The authors thank Christiaan Zeelenberg for his assistance during the SIM imaging. JW acknowledges the China Scholarship Council for a PhD fellowship and thanks Irene Stellingwerf for her help during the primary stage of imaging.

1. B. subtillis&#160;Sporulation (Timing: 7 Days Before Microscopic Observation)
<ol>
	<li>Day 1
	<ol>
		<li>Streak a bacterial culture on a Luria-Bertani Broth (LB) agar plate (1% tryptone, 0.5% yeast extract, 1% NaCl, 1% agar)13 and incubate overnight at 37 &#176;C to obtain single colonies. Use the B. subtilis KGB80 (PS4150 gerKA gerKC gerKB-mCherry cat, gerD-gfp kan) strain and its parent background strain B. subtilis PS4150 (PS832 &#916;gerE::spc, &#916;cotE::tet) as described previously7. 
		NOTE: The use of spores with the &#916;cotE&#916;gerE background is essential in germinosome visualization, in order to minimize the autofluorescence of the spore coat layers7.</li>
		<li>Sterilize all media, tubes, pipets and other culture materials to be used with appropriate methods.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Inoculate a single colony into 5 mL of LB medium early in the morning and incubate the culture under continuous agitation in a screw cap tube at 200 rpm/min and 37 &#176;C until the OD600 reaches 0.3-0.4 (approximately 7 h).</li>
		<li>Make 500 mL of MOPS medium (pH 7.5) containing 3 nM (NH4)6Mo7O24&#183;4H2O, 0.4 &#956;M H3BO3, 30 nM CoCl2&#183;6H2O, 10 nM CuSO4&#183;5H2O, 10 nM ZnSO4&#183;7H2O, 1.32 mM K2HPO4, 0.4 mM MgCl2&#183;6H2O, 0.276 mM K2SO4, 0.01 mM FeSO4&#183;7H2O, 0.14 mM CaCl2&#183;2H2O, 80 mM MOPS, 4 mM Tricine, 0.1 mM MnCl2&#183;2H2O, 10 mM D-glucose-monohydrate, and 10 mM NH4Cl.</li>
		<li>Prepare in screw cap tubes 10-1- 10-7 serial dilutions of the LB culture in 5 mL MOPS medium each and incubate the cultures overnight under continuous agitation at 200 rpm/min and 37 &#176;C. 
		NOTE: The serial dilutions prepared here aim to obtain a dilution in early exponential phase in the next morning. This step and the next step are necessary to allow the cells to adapt to the MOPS buffered growth medium.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Select one of the MOPS dilutions with an OD600 of 0.3-0.4. Inoculate 0.2 mL of the culture to pre-warmed (37 &#176;C) 20 mL of MOPS medium in a conical 250 mL flask and incubate under continuous agitation until the OD600 reaches 0.3-0.4 (~7 h).</li>
		<li>Inoculate the MOPS based sporulation medium (250 mL) with 1% (v/v) pre-culture from step 1.3.1 and incubate for 3 days at 37 &#176;C in a conical liter flask under continuous agitation. For FM4-64 staining of PS4150 spores, add 2 &#181;g/mL FM4-64 probe (see Table of Materials) to the sporulation medium 1 or 2 h after reaching the peak OD600 value of vegetative growth (generally approximately 2) and allow the culture to subsequently sporulate whilst protecting it from light5.</li>
	</ol>
	</li>
	<li>Day 7: Harvesting of spores
	<ol>
		<li>Determine the sporulation yield (spores vs vegetative cells) using phase contrast microscopy at 100X magnification to distinguish the phase bright spores from phase dark cells and possibly non-mature spores. A 90 % phase bright spore yield is expected.</li>
		<li>Pellet the spores at 4,270 x g for 15 min at 4 &#176;C in round bottom centrifuge tubes. Wash the spore pellet 2-3 times with 40 mL of sterile ultra-pure Type 1 demineralized water in 50 mL conical centrifuge tubes (see Table of Materials). Spin-down at each wash at 4,270 x g for 15 min (4 &#176;C).</li>
	</ol>
	</li>
	<li>Day 7: Spore purification
	<ol>
		<li>Spore purification: Suspend the spore pellet in 750 &#181;L of 20% nonionic density gradient medium (see Table of materials) and load onto 800 &#181;L of 50% nonionic density gradient medium in sterilized microcentrifuge tubes. Centrifuge for 60 min at 21,500 x g. The pellet obtained contains the free spores. Suspend the spore pellet in 200 &#181;L of 20% nonionic density gradient medium and load onto 1 mL of 50% nonionic density gradient medium in a microcentrifuge tube, and centrifuge for 15 min at 21,500 x g.</li>
		<li>Washing the final spore preparation and storage: The pellet obtained contains the purified dormant spores. Wash the spore pellet 2-3 times with 1.5 mL of sterile ultra-pure Type 1 water (see Table of Materials) and spin-down in between at 9,560 x g for 15 min at 4 &#176;C. Finally, suspend the pellet in sterile ultra-pure Type 1 water to a final OD600 of approximately 30. Aliquots of the spores can be stored at -80 &#176;C for 8 weeks14.</li>
	</ol>
	</li>
</ol>
2. Decoating
<ol>
	<li>Treat PS4150 spores with 0.1 M NaCl/0.1 M NaOH/1% sodium dodecyl sulfate (SDS)/0.1 M dithiothreitol (DTT) at 70 &#176;C for 1 h. Wash the spores 10 times with sterilized ultra-pure Type 1 water15,16. By doing so, any adsorbed FM4-64 probe in spore&#8217;s outer membrane and outer layers will be removed.</li>
</ol>
3. Coverslip and Slide Preparation11 (Timing: 1 H Before Observation)
<ol>
	<li>Pre-clean high precision coverslips (see Table of materials) with 1 M HCl for 30 min in a gently shaking water bath. Wash the coverslips twice in ultra-pure Type 1 water, and store them in 100% (vol/vol) EtOH. Let them dry and verify their clarity before use. Pre-clean the glass slides in 70% EtOH. Let them dry and verify their clarity before use.</li>
</ol>
4. Sampling Fluorescent Microspheres or Spores in the Gene Frame Slide10 (Timing 15 Min)
<ol>
	<li>Pre-warm two 70% EtOH cleaned and air dried glass slides (see Table of materials) for several seconds on a 70 &#176;C heating block, drop 65 &#956;L of sterilized 2% agarose held at 70 &#176;C on top of one glass slide and place the other glass slide on top to spread the agarose between the slides. The agarose patch will dry in approximately 5 min.</li>
	<li>Cut the agarose patch into a 1 x 1cm section after removing one of the glass slides, add 0.4 &#956;L of sample (fluorescent microspheres or spores of ~108/mL), and transfer the patch onto the high precision coverslip by placing the coverslip onto the patch and sliding it off.</li>
	<li>Fix a Gene Frame (1.5 x 1.6 cm2, 65 &#181;L) onto the dried slide, onto which the coverslip is placed closing all corners of the frame, thus completing the slide for use in microscopy.</li>
</ol>
5. Imaging11,17(Timing: 1 H)
<ol>
	<li>Capture the transmission and fluorescence images of spores as well as a mixture of red and yellow-green carboxylate-modified fluorescent microspheres on a Structured Illumination Microscope (see Table of materials) equipped with a 100x oil objective (Numerical Aperture = 1.49), a CCD camera and image analysis software (see Table of materials). Generate all images at room temperature without the disturbance of ambient light. Make sure to always clean the 100x objective and the slide with 75% ethanol before imaging.</li>
	<li>Focus on 100 nm (diameter) fluorescent microspheres and optimize the point spread function (psf) by adjusting the correction ring on the 100x objective until a symmetric psf is obtained, thus minimizing blurring of the image. The psf is the impulse response or the response of an imaging system to a point source or point object.</li>
	<li>Select a field of view with approximately 10 round fluorescent microspheres. Apply a grating focus adjustment for both 561 nm and 488 nm excitation wave lengths as the guide for the image analysis software.</li>
	<li>Focus the spores with the transmission light and capture a transmission light image in the 16&#215; average mode with 200 ms exposures for each image.</li>
	<li>Capture 3D-SIM raw fluorescent images of the spores with the illumination mode &#8220;3D-SIM&#8221;, the camera settings to readout mode Electron Multiplying (EM) Gain 10MHz at 14-bit, and EM gain at 175. Excite the FM4-64 probe in PS4150 spores with 561 nm laser light at 20% laser power, and an illumination time of 400 ms.</li>
	<li>Excite the GerKB-mCherry and GerD-GFP in KGB80 spores with, respectively, 561 nm laser light at 30% laser power for 1 s, and 488 nm laser light at 60% laser power for 3 s. Z-Stack settings are in the top to bottom mode, 0.2 &#181;m/ step, 7 steps and 20 steps for germinosome and IM analysis, respectively. 
	NOTE: These laser parameters were applied in order to assure a maximum brightness value of the histogram window of around 4,000.</li>
</ol>
6. Reconstruct 3D-SIM Raw Images of FM4-64 Stained PS4150 Spores
<ol>
	<li>Perform the N-SIM Slice Reconstruction for the FM4-64 stained PS4150 spores&#8217; raw data. Click the Param button for Reconstruct Slice on the N-SIM pad tab sheet to open the N-SIM Slice Reconstruction window.</li>
	<li>Set the reconstruction parameters in the N-SIM Slice Reconstruction window. To get perfect reconstructed images, follow the suggestion of the N-SIM instructions and click on the appropriate controls in the N-SIM Slice Reconstruction window to set the Illumination Modulation Contrast (IMC) to Auto, High Resolution Noise Suppression (HRNS) to 1.00 and Out of the Focus Blur Suppression (OFBS) to 0.05 as starting points.</li>
	<li>Click the Reconstruct Slice button in the N-SIM Slice Reconstruction window to reconstruct the image. Evaluate the quality of the reconstructed images by the Fast Fourier Transformed (FFT) images and reconstruction score, which display after reconstruction12.</li>
	<li>Adjust the HRNS from 0.10 to 5.00 and OFBS from 0.01 to 0.50 by clicking on the appropriate controls in the N-SIM Slice Reconstruction window until the best parameter settings are obtained.</li>
	<li>Click the Apply button in the N-SIM Slice Reconstuction window to apply changed parameters. Click Close button to close the window.</li>
	<li>Make a FM4-64 stained PS4150 spores raw image active and click the Reconstruct Slice button on the N-SIM Pad tab sheet to execute Slice Reconstruction. Save the reconstructed image.</li>
</ol>
7. Image Analysis
<ol>
	<li>Convert Pseudo-Widefield images of the KGB80 germinosome
	<ol>
		<li>Convert 3D-SIM raw images of KGB80 into Pseudo-Widefield images by activating with a left click the ImageJ plugin SIMcheck utility Raw Data SI to Pseudo-Widefield12. Pseudo-Widefield averages images from raw SIM data and assembles, for comparison, an image equivalent to conventional widefield illumination12. For ImageJ itself see https://imagej.net/Welcome. Randomly select ~25 spores in each inverted transmission image for later germinosome analysis in the fluorescent Pseudo-Widefield images.</li>
		<li>Select in total approximately 350 (in this example, 346) KGB80 spores in 14 fields of view from two slides. The GerD-GFP and GerKB-mCherry fluorescent foci numbers in selected KGB80 spores should be counted independently by two researchers. The researcher can refer back to the 3D-SIM raw images whenever in doubt of the presence of separate fluorescent germinosome foci in spores.</li>
		<li>Assess the maximum intensities of each GerD-GFP and GerKB-mCherry focus and the integrated intensity of each KGB80 spore&#8217;s 3D image with ImageJ.</li>
	</ol>
	</li>
	<li>Analyze Pseudo-Widefield images of the KGB80 germinosome
	<ol>
		<li>Use the mean integrated intensity value of 7 stacks as the integrated signal intensity of the KGB80 spore. Determine background intensities by imaging the background strain PS4150 using identical settings. Regard fluorescent spots in individual KGB80 spores as germinosome foci when they are clearly distinguishable from the background.</li>
		<li>Apply one-way ANOVA-tests for significance determination with software Origin 9.0. considering P values &#60;0.05 as statistically significant. Use the Spearman&#8217;s rank correlation coefficient18 to evaluate the correlation of GerD-GFP and GerKB-mCherry foci number and the measurements of the integrated signal intensity.</li>
	</ol>
	</li>
</ol>

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No conflicts of interest declared.

&#160;The protocol presented contains a standard 3D-SIM procedure for analysis of FM4-64 stained B. subtilis spores that includes sporulation, slide preparation and imaging processes. In addition, the protocol describes a modified SIMcheck (ImageJ)-assisted 3D imaging process for SIM microscopy of B. subtilis spore germinosomes labeled with fluorescent reporters. The latter procedure allowed us to observe this dim substructure with enhanced contrast. By coupling two imaging procedures, it is possible to...

Spores of the orders Bacillales and Clostridiales are metabolically dormant and extraordinarily resistant to harsh decontamination regimes, but unless they germinate, cannot cause deleterious effects in humans1. In nutrient germinant triggered germination of Bacillus subtilis (B. subtilis) spores, the initiation event is germinant binding to germinant receptors (GRs) located in the spore&#8217;s inner membrane (IM). Subsequently, the GRs transduce signals to the SpoVA channel protein also located in the IM. This results in the onset of the exchange of spore core pyridine-2,6-dicarboxylic acid (dipicolinic acid; DPA; comprising 20% of spore core dry wt) for water via the SpoVA channel. Subsequently, the DPA release triggers the activation of cortex peptidoglycan hydrolysis, and additional water uptake follows2,3,4. These events lead to mechanical stress on the coat layers, its subsequent rupture, the onset of outgrowth and, finally, vegetative growth. However, the exact molecular details of the germination process are still far from resolved.
A major question about spore germination concerns the biophysical properties of the lipids surrounding the IM germination proteins as well as the IM SpoVA channel proteins. This largely immobile IM lipid bilayer is the main permeability barrier for many small molecules, including toxic chemical preservatives, some of which exert their action in the spore core or vegetative cell cytoplasm5,6. The IM lipid bilayer is likely in a gel state, although there is a significant fraction of mobile lipids in the IM5. The spore&#8217;s IM also has the potential for significant expansion5. Thus, the surface area of the IM increases 1.6-fold upon germination without additional membrane synthesis and is accompanied by the loss of this membrane&#8217;s characteristic low permeability and lipid immobility5,6.
While the molecular details of the activation of germination proteins and organization of IM lipids in spores are attractive topics for study, the small size of B. subtilis spores and the relatively low abundance of germination proteins, pose a challenge to microscopic analyses. Griffiths et al. compelling epifluorescence microscope evidence, using fluorescent reporters fused to germination proteins, suggests that in B. subtilis spores the scaffold protein GerD organizes three GR subunits (A, B and C) for the GerA, B and K GRs, in a cluster7. They coined the term &#8216;germinosome&#8217; for this cluster of germination proteins and described the structures as ~300 nm large IM protein foci8. Upon initiation of spore germination, fluorescent germinosome foci ultimately change into larger disperse fluorescent patterns, with &#62;75% of spore populations displaying this pattern in spores germinated for 1 h with L-valine8. Note that the paper mentioned above used averaged images from dozens of consecutive fluorescent pictures, to gain statistical power and overcome the hurdle of low fluorescent signals observed during imaging. This visualization of these structures in bacterial spores was at the edge of what is technically feasible with classical microscopic tools and neither an evaluation of the amount of foci in a single spore nor their more detailed subcellular localization was possible with this approach.
Here, we demonstrate the use of Structured Illumination Microscopy (SIM) to obtain a detailed visualization and quantification of the germinosome(s) in spores of B. subtilis, as well as of their IM lipid domains9. The protocol also contains instructions for the sporulation, slide preparation and image analysis by SIMcheck (v1.0, an imageJ plugin) as well as ImageJ10,11,12.

<table>
	<tbody>
		<tr>
			<td>Air dried glass slides</td>
			<td>Menzel Gl&#228;ser</td>
			<td>630-2870</td>
			<td></td>
		</tr>
		<tr>
			<td>APO TIRF N20R8 100&#215; oil objective (NA=1.49)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B. subtilis KGB80 (PS4150 gerKA gerKC gerKB-mCherry cat, gerD-gfp kan)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B. subtilis PS4150 (PS832 &#916;gerE::spc, &#916;cotE::tet)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks 1 L</td>
			<td>Sigma-Aldrich</td>
			<td>Z567868</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks 250 mL</td>
			<td>Sigma-Aldrich</td>
			<td>Z723088</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoSpheres carboxylate-modified microspheres</td>
			<td>Invitrogen, 0.1 &#956;m</td>
			<td>F8803</td>
			<td></td>
		</tr>
		<tr>
			<td>FM4-64</td>
			<td>Thermo Fisher Scientific</td>
			<td>F34653</td>
			<td></td>
		</tr>
		<tr>
			<td>Histodenz nonionic density gradient medium</td>
			<td>Sigma-Aldrich</td>
			<td>D2158</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iXON3 DU-897 X-6515 CCD camera</td>
			<td>Andor Technology</td>
			<td></td>
			<td>https://imagej.net/Welcome</td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Sigma-Aldrich</td>
			<td>L2897</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge tubes 1.5 mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>3451PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope imaging software</td>
			<td>Nikon, Japan</td>
			<td>NIS-Element AR 4.51.01</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliQ Ultrapure Deminerilzed Water</td>
			<td>Millipore</td>
			<td>Milli-Q IQ 7003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Screw Cap Bottle 180 mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>75003800</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Coverslips</td>
			<td>Paul Marienfeld</td>
			<td>117650</td>
			<td></td>
		</tr>
		<tr>
			<td>Round Bottom tubes 15 mL</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Nunc TM</td>
		</tr>
		<tr>
			<td>Screw cap tubes 50 mL</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Nunc TM</td>
		</tr>
	</tbody>
</table>

visualization of germinosomes and the inner membrane in bacillus subtilis spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

The current protocol presents a SIM microscope imaging procedure for bacterial spores. The sporulation and slide preparation procedures were carried out as shown in Figure 1 before imaging. Later, the imaging and analysis procedures were applied both for dim (fluorescent protein labeled germination proteins) and bright (lipophilic probe stained IM) spore samples as shown in the following text.
<stro...

Watch this Scientific Journal Video about Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores at JoVE.com

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

visualization-germinosomes-inner-membrane-bacillus-subtilis

Research

JoVE Journal

Bioengineering

9.6K Views.  University of Amsterdam. Germinant receptor proteins cluster in ‘germinosomes’ in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

Video: Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane technology. Understanding the fouling process could lead to optimization and higher efficiency of membrane based filtration. Here we show the design and fabrication of an automated three-dimensionally (3-D) printed microfluidic cross-flow filtration system that can test up to 4 membranes in parallel. The microfluidic cells were printed using multi-material photopolymer 3-D printing technology, which used a transparent hard polymer for the microfluidic cell body and incorporated a thin rubber-like polymer layer, which prevents leakages during operation. The performance of ultrafiltration (UF), and nanofiltration (NF) membranes were tested and membrane fouling could be observed with a model foulant bovine serum albumin (BSA). Feed solutions containing BSA showed flux decline of the membrane. This protocol may be extended to measure fouling or biofouling with many other organic, inorganic or microbial containing solutions. The microfluidic design is especially advantageous for testing materials that are costly or only available in small quantities, for example polysaccharides, proteins, or lipids due to the small surface area of the membrane being tested. This modular system may also be easily expanded for high throughput testing of membranes.&#160;

Design and fabrication of a three-dimensionally (3-D) printed microfluidic cross-flow filtration system is demonstrated. The system is used to test performance and observe fouling of ultrafiltration and nanofiltration (thin film composite) membranes.

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Three-Dimensional Echocardiographic Method for the Visualization and Assessment of Specific Parameters of the Pulmonary Veins

The dimensions of the pulmonary veins are important parameters when planning pulmonary vein isolation (PVI), especially with the cryoballoon ablation technique. Acknowledging the dimensions and anatomical variations of the pulmonary veins (PVs) may improve the outcome of the intervention. Conventional 2D transoesophageal echocardiography can only provide limited data about the dimensions of the PVs; however, 3D echocardiography can further evaluate relevant diameters and areas of the PVs, as well as their spatial relationship to surrounding structures. In previous literature data, parameters influencing the success rate of PVI have already been identified. These are the left lateral ridge, the intervenous ridge, the ostial area of the PVs and the ovality index of the ostium. Proper imaging of the PVs by 3D echocardiography is a technically challenging method. One crucial step is the collection of images. Three individual transducer positions are necessary to visualize the important structures; these are the left lateral ridge, the ostium of the PVs and the intervenous ridge of the left and right PVs. Next, 3D images are acquired and saved as digital loops. These datasets are cropped, which result in the en face views displaying spatial relationships. This step can also be employed to determine the anatomical variations of the PVs. Finally, multiplanar reconstructions are created to measure each individual parameter of the PVs.
Optimal quality and orientation of the acquired images are paramount for the appropriate assessment of PV anatomy. In the present work, we examined the 3D visibility of the PVs and the suitability of the above method in 80 patients. The aim was to provide a detailed outline of the essential steps and potential pitfalls of PV visualization and assessment with 3D echocardiography.

The dimensions of the pulmonary veins (PV) are important parameters when planning pulmonary vein isolation. 2D transoesophageal echocardiography can only provide limited data about the PVs; however, 3D echocardiography can evaluate relevant diameters and areas of the PVs, as well as their spatial relationship to surrounding structures.

The dimensions of the pulmonary veins are important parameters when planning pulmonary vein isolation (PVI), especially with the cryoballoon ablation ...