onder meer beeldvorming van sperma onder fysi...

Video: Auteur in de schijnwerpers: een live celbeeldvormingstechniek om calciumsignalering en acrosoomexocytose in muizensperma te bestuderen 

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Neuroscience

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Biology

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Tracking Calcium in the Early Developmental Phases of C. elegans

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 &#176;C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter &#34;CaFE&#34; for Calcium during Fertilization in C. elegans. The CaFE reporter was&#160;engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.

Here, we present protocols and tools for visualizing calcium during fertilization and early embryogenesis using a genetically encoded calcium reporter expressed in the germline of the model nematode C. elegans.

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Author Spotlight: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

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

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