This study was partially supported by JST Mirai program, Grant Number JPMJMI17DD.

The methods described here have been approved by the Human Research Ethics Committee of Tokyo Institute of Technology.
1. SAW Device Preparation and Checking Impedance
<ol>
	<li>Prepare a 10 MHz SAW device on a LiNbO3 substrate [128o-rotated Y-cut, X propagation, with 21 finger pairs of an IDT (inter-digitated transducer)], together with reflectors made of 32 finger pairs on one IDT side, as shown in Figure 1a. 
	Note: Figure 1b shows the principle of atomization. The SAW is converted to a longitudinal wave at the liquid droplet. Mist is generated due to acoustic streaming if the SAW energy is sufficiently large. 
	Note: The SAW device was fabricated by the manufacturer using typical photo lithography according to the electrode pattern that the authors designed. The piezoelectric substrate mentioned above was selected due to a high electromechanical coupling coefficient.</li>
	<li>Mount the SAW device using aluminum foil and conductive paste on a printed circuit board made of aluminum (designed for this SAW device) where the SMA connector is attached (Figure 2). 
	Note: A printed circuit board made of aluminum is effective for heat radiation.</li>
	<li>Measure the frequency characteristic of impedance using a network analyzer. The SAW device is connected to the analyzer via a coaxial cable connection from the printed circuit board. Frequency characteristics should show frequencies where imaginary parts of the device admittance go to zero, which are the SAW device resonance frequencies. 
	Note: When acoustic loss in the device is large, atomization does not occur. The acoustic loss can be checked by measuring the frequency characteristic of impedance. When the SAW device is coated with Teflon film, the difference between the frequency characteristics before and after coating should be monitored to check if the film is too thick.</li>
</ol>
2. Silanization
<ol>
	<li>Prepare the amino-based silane coupling agent (3-ahhminopropyltriethoxy silane). Adjust its concentration to 0.5 % (v/v) in water using a pipette. 
	Note: Silanization is mandatory to enhance the adhesion of amorphous Teflon coating. Teflon coating is removed during atomization if silanization is not performed.</li>
	<li>Clean the surface of the SAW device using a cotton swab soaked with acetone.</li>
	<li>Set the device at a dip coater (Figure 3). 
	Note: A printed circuit board where the SAW device is fixed using mending tape is attached to the dip coater, since the thickness of the SAW device (0.5 mm) is too thin to be attached to the dip coater directly.</li>
	<li>Pull down the device so that the atomization area can be immersed in the solution at a speed of 0.2 mm/s. Keep the device in solution for 5 min.</li>
	<li>Pull up the device at a speed of 1.7 mm/s. Keep the device in the air for 5 min.</li>
	<li>Rinse the device in pure water for 1 min.</li>
	<li>Keep the device in the air for 30 min.</li>
</ol>
3. Amorphous Teflon Coating
<ol>
	<li>Prepare the amorphous Teflon material and solvent for dilution. Adjust the concentration of the amorphous Teflon solution to 3% (v/v) using the solvent.</li>
	<li>Set the device at a dip coater (Figure 3). 
	Note: Dip coating was adopted here since the uniform coating is indispensable. Rough coating as well as coating that is too thick can cause deterioration of the atomization efficiency due to SAW attenuation.</li>
	<li>Pull down the device so that atomization area is immersed in the solution at a speed of 0.2 mm/s. Keep the device in solution for 15 s.</li>
	<li>Pull up the device at the speed of 1.7 mm/s. Keep the device in the air for 5 min.</li>
	<li>Pull down the device so that atomization area is immersed in the solution at a speed of 0.2 mm/s. Keep the device in solution for 15 s.</li>
	<li>Pull up the device at the speed of 1.7 mm/s. Keep the device in the air for 30 min.</li>
	<li>Bake the device at 180 &#176;C for 60 min using a hot plate. 
	Note: Thickness of the coating was approximately 400 nm according to quartz crystal microbalance (QCM) measurement.</li>
</ol>
4. Experimental Set-up for Atomization
<ol>
	<li>Mount the SAW device on a printed circuit board. 
	Note: Since the thickness of the SAW device is 0.5 mm, it can be easily broken. Thus, it is necessary to support it mechanically.</li>
	<li>Measure the frequency characteristic of the SAW device impedance using a network analyzer. The SAW device is connected to the analyzer via a coaxial cable connection of the printed circuit board. Frequency characteristics should show frequencies where imaginary parts of the device admittance go to zero, which are the SAW device resonance frequencies 
	Note: Check the RF attenuation at the SAW device. The loss of the SAW device increases when it is inappropriately coated. This increase in loss typically occurs due to coating inhomogeneity or excessive thickness of coating; thus, the impedance characteristics before and after coating should be compared. The atomization cannot be performed if the SAW attenuation is too much.</li>
	<li>Connect the SAW device to a function generator through an RF power amplifier.</li>
	<li>Set the waveform of the RF burst signal at a function generator (Figure 4a). The burst signal for the SAW device should be a sine wave, and its duty cycle should be 10%. The wave frequency should also be set to the SAW device resonance frequency obtained from the impedance characteristic measurements.</li>
	<li>Connect a burst square wave generator to a solenoid valve [i.e., micro-dispenser through a driving circuit (Figure 5)] so that a 24 V pulse signal can be supplied to the dispenser, which is also mentioned in the discussion18,19. 
	Tip: For driving solenoid valves, a transistor array is convenient. Up to eight solenoid valves can be driven using the transistor array in this study.</li>
	<li>Set the micro-pump to apply pressure to flow the liquid to the micro-dispenser (Figure 5). The micro-pump supports the self-priming capability of the micro-dispenser20.</li>
	<li>Use an infrared thermometer to measure the temperature of the SAW device, if necessary. 
	Note: The temperature at the SAW device surface typically reaches around 45 &#176;C when the RF burst signal (85 Vp-p and 10% duty cycle) is applied for 5 min.</li>
</ol>
5. Atomization
<ol>
	<li>Put the liquid (i.e., fragrance or chemical diluted with ethanol) into a vial.</li>
	<li>Set the waveform of the pulse signal applied to a micro-dispenser (Figure 4b). The pulse signal is a square wave pulse sequence with a 10% duty cycle and is generated with a function generator.</li>
	<li>Apply the pulse signal to the micro-dispenser to jet a liquid droplet to the SAW device18. Since a single droplet from the micro-dispenser is only a few nanoliters, a pulse sequence is needed to form a larger droplet for atomization.</li>
	<li>Apply the RF burst signal to the SAW device to atomize liquid droplets17. The burst signal is applied from a function generator through an amplifier after formation of the liquid droplets. The signal should be applied for as long as vapor is still generated from the atomization process. 
	Note: The RF burst signal is used to adjust the average RF power. The SAW device may have a crack if the RF power is much larger than 2 W.</li>
	<li>Observe the surface of SAW device to inspect the remaining liquid droplet.</li>
	<li>Perform the same procedure as done in steps 4.1-4.7 and 5.1-5.5 for a bare SAW device. Then, compare the amount of remaining liquid droplet on the coated substrate with that from bare one.</li>
</ol>
6. Detecting Scents
<ol>
	<li>Put the liquid into a vial as done in step 5.1.</li>
	<li>Adjust the height of the SAW atomizer using a jack so that its height remains equal to the participant&#8217;s nose.</li>
	<li>Dispense the liquid onto the SAW device.</li>
	<li>Turn on the fan.</li>
	<li>Allow the participant to detect the scent. 
	Note: The authors did the sensory test instead of VOC analyzer used in the previous work since the perceived intensity rather than vapor concentration should be evaluated.</li>
</ol>

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The authors have nothing to disclose.

One of the key components in this study is the micro-dispenser made of a high-speed solenoid valve18,19. Figure 8a shows the principle of this micro-dispenser. The plunger was driven by an electromagnetic coil. Its outlet is completely closed by the plunger during the OFF phase. The plunger rapidly moves to draw liquid in front during a short ON phase, then it moves back to the original location and jets a tiny liquid droplet from an orifice of the solenoid valve, which is driven by the circuit shown in Figure 8b. The amount of one liquid droplet is a few nanoliters. The frequency of the valve is between 1 and 1000 Hz, its minimum pulse width is 0.5 ms, and it works much faster than a typical solenoid valve. The typical distance between the orifice of the solenoid valve and the substrate was 15 mm. This study showed that the amount of liquid is precise and reproducible; moreover, it is robust against bubbles.
Smell persistence can be drastically reduced owing to amorphous Teflon coatingwhen an olfactory display based upon a SAW atomizer is used21. It can be further improved when a channel dedicated to deliver solvents to the substrate surface for cleaning is used.
The critical step in protocol is manually adjusting the excitation frequency of the atomizer when it deviates from the optimal one. This should be automatically performed in the future. A modification from the initial protocol was to include the silanization process since Teflon coating itself without silanization was atomized.
There are two remaining issues which limit this technique, one being the standing wave problem. The standing wave is generated when reflection occurs at the edge of the substrate. Since antinode and node appears periodically, atomization becomes weak at the node. Although we use a silicone gel to suppress the standing wave, this is not sufficient. A better material to absorb the acoustic energy is necessary.
The second limitation is the durability of the Teflon coating. The Teflon coating is partially removed after atomizing a liquid many times. Since the current condition for coating has not been studied extensively, the authors can optimize it to extend the durability of Teflon coating.
Nevertheless, the significance of the protocol with respect to existing methods is the reduction of remaining liquid after atomization on a surface with coating compared to without coating. Thus, the smell persistence is drastically reduced as is described elsewhere17. Using this SAW device, demonstration of the olfactory display was performed. The eight-component olfactory display to demonstrate cassis, orange, whiskey, and their mixture were presented to a user with Head Mount Display (Figure 9)19. In this situation, a SAW device with the proposed coating works well to suppress smell persistence, which otherwise can considerably deteriorate the quality of scent presentation.
The technique described here is important for olfactory display. Moreover, the SAW atomizer is applicable to a nebulizer for medical use and electrospray ionization for mass spectrometry. The atomization efficiency is also required in those applications.

Although devices for stimulating visual and auditory senses are popular, we cannot present all sensations that we perceive; though, we can ordinarily present a sensation using only these two senses. An olfactory display is a gadget that can present a scent, and it is used in virtual reality so that a user can perceive scents1,2,3,4,5,6,7. Since olfaction contributes to emotions greatly, an olfactory stimulus is indispensable for enhancing reality. We have previously studied movies, animations, and games with scents8,9.
Several researchers have studied olfactory displays; for example, Yanagida has studied a scent projector that delivers a scent to a specified person even when no one around him or her perceives it1. Yamada et al. have studied an odor source localization in virtual space using a simple Gaussian distribution model of odor concentration2. Kim et al. have proposed the concept of two-dimensional arrays of odor-releasing devices 3. Moreover, simple wearable olfactory displays and the ultrasonic phased array for controlling the direction of these scents have been proposed4,5,6.
One of the problems in olfactory display is smell persistence. A user may detect the smell even after it is intended to be changed to the air or another scent. Since it is preferable to switch between scents as quickly as possible in virtual reality, the smell persistence problem should be studied.
We have studied the olfactory display with a function of blending many ingredients. We previously developed this system using solenoid valves with high-speed switching10. Although it stably blends many ingredients, we could not yet solve the problem of smell persistence. Thus, we have since developed the olfactory display using micro-dispensers and a SAW atomizer11. Although similar techniques have been used to manipulate liquid droplets12,13,14, we applied it to scent generation. The SAW device is suitable for atomizing liquid droplets since it can atomize liquid droplets instantaneously15,16; however, we have found that tiny liquid droplets stay on a piezoelectric substrate after the atomization. These tiny liquid droplets cause smell persistence, even if most of the liquid is atomized.
Typically, a perfume is dissolved into a solvent such as ethanol to reduce viscosity. However, diluted perfume spreads onto the surface of a piezoelectric substrate due to its hydrophilic nature, and the atomization efficiency deteriorates when thin film spreads. Thus, a part of the liquid remains even after atomization, which cannot be removed even if RF power increases. Since the solvent evaporates soon after, only the perfume remains on and sticks to the substrate.
In this study, we coat the surface of a piezoelectric substrate with thin amorphous Teflon film so it becomes hydrophobic in nature. Since we can keep the droplet-like sphere on the hydrophobic surface, the energy required for detaching liquid from the substrate surface decreases. It is expected that an atomization efficiency is improved when the surface of SAW device becomes hydrophobic. The overall goal of this method is to improve the atomization efficiency so that a scent is immediately presented and can quickly disappear after its presentation, ultimately for application to olfactory display. In this paper, we show how a SAW device is coated with amorphous Teflon film and demonstrate the improvement of atomization efficiency and its experimental results were described in the reference17.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SAW device</td>
			<td style="width:71px;">Lightom</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Custom-made</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Network analyzer</td>
			<td style="width:71px;">SDR-kits</td>
			<td style="width:119px;">DG84AQ VNWA 3E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dip coater</td>
			<td style="width:71px;">Aiden</td>
			<td style="width:119px;">DC4300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Silane coupling agent</td>
			<td style="width:71px;">Shin-etsu Chemical</td>
			<td style="width:119px;">KBE 903</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytop amorphous teflon coating</td>
			<td style="width:71px;">Asahi glass</td>
			<td style="width:119px;">CT107MK</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Solvent for diluting cytop coating</td>
			<td style="width:71px;">Asahi glass</td>
			<td style="width:119px;">CT-SOLV100K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Solenoid valve</td>
			<td style="width:71px;">Lee</td>
			<td style="width:119px;">INKA2438510H</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Transistor array</td>
			<td style="width:71px;">Texas Instrument</td>
			<td style="width:119px;">ULN2803A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RF power amplifier</td>
			<td style="width:71px;">Mini-Circuits</td>
			<td style="width:119px;">ZHL-5W-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Digital camera&#160;</td>
			<td style="width:71px;">Panasonic Corp</td>
			<td style="width:119px;">DMC-FZ300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Head Mount Display</td>
			<td style="width:71px;">Occulus</td>
			<td style="width:119px;">Occulus Rift Headset</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hot plate</td>
			<td style="width:71px;">As One</td>
			<td style="width:119px;">HHP-170A</td>
			<td></td>
		</tr>
	</tbody>
</table>

enhancement method of surface acoustic wave-atomizer efficiency for olfactory display

Since olfaction is an important sense in human interfaces, we have developed an olfactory display using a surface acoustic wave (SAW) atomizer and micro-dispensers. In this olfactory display, the efficiency of atomization is important in order to avoid smell persistence problems often encountered in human olfactory interfaces. Thus, the SAW device is coated with amorphous Teflon film to change the substrate nature from hydrophilic to hydrophobic. It is also necessary to silanize the piezoelectric substrate surface prior to Teflon coating to enhance the adhesion of the film. A dip coating method was adopted to obtain uniform coating on the substrate. The high-speed solenoid valve was used as micro-dispenser to spout a liquid droplet to the SAW device surface since its accuracy and reproducibility were high. Then, the atomization became easier on the hydrophobic substrate. In this study, the amorphous Teflon coating for minimizing the remaining liquid on the substrate after atomization was studied. The goal of the protocol described here is to show the methods for coating a SAW device surface with amorphous Teflon film and generating the smell using the SAW atomizer and a micro-dispenser, followed by a sensory test.

One microliter of ethanol was placed on both bare and coated LiNbO3 substrates (ethanol was typically used as a solvent for perfume). A thin film of ethanol solution was formed after it spread onto the substrate (Figure 6a); on the other hand, the sphere-like shape was kept on the coated substrate (Figure 6b). The contact angle of the one microliter of water increased from 50 to 110 degrees after amorphous Teflon coating (Figure 6c and 6d). It was found that the amorphous Teflon coating enhanced the hydrophobic nature. The sphere-like shape of the droplet was kept on the coated substrate, whereas the liquid spread into a thin film on the bare substrate.
Next, the experiment with atomization of 200 nL of lavender was performed (Figure 7). The subsequent images of atomization without and with coating are shown in Figure 7a and 7b, respectively. Timescale in the photos was obtained from the number of frames recorded by a digital camera. Lavender was diluted with ethanol (dilution ratio: 50:1 v/v). On the bare substrate, the liquid spread immediately after it was dispensed. At 33 ms, strong atomization occurred at the center of liquid, whereas only limited mist was generated at the liquid edge within the circle as shown in Figure 7a. At 100 ms, atomization stopped; so, although atomization occurred at first, it stopped soon after. Then, part of the liquid remained. While the solvent evaporated rapidly, partial solute remained on the substrate surface; thus, the remaining solute caused the serious smell persistence. On the other hand, the sphere-like shape with its contact angle of more than 90 degrees was maintained on the coated substrate after it was dispensed (Figure 7b). A concentrated mist was generated during atomization. After atomization, much less liquid in a smaller area was left in the smaller area compared to the bare substrate. Since the remaining liquid was not a smooth and complete surface but instead formed small single droplets, it is difficult to accurately calculate the coverage of droplets on Teflon coating. Roughly speaking, the remaining liquid on the hydrophobic surface was at most 10% of that on the hydrophilic surface.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58409/58409fig1.jpg" /> 
Figure 1: SAW atomizer. (a) Configuration of the SAW device and (b) principle of the SAW atomizer. Its electrode consists of gold and chromium. Figure 1a is reprinted with permission20. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58409/58409fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58409/58409fig2.jpg" /> 
Figure 2: SAW device on a printed circuit board.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58409/58409fig3.jpg" /> 
Figure 3: Dip coater used in this study.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58409/58409fig4.jpg" /> 
Figure 4: Time sequences. (a) Waveform of the RF burst signal. The typical values of Vpp and Tr are 85 Vp-p and 1 s, respectively. A typical duty cycle such as TH/Tr is 10%. (b) Waveform applied to a micro-dispenser. The typical Tw, T, and N are 1 ms, 10 ms, and 70 pulses, respectively.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58409/58409fig5.jpg" /> 
Figure 5: Experimental setup for atomizing liquid droplet. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58409/58409fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58409/58409fig6.jpg" /> 
Figure 6: Comparison of liquid droplet shape between bare and coated subtrates. Shown are a (a) top view of the thin film on the bare lithium niobate surface and (b) side view of the liquid droplet on the coated one. One microliter of ethanol was used in both (a) and (b). Here, (c) and (d) show side views of one microliter of water on the bare and coated substrates, respectively. This figure was reproduced with permission17. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58409/58409fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58409/58409fig7.jpg" /> 
Figure 7: Atomization of the liquid droplet. Shown are the (a) hydrophilic surface (bare lithium niobate) and (b) hydrophobic surface (substrate coated with amorphous Teflon). The sample is 200 nL of lavender. This figure was reproduced with permission17. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58409/58409fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58409/58409fig8.jpg" /> 
Figure 8: Micro-dispenser based upon a solenoid valve. Shown are (a) the micro-dispenser&#39;s principle and (b) a driver circuit for a single channel. This figure is reprinted with permission20. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58409/58409fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/58409/58409fig9.jpg" /> 
Figure 9: Application of the SAW atomizer coated with amorphous Teflon film. Research demonstration from the Intelligent User Interfaces (IUI) Symposium 2018 in Tokyo, Japan.

Watch this Scientific Journal Video about Enhancement Method of Surface Acoustic Wave-Atomizer Efficiency for Olfactory Display at JoVE.com

Enhancement Method of Surface Acoustic Wave-Atomizer Efficiency for Olfactory Display

We establish here a method for coating the surface of a surface acoustic wave (SAW) device with amorphous Teflon film to improve the atomization efficiency required for application to an olfactory display.

Since olfaction is an important sense in human interfaces, we have developed an olfactory display using a surface acoustic wave (SAW) atomizer and ...

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Research

JoVE Journal

Immunology and Infection

8.0K Views.  Tokyo Institute of Technology. We establish here a method for coating the surface of a surface acoustic wave (SAW) device with amorphous Teflon film to improve the atomization efficiency required for application to an olfactory display.

Video: Enhancement Method of Surface Acoustic Wave-Atomizer Efficiency for Olfactory Display

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular localization can provide insights into the functional role of bacterial proteins. Surface localization of bacterial proteins is a key step towards identification of virulence factors involved in mechanisms of pathogenicity. 
Methods for fractionating leptospiral membranes 2-5 may be selective for a certain class of outer-membrane proteins (OMPs), such as lipoproteins vs. transmembrane OMPs, and therefore lead to misclassification. This likely is due to structural differences and how they are associated to the outer membrane. Lipoproteins are associated with membranes via a hydrophobic interaction between the N-terminal lipid moiety (three fatty acids) and the lipid bilayer phospholipids 6, 7. In contrast, transmembrane OMPs are typically integrated into the lipid bilayer by amphipathic &beta;-sheets arranged in a barrel-like structure 8, 9. In addition, presence of a protein in the outer-membrane does not necessarily guarantee that the protein or its domains are exposed on the surface. Spirochetal outer membranes are known to be fragile and therefore necessitate methods involving gentle manipulation of cells and inclusion of sub-surface protein controls to assess the integrity of the outer membrane.
Here, we present an immunofluorescence assay (IFA) method to directly assess surface exposure of proteins on intact leptospires. This method is based on recognition of leptospiral surface proteins by antigen-specific antibodies. Herein, antibodies specific for OmpL5410 are detetcted aftero binding to native, surface exposed epitopes. Comparison of antibody reactivity to intact versus permeabilized cells enables evaluation of cellular distribution and whether or not a protein is selectively present on leptospiral surface. The integrity of outer membrane should be assessed using antibody to one or more subsurface proteins, preferably located in the periplasm.
The surface IFA method can be used to analyze surface exposure of any leptospiral protein to which specific antibodies are available. Both the usefulness and limitation of the method depends on whether the antibodies employed are able to bind to native epitopes. Since antibodies often are raised against recombinant proteins, epitopes of native, surface-exposed proteins may not be recognized. Nevertheless, the surface IFA method is a valuable tool for studying components of intact bacterial surfaces. This method can be applied not only for leptospires but also other spirochetes and gram-negative bacteria. For stronger conclusions regarding surface-exposure of OMPs, a comprehensive approach involving several cell localization methods is recommended 10.

An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

Particle Agglutination Method for Poliovirus Identification

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute flaccid paralysis (AFP) cases. In the World Health Organization (WHO) Global Polio Laboratory Network, PV isolation and identification are currently being performed by using cell culture system and real-time RT-PCR, respectively. In the post-eradication era of PV, simple and rapid identification procedures would be helpful for rapid confirmation of polio cases at the national laboratories. In the present study, we will show the procedure of novel PA assay developed for PV identification. This PA assay utilizes interaction of PV receptor (PVR) molecule and virion that is specific and uniform affinity to all the serotypes of PV. The procedure is simple (one step procedure in reaction plates) and rapid (results can be obtained within 2 h of reaction), and the result is visually observed (observation of agglutination of gelatin particles).

A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

A Method for Targeted 16S Sequencing of Human Milk Samples

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as with all these technologies, reproducible results depend on a laboratory workflow that incorporates appropriate precautions and controls. This is particularly important with low-biomass samples where contaminating bacterial DNA can generate misleading results. This article details a semi-automated workflow to identify microbes from human breast milk samples using targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region on a low- to mid-throughput scale. The protocol describes sample preparation from whole milk including: sample lysis, nucleic acid extraction, amplification of the V4 region of the 16S rRNA gene, and library preparation with quality control measures. Importantly, the protocol and discussion consider issues that are salient to the preparation and analysis of low-biomass samples including appropriate positive and negative controls, PCR inhibitor removal, sample contamination by environmental, reagent, or experimental sources, and experimental best practices designed to ensure reproducibility. While the protocol as described is specific to human milk samples, it is adaptable to numerous low- and high-biomass sample types, including samples collected on swabs, frozen neat, or stabilized in a preservation buffer.

A semi-automated workflow is presented for targeted sequencing of 16S rRNA from human milk and other low-biomass sample types.

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...