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>

<ol><li>Yanagida, Y. <a target="_blank" href="https://ieeexplore.ieee.org/document/6411380/">A survey of olfactory displays: Making and delivering scents.</a> IEEE Sensors. , 1-4 (2012).</li>
<li>Yamada, T., Yokoyama, S., Tanikawa, T., Hirota, K., Hirose, M. <a target="_blank" href="http://ieeexplore.ieee.org/document/1667645/">Wearable Olfactory Display: Using Odor in Outdoor Environment.</a> IEEE Virtual Reality. , 199-206 (2006).</li>
<li>Kim, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+X-Y+addressablematrix+odor-releasing+system+using+an+on-off+switchable+device%5BTitle%5D)+AND+%22Angewandte+Chemie%22%5BJournal%5D)">An X-Y addressablematrix odor-releasing system using an on-off switchable device.</a> Angewandte Chemie. 123 (30), 6903-6907 (2011).</li>
<li>Amores, J., Maes, P. <a target="_blank" href="http://edithlaw.ca/cs889/2018/reading/Sensing/Paper8.pdf">Essence: olfactory interfaces for unconscious influence of mood and cognitive performance.</a> CHI Conference on Human Factors in Computing Systems. , 28-34 (2017).</li>
<li>Dobbelstein, D., Herrdum, S., Rukzio, E. <a target="_blank" href="https://www.semanticscholar.org/paper/inScent%3A-a-wearable-olfactory-display-as-an-for-Dobbelstein-Herrdum/49509328138f483de3510d5fbbbba101a09f2066">inScent: A Wearable Olfactory Display as an Amplification for Mobile Notifications.</a> International Symposium on Wearable Computers. , 28-34 (2017).</li>
<li>Hasegawa, K., Qiu, L., Shinoda, H. <a target="_blank" href="https://ieeexplore.ieee.org/stamp/stamp.jsp?arnumber=8260917">Midair Ultrasound Fragrance Rendering.</a> IEEE Transaction ON VISUALIZATION AND COMPUTER GRAPHICS. 24 (4), 1477-1485 (2018).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Essentials+of+machine+olfaction+and+taste%22">Essentials of machine olfaction and taste</a>. Nakamoto, T. , Wiley. 247-314 (2016).</li>
<li>Nakamoto, T., Yoshikawa, K. <a target="_blank" href="https://ieeexplore.ieee.org/document/1667665/">Movie with scents generated by olfactory display using solenoid valves.</a> Institute of Electronics, Information and Communication Engineers. E89-A (11), 3327-3332 (2006).</li>
<li>Nakamoto, T., Otaguro, S., Kinoshita, M., Nagahama, M., Ohnishi, K., Ishida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cooking+up+an+interactive+olfactory+game+display%5BTitle%5D)+AND+%22IEEE+Computer+Graphics+and+Application%22%5BJournal%5D)">Cooking up an interactive olfactory game display.</a> IEEE Computer Graphics and Application. 28 (1), 75-78 (2008).</li>
<li>Nakamoto, T., Minh, P. H. D. <a target="_blank" href="https://ieeexplore.ieee.org/document/4161021/">Improvement of olfactory display using solenoid valves.</a> Proceedings of IEEE Virtual Reality Annual International Symposium. , 171-178 (2007).</li>
<li>Hashimoto, K., Nakamoto, T. <a target="_blank" href="https://ieeexplore.ieee.org/document/7447720/">Tiny Olfactory Display Using Surface Acoustic Wave Device and Micropumps for Wearable Applications.</a> IEEE Sensors Journal. 16 (12), 4974-4980 (2016).</li>
<li>Beyssen, D., Brizoual, L. L., Elmazria, O., Alnot, P. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/S0925400506003212">Microfluidic device based on surface acoustic wave.</a> Sensors and Actuators B: Chemical. 118 (1-2), 380-385 (2006).</li>
<li>Wixforth, A., Strobl, C., Gauer, C., Toegl, A., Scriba, J., Guttenberg, Z. V. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/15257426">Acoustic manipulation of small droplets.</a> Analytical and Bioanalytical Chemistry. 379 (7-8), 982-991 (2004).</li>
<li>Heron, S. R., Wilson, R., Shaffer, S. A., Goodlett, D. R., Cooper, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface+Acoustic+Wave+Nebulization+of+Peptides+as+a+Microfluidic+Interface+for+Mass+Spectrometry%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Surface Acoustic Wave Nebulization of Peptides as a Microfluidic Interface for Mass Spectrometry.</a> Analytical Chemistry. 82 (10), 3985-3989 (2010).</li>
<li>Kurosawa, M., Watanabe, T., Higuchi, T. <a target="_blank" href="http://www.kurosawa.ip.titech.ac.jp/publications/international_conf/mems95mk.pdf">Surface acoustic wave atomizer with pumping effect.</a> IEEE Micro Electro Mechanical Systems (MEMS). , 25-30 (1999).</li>
<li>Shiokawa, S., Matsui, Y., Ueda, T. <a target="_blank" href="https://ieeexplore.ieee.org/document/67063/">Liquid streaming and droplet formation caused by leaky Rayleigh waves.</a> Ultrasonics Symposium:Proceedings of the IEEE. 1, 643-646 (1989).</li>
<li>Li, H., Qi, G., Kato, S., Nakamoto, T. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/S0925400518302697">Investigation and Improvement of Atomization Efficiency based on SAW Device Coated with Amorphous Fluoropolymer Film for Olfactory Display.</a> Sensors and Actuators B: Chemical. 263 (15), 266-273 (2018).</li>
<li>Kato, S., Nakamoto, T. <a target="_blank" href="https://www.jstage.jst.go.jp/article/ieejsmas/138/6/138_220/_article/-char/en">Control system of micro droplet using micro-dispenser based on solenoid valve and its application for highly efficient SAW atomizer.</a> IEEJ Transactions on Sensors and Micromachines. 138 (6), 220-225 (2018).</li>
<li>Itou, S., Iseki, M., Kato, S., Nakamoto, T. <a target="_blank" href="https://dl.acm.org/citation.cfm?doid=3180308.3180331">Olfactory and visual presentation using olfactory display using SAW atomizer and solenoid valves.</a> Proceedings of the 2018 Conference on Intelligent User Interface. , (2018).</li>
<li>Nakamoto, T., Ito, S., Kato, S., Qi, G. <a target="_blank" href="https://ieeexplore.ieee.org/document/8357566/">Multicomponent olfactory display using solenoid valves and SAW atomizer and its blending-capability evaluation.</a> IEEE Sensors Journal. , (2018).</li>
<li>Kato, S., Nakamoto, T. Olfactory Display Based on Sniffing Action. IEEE Conference on Virtual Reality. , (2018).</li>
</ol>

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><tbody><tr><td>SAW device</td><td>Lightom</td><td></td><td>Custom-made</td></tr><tr><td>Network analyzer</td><td>SDR-kits</td><td>DG84AQ VNWA 3E</td><td></td></tr><tr><td>Dip coater</td><td>Aiden</td><td>DC4300</td><td></td></tr><tr><td>Silane coupling agent</td><td>Shin-etsu Chemical</td><td>KBE 903</td><td></td></tr><tr><td>Cytop amorphous teflon coating</td><td>Asahi glass</td><td>CT107MK</td><td></td></tr><tr><td>Solvent for diluting cytop coating</td><td>Asahi glass</td><td>CT-SOLV100K</td><td></td></tr><tr><td>Solenoid valve</td><td>Lee</td><td>INKA2438510H</td><td></td></tr><tr><td>Transistor array</td><td>Texas Instrument</td><td>ULN2803A</td><td></td></tr><tr><td>RF power amplifier</td><td>Mini-Circuits</td><td>ZHL-5W-1</td><td></td></tr><tr><td>Digital camera&amp;nbsp;</td><td>Panasonic Corp</td><td>DMC-FZ300</td><td></td></tr><tr><td>Head Mount Display</td><td>Occulus</td><td>Occulus Rift Headset</td><td></td></tr><tr><td>Hot plate</td><td>As One</td><td>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/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/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/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/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/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.

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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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8.1K Views.  Tokyo Institute of Technology. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in de menselijke interface veld. Bijvoorbeeld, veel met betrekking tot olfactorische virtual reality. In ons systeem, een micro-dispenser jets een paar nano-liter druppels op een oppervlak.Dan verdeelt een oppervlakte akoestische golf, vernevelt die druppel om snel een geur voor te stellen. Visuele demonstratie van deze methode is van cruciaal belang om het optimalisatiegedrag weer te geven. Bereid het oppervlak akoest...

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

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

Neuroscience

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Olfactory Context Dependent Memory: Direct Presentation of Odorants

Information is retrieved more effectively when the retrieval occurs in the same context as that in which the information was first encoded. This is termed context dependent memory (CDM). One category of cues that have been shown to effectively produce CDM effects are odors. However, it is unclear what the boundary conditions of these CDM effects are. In particular, given that olfaction has been called an implicit sense, it is possible that odors are only effective mnemonic cues when they are presented in the background. This assertion seems even more likely given that previous research has shown odors to be poor cues during paired associate memory tests, where odors are in the focus of attention as mnemonic cues for other information. In order to determine whether odors are only effective contextual mnemonic cues when presented outside the central focus of an observer, an olfactory CDM experiment was performed in which odorants were presented directly, rather than ambiently. Direct presentation was accomplished with the aid of an olfactometer. The olfactometer not only allows for direct presentation of odorants, but provides other methodological benefits, including the allowance of trial by trial manipulations of odorant presentations and, relatedly, time-specific releases of odorants. The presence of the same odor during both encoding and retrieval enhanced memory performance, regardless of whether the odor was presented ambiently or directly. This finding can serve as a basis for future olfactory CDM research which can utilize the benefits of direct presentation.

The use of an olfactometer for directly presenting odorants opens exciting opportunities for researchers of olfactory memory. The current paper discusses issues related to this methodology as related to a previously published experiment on olfactory context dependent memory.

Information is retrieved more effectively when the retrieval occurs in the same context as that in which the information was first encoded. This is termed ...

Behavior

Vertical T-maze Choice Assay for Arthropod Response to Odorants

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, various technologies are being developed as control tools. A subset of these tools focuses on modifying the behavior of arthropods by attraction or repulsion. Therefore, arthropods are often the focus of behavioral investigations. Various tools have been developed to measure arthropod behavior, including wind tunnels, flight mills, servospheres, and various types of olfactometers. The purpose of these tools is to measure insect or arachnid response to visual or more often olfactory cues. The vertical T-maze oflactometer described here measures choices performed by insects in response to attractants or repellents. It is a high throughput assay device that takes advantage of the positive phototaxis (attraction to light) and negative geotaxis (tendency to walk or fly upward) exhibited by many arthropods. The olfactometer consists of a 30 cm glass tube that is divided in half with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the test subjects to make a choice between two potential odor fields in assays involving attractants. In assays involving repellents, lack of normal response to known attractants can also be measured as a third variable.

A vertical, T-maze olfactometer is described for assaying the behavioral response of arthropods. The olfactometer allows the experimenter to measure choices performed by test subjects when subjected to two potential odor fields. Both attraction to and repulsion from odorants can be measured with this device.

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, ...

Fabrication and Characterization of Thickness Mode Piezoelectric Devices for Atomization and Acoustofluidics

We present a technique to fabricate simple thickness mode piezoelectric devices using lithium niobate (LN). Such devices have been shown to atomize liquid more efficiently, in terms of &#64258;ow rate per power input, than those that rely on Rayleigh waves and other modes of vibration in LN or lead zirconate titanate (PZT). The complete device is composed of a transducer, a transducer holder, and a &#64258;uid supply system. The fundamentals of acoustic liquid atomization are not well known, so techniques to characterize the devices and to study the phenomena are also described. Laser Doppler vibrometry (LDV) provides vibration information essential in comparing acoustic transducers and, in this case, indicates whether a device will perform well in thickness vibration. It can also be used to &#64257;nd the resonance frequency of the device, though this information is obtained more quickly via impedance analysis. Continuous &#64258;uid atomization, as an example application, requires careful &#64258;uid flow control, and we present such a method with high-speed imaging and droplet size distribution measurements via laser scattering.

Fabrication of piezoelectric thickness mode transducers via direct current sputtering of plate electrodes on lithium niobate is described. Additionally, reliable operation is achieved with a transducer holder and &#64258;uid supply system and characterization is demonstrated via impedance analysis, laser doppler vibrometry, high-speed imaging, and droplet size distribution using laser scattering.

We present a technique to fabricate simple thickness mode piezoelectric devices using lithium niobate (LN). Such devices have been shown to atomize liquid ...

Engineering

Designing a Flexible, Cost-Effective Olfactometer for Rodent Olfactory Experiments

Constructing an Olfactometer for Rodent Olfactory Behavior Studies

The use of olfactometers to study rodent behavior and brain activity during olfactory tasks is crucial for understanding brain circuits. These sophisticated devices allow researchers to precisely control and deliver odor stimuli, enabling the investigation of complex olfactory processes in rodents. Although commercially available olfactometers are convenient, they present challenges when technical issues arise, often requiring costly assistance and potentially disrupting research timelines. This article details the construction of a custom olfactometer specifically designed for mouse olfactory behavior experiments, providing a comprehensive list of parts and step-by-step instructions. The olfactometer is controlled through MATLAB, offering a user-friendly interface for researchers. Importantly, the open-source code allows users to modify and adapt the system, tailoring behavioral tasks to meet specific experimental needs. Building a customized olfactometer empowers users with the knowledge and capability to perform custom experimental design and troubleshooting independently, saving both time and resources. This approach not only enhances research flexibility but also fosters a deeper understanding of the equipment's functionality, ultimately leading to more robust and reliable olfactory studies in rodents.

Constructing an Olfactometer for Rodent Olfactory Behavior Studies Near-Infrared Spectroscopy Hyperscanning Study in Psychological Counseling

This protocol describes the construction of an olfactometer for go/no-go olfactory behavior experiments. Step-by-step instructions, along with images, are provided to ensure the successful construction of the olfactometer. Information for troubleshooting issues encountered during the process is also included.

The use of olfactometers to study rodent behavior and brain activity during olfactory tasks is crucial for understanding brain circuits. These ...

Modeling and Simulations of Olfactory Drug Delivery with Passive and Active Controls of Nasally Inhaled Pharmaceutical Aerosols

There are many advantages of direct nose-to-brain drug delivery in the treatment of neurological disorders. However, its application is limited by the extremely low delivery efficiency (&#60; 1%) to the olfactory mucosa that directly connects the brain. It is crucial to develop novel techniques to deliver neurological medications more effectively to the olfactory region. The objective of this study is to develop a numerical platform to simulate and improve intranasal olfactory drug delivery. A coupled image-CFD method was presented that synthetized the image-based model development, quality meshing, fluid simulation, and magnetic particle tracking. With this method, performances of three intranasal delivery protocols were numerically assessed and compared. Influences of breathing maneuvers, magnet layout, magnetic field strength, drug release position, and particle size on the olfactory dosage were also numerically studied.
From the simulations, we found that clinically significant olfactory dosage (up to 45%) were feasible using the combination of magnet layout and selective drug release. A 64 -fold higher delivery of dosage was predicted in the case with magnetophoretic guidance compared to the case without it. However, precise guidance of nasally inhaled aerosols to the olfactory region remains challenging due to the unstable nature of magnetophoresis, as well as the high sensitivity of olfactory dosage to patient-, device-, and particle-related factors.

This manuscript reviews the modeling and simulations of different protocols to deliver medications to the olfactory region in image-based nasal airway models. Multiple software modules are used to develop the anatomically accurate nose model, generate computational mesh, simulate nasal airflows, and predict particle deposition at the olfactory region.

There are many advantages of direct nose-to-brain drug delivery in the treatment of neurological disorders. However, its application is limited by the ...

Medicine

A Free-breathing fMRI Method to Study Human Olfactory Function

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, evaluation of the functions of the human central olfactory system with functional magnetic resonance imaging (fMRI) is still a challenge because of several technical difficulties. There are some significant variables to take into account when considering an effective method for mapping the function of the central olfactory system using fMRI, including proper odorant selection, the interaction between odor presentation and respiration, and potential anticipation of or habituation to odorants. An event-related, respiration-triggered olfactory fMRI technique can accurately administer odorants to stimulate the olfactory system while minimizing potential interference. It can effectively capture the precise onsets of fMRI signals in the primary olfactory cortex using our data post-processing method. The technique presented here provides an efficient and practical means for generating reliable olfactory fMRI results. Such a technique can ultimately be applied in the clinical realm as a diagnostic tool for diseases associated with olfactory degeneration, including Alzheimer&#39;s and Parkinson&#39;s disease, as we begin to further understand the complexities of the human olfactory system.

We present the technical challenges and solutions for obtaining reliable functional magnetic resonance imaging (fMRI) data from the human central olfactory system. This includes special considerations in olfactory fMRI paradigm design, descriptions of fMRI data acquisition with an MRI-compatible olfactometer, odorant selection, and a special software tool for data post-processing.

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, ...

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

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

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