Methods for building an olfactometer with known concentration outcomes

Journal of Neuroscience Methods 160 (2007) 231–245

Methods for building an olfactometer with known concentration outcomes Bradley N. Johnson a,∗ , Noam Sobel b a

Helen Wills Neuroscience Institute at UC Berkeley, UC Berkeley/UC San Francisco Joint Graduate Group in Bioengineering, USA b Department of Neurobiology, Weizmann Institute of Science, Rehovot 7600, Israel Received 21 July 2006; received in revised form 11 September 2006; accepted 12 September 2006

Abstract We provide detailed instructions and part selections for construction of a five-channel air dilution olfactometer capable of generating neat odorants and binary mixtures at a range of known concentrations. At the heart of the olfactometer is an odorant canister that is (1) cheap and readily available, (2) safe and durable, (3) has minimal odor adherence, (4) is easily incorporated into any olfactometer, and critically (5) produces a highly consistent stimulus. By flowing a given carrier gas at a given flowrate through a given odorant in this canister, the same end-vapor is achieved. Flow/concentration outcomes are provided for several odorants routinely used in olfactometry. This tool will enable researchers to generate known concentrations without expensive analytical machinery. © 2006 Elsevier B.V. All rights reserved. Keywords: Olfactometer; Psychophysics; fMRI; Olfaction; Methods

1. Introduction Several theoretical design features of olfactometry have been developed and published (Benignus and Prah, 1980; deWijk et al., 1996; Kobal, 1985; Lorig et al., 1999; Prah et al., 1995; Slotnick, 1990; Vigouroux et al., 1988). Whereas these design features have been described in principal, a text akin to a construction manual remains unavailable. It is this void that we aim to fill in this manuscript. In other words, this manuscript is designed as a tool to aid the new practitioner of olfactometry. For example, here we set out not only to note that “a valve should be used”, but rather specify what type of valve, what manufacturers provide appropriate solutions, and what specific part number served best in this function. In this regard, the purpose of this manuscript is to save time and money for those building their first odorant generating device. A second goal of this manuscript is to suggest a basis for uniform odorant generation that will aid in comparison of results across researchers. The sciences of vision and audition have followed a path whereby a rich history of psychophysics pro-

∗

Corresponding author at: Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720, USA. Tel.: +1 510 643 0131; fax: +1 510 643 0132. E-mail addresses: [email protected] (B.N. Johnson), [email protected] (N. Sobel). 0165-0270/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2006.09.008

vided the foundation for probing physiology. For example, psychophysical studies in vision demonstrated that all perceived colors can result from the mixing of just three colors, and that these perceived colors are largely determined by the wavelength of the impinging light. This psychophysical trichromatic model of color perception then guided physiologists in their discovery of the three receptor types in the retina that provide the basis for color perception (De Valois and De Valois, 1993). In other words, a conceptual framework of the sensory process was obtained through psychophysics, and the details were then filled in through physiological studies. By contrast, olfaction has taken an opposite path whereby today we know a lot more about the detailed physiology of olfaction (e.g., receptor transduction) than we do about how the sense of smell functions as a whole (Zelano and Sobel, 2005). One reason for the poor overall characterization of human olfaction has to do with the difficulty in comparing results across labs, or in other words, lack of standards. Such standards do exist for olfactory identification, namely the University of Pennsylvania Smell Identification Test (UPSIT) (Doty et al., 1984), and have recently been introduced for olfactory detection thresholds, namely Sniffin Sticks (Hummel et al., 1997) and “The Smell Threshold Test” (Doty, 2006), but odor generating devices (olfactometers) remain largely lab-specific. Thus, whereas a wavelength or frequency are understood to be the same whether generated in Yokohama or Yuba City, there is no equivalent uniformity in the field of olfaction. Odor con-

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centrations are usually reported as the liquid concentration in a saturation canister rather than the vapor concentration at the subject’s nose. Considering that the shape of the canister, the identity of the carrier (nitrogen, oxygen, breathable air) and the properties of the flow (rate, temperature, humidity, pressure) will all dramatically alter the end concentration, it is difficult to compare results across labs. A solution to this problem could be an analytical measurement of vapor concentration in every psychophysical or imaging report. However, analytical devices such as flame or photo ionization detectors are quite expensive, have strict calibration demands, and are often intrinsically precise but not ultimately accurate. An alternative solution would be use of a standardized odor canister that offers known flow to concentration outcomes. In a standard canister, a given flow (carrier, rate, temperature, humidity, pressure) through the canister containing a given odorant should always produce the same end vapor (with negligible variance related to barometric pressure at site of operation). For a canister to become a standard it would ideally satisfy the following criteria: 1, safe; 2, cheap; 3, readily available; 4, have minimal odor adherence; 5, be easily integrated into olfactometers of various kinds; 6, liquid and solid odorant compatible; 7, durable; 8, easily calibrated. In this manuscript we describe and characterize a canister that satisfies the above criteria, and that we propose as a standard odor canister (SOC) for olfactometry. 2. Materials and methods 2.1. Olfactometer design and construction Olfactometers can be conceptually divided into three subsystems. The first subsystem is (A) an airflow preparation apparatus. Here a carrier gas, typically medical grade breathable air, is brought to a desired flow velocity, temperature, and humidity. This subsystem also selects the desired odorant by directing the airflow through one odorant canister or another, sets the concentration by diluting the resultant odorized airflow with a clean airflow, and finally controls various vacuum lines that evacuate the stimulus. The second subsystem is (B) an odor-sourcing apparatus, or canister. Here odorant molecules are introduced into the previously prepared carrier gas. The third subsystem is (C) a delivery apparatus. Here the stimulus is delivered to the subject’s nose, where the change from clean air to odorized air ideally occurs without accompanying non-olfactory cues, and with well defined and rapid temporal characteristics. These three subsystems are then incorporated into one olfactometer. In the following pages we provide detailed advice on construction and part selection for a five-chanel olfactometer. In brief, the olfactometer is designed to alternate between constant odorized and non-odorized airflows of the same volumetric rate and characteristics. Up to five different odorants can be used within a given experiment. The olfactometer can deliver clean air and/or any of the five odorants at a nearly continuous range of concentrations, either individually, or in any binary mixture, to either one or both nostrils. When delivering odorant to one nostril, the opposite nostril can receive clean air at flow parame-

ters identical to the odorized nostril if desired. All airflows to the nose are constantly vacuumed away at the same rate, preventing any accumulation or lingering of odor. Finally, it is important that we state up front that we have no commercial relationship with any of the manufacturers and suppliers listed throughout the text, nor any commercial interests in this manuscript. We do not sell olfactometers. Our vendor and product recommendations, when made, reflect our own experiences, that in many cases followed experimentation with several alternatives. 2.1.1. Airflow preparation apparatus 2.1.1.1. General layout of olfactometer. The olfactometer was built on a cart to assure maximum mobility (Fig. 1). Portability is essential for an imaging olfactometer as space is often a limited commodity in MRI and PET facilities. For this purpose we used a 36 in. L, 24 in. W, 60 in. H stock cart (Edsal, part # ST9003 http://www.edsal.com). For added stability when parked we bolted an angle-iron on each lower side, welded 1 in. NPT × 2 in. nut to the inside of the angle-iron, and threaded 1 in. bolts that served as supporting legs (Fig. 1b). We placed shelves at ground (Fig. 1c), 24 in. (Fig. 1d), 34 in. (a double shelf for insulation) (Fig. 1e), a half-shelf at 47 in. (Fig. 1f), and a double shelf on top (Fig. 1g). At two diagonal corners of the first and second levels we placed 4 in. 110 V cooling fans (Fulltech, part # UF80A12 http://www.fulltech.com.tw) (Fig. 1h). Finally, we fashioned Plexiglas panels to enclose the olfactometer all around. General layout was as follows: on the ground level we rested the humidifier canister, vacuum tank (Fig. 1i), two computers (Fig. 1j), inline air heater (Fig. 1k), and 110 V ac uninterruptible power supply (Fig. 1l). On the second shelf we placed a photo-ionization detector (Fig. 1m), humidity probe controller module (Fig. 1n), a dc power supply providing +5, +15, −15, and +24 V (Fig. 1o), three temperature controllers (Fig. 1p), and two National Instrument connector blocks. On the third shelf we placed all the odor flow valves, mass flow controllers (MFCs), and odorant canisters. On the half-shelf we placed the MFCs for the vacuum systems. On the top shelf we placed two spirometers and an eight channel analog-to-digital amplifier. On the front panel we mounted two flat-panel monitors (Fig. 1q), keyboard and mouse (Fig. 1r), USB switchbox, and additional laptop (Fig. 1s). 2.1.1.2. Plumbing general. All plumbing within the olfactometer was with 0.25 in. medical grade stainless steel tubing (Duhig & Co., http://www.duhig.com) and 316 stainless-steel compression fittings (Hamlet Inc. http://www.ham-let.com/) and check-valves (Hoke Inc., http://www.hoke.com). All plumbing between the olfactometer and the delivery-line was with 0.3125 in. Teflon tubing and compression fittings (TEQCOM, http://www.teqcom.com) for clean and odorized air lines, and 0.3125 braided nylon hose and fittings for vacuum. Connections between the olfactometer and the delivery-line were with Twintec connectors (http://www.twintecinc.com). A schematic of the plumbing is shown in Fig. 2. Throughout the proceeding text there are callout numbers (C01, C02, etc.) that refer to locations within the schematic. The schematic reads from the bottom to the top.

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Fig. 1. The self-contained portable olfactometer. Side panels were removed for visibility. The following components are visible: parking legs (a and b), individual shelves (c–g), cooling fans (h), vacuum tank (i), two computers (j), inline air heater (k), and 110 V ac uninterruptible power supply (l), photo-ionization detector (m), humidity probe controller module (n), a dc power supply providing +5, +15, −15, and +24 V (o), three temperature controllers (p), two flat-panel monitors (q), keyboard and mouse (r), and additional laptop (s).

2.1.1.3. Plumbing—olfactometer vacuum. We recommend reading the following sections with the schematic Fig. 2 in hand. Appendix 1 in supplementary data is a consolidated table of the “output” connections on the olfactometer cart and also useful to have in hand when reading the following sections. Vacuum is supplied (600–700 mmHg) through a vacuum quick-connect plug (C1) (Colder part # LC-SST http://www.colder.com) situated at the front lower panel of the olfactometer. If central vacuum is unavailable, a local pump can supply sufficient vacuum (Welch part #2585B-01 http://www.welchvacuum.com). From the plug, vacuum is pulled through a 6 gal air tank (C2) (http://www.grainger.com; item #5Z374 or equivalent) placed in-line to dampen any vacuum pulsation related to pump action. From the tank vacuum is pulled through a vacuum regulator (C3) (vacuum gauge and bleeder valve, http://welchvacuum.com) and then from the top half-shelf where it pulls on MFCs 8 (C4), 9 (C5) and 10 (C6) (all MFCs in the olfactometer are MKS Instruments (http://www.mksinst.com) model M100B (M100B34CS1BV), with full-range output from 0.3 to 30 LPM). From MFC8 (C4) vacuum pulls on three-way valve (C7), where it is diverted to either line 1 (C8) or 2 (C9) of TWINTEC1 (C41) that connect to tubes 1 and 2 of the delivery-line. Vacuum through MFCs 9 (C5) and 10 (C6) form lines 3 (C10) and 4 (C11) of the TWINTEC1 connector (C41) that then connect to tubes 3 and 4 of the delivery-line. These last two evacuate the odorant from the left and right sides of the subject’s mask, and with a mask

septum allow the creation of a separate olfactory environment for each nostril. 2.1.1.4. Plumbing—olfactometer carrier air. Air is supplied (∼50 psi, either cylinder or pump + tank) through an air quick-connect plug situated at the front lower panel of the olfactometer (C12). From the plug air flows to two positive shutoff valves (C13 and C14). From each valve, air flows through individual pressure regulators (C15 and C16). These two lines feed the olfactometer main line (C18) and pneumatic control line (C17), respectively. The pneumatic control line is an optional feature for controlling a set of three-way Teflon valves (M443WPFS-T TEQCOM, http://www.teqcom.com) that are situated at the subject’s nose, and are used as diverter valves only in experiments where odor is to be directed to one nostril and not the other. Activating these valves diverts odor to one nostril, and clean air to the other. In the pneumatic control line, air flows from the regulator to two three-way control valves (C19) (Humprey H110 series). The four air-pressure lines (C20) coming out of the two threeway control valves terminate at TWINTEC2 (C42). These four lines then continue off the olfactometer to form tubes 8 to 11 of the delivery-line. These four tubes activate the three-way Teflon valves at the subject’s nose. The olfactometer main line (C18) air is divided from the regulator into two lines. One line feeds the photo ionization detector (C21) as a “zero” gas source. The second line flows

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Fig. 2. Schematic of olfactometer plumbing. See text for callout descriptions. Circles-with-line represent manual shut-off valves. Black-triangles represent one-way flow valves.

through a stainless steel inline air heater (C22) (Osram-Sylvania Process, Inline air heater #038824), passed a temperature probe (C23) (K-type thermocouple, Newport Electronics, Santa Ana, CA), on to MFCs 6 (C24) and 7 (C25). These two MFCs form the humidity control loop. Air from MFC7 flows into the humidifier

(C26). Air from MFC6 (C24) flows into a T connector (C27) that receives an additional input from the humidifier output (C28). The now combined airflows (dry and humidified) continue to feed MFCs 1–5. MFC5 (C29) is the clean air line. Here air flows through four SOCs in series (that can contain distilled deionized

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water), through the humidity and temperature sensor (C30), and to an air quick-connect plug on the outside of the olfactometer (C31) where it will connect to tube 6 of the delivery-line. MFC4 (C32) is the clean air line for the opposing nostril in experiments where odor is to go to one nostril only. It flows through four SOCs in series (that can contain distilled deionized water) to an air quick-connect plug on the outside of the olfactometer (C33) where it will connect to tube 7 of the delivery-line. MFC3 (C34) is the clean air line for the dilution. It flows through four SOCs in series (that can contain distilled deionized water) to the six-port Teflon mixing manifold (C35). MFCs 1 (C36) and 2 (C37) each feed a five-valve Teflon manifold (M446W2DFSHT TEQCOM, http://www.teqcom.com) (C38 and 539). Each pair of corresponding valves from the two manifolds join to form a single line. For example valve 3 from manifold 1 joins with valve 3 from manifold 2 to make one joint line. Each of the five joint lines flows through four SOCs in series (that will contain odorants) to the six-port Teflon mixing manifold (C35). This design, whereby each SOC line is served with air from either one five-valve-manifold or the other, enables generating any binary mixture of the five available odorants. The mixture capacity of the olfactometer (e.g. ternary, quaternary, etc.) can be increased by expanding the number of five-valve manifolds serving the SOCs. A cheaper and more simple but less flexible alternative to increasing mixture capacity is to put different odorants within the four serial SOCs; this has the drawback that composition percentages cannot be controlled by air-dilution. The six-port Teflon dilution manifold out-port (C40) then branches to the sampling port of the gas-analyzer (C21) and to TWINTEC1 on the outside of the olfactometer (C41) where it connects to tube 5 of the delivery-line. As a safety feature, a tee with a 3 psi pressure relief valve (C47) prevents excess pressure in the SOCs if the external tubing becomes occluded. 2.1.1.5. Humidity and pressure. Humidification is achieved by sparging the MFC7 (C25) air stream through deionized water in a stainless steel canister (C26). The canister is made of a 3 ft long, 3 in. diameter stainless steel pipe with (1/8) in. wall diameter along with stainless steel end caps. We drilled and tapped two 0.75 in. NPT holes in each of the caps. In the bottom cap we inserted a stainless steel screw-plug type immersion heater (C43) (http://www.gaumer.com) in the first hole. In the second hole, we connected a 90◦ elbow followed by a tee that was orientated up-and-down. A ball-valve was attached to the down-pointing threads to serve as a drain for the humidity canister. To the up-pointing threads we attached a second ball-value that was then connected to a vertical length of clear plastic tube for both monitoring the liquid level in humidifier canister and filling the humidifier canister. In the top cap we inserted a 0.5 in. diameter 2.5 ft long stainless steel tube for air inlet (from MFC7), and a compression fitting for humidified air outlet (C28). The humidified air outlet feeds the olfactometer (C27), a manual pressure release valve (C44), a 35 psi automatic pressure relief Hoke valve in parallel with a (1/3) psi automatic vacuum relief Hoke valve (C45), and a pressure sensor (C46) (Cole Palmer EW-68075-46, http://www.coleparmer.com). Both the inlet and outlet have Hoke one-way flow valves that are critical

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for preventing water backflow into the olfactometer after pressure buildup. The desired final humidity is achieved by varying the ratio of flow from MFC7 (humid) and MFC6 (dry) within the final flow. The ratio is constantly updated based on values from the inline humidity sensor located at the flow output of the olfactometer (C30, immediately before the air quick-connect). A second software PID loop regulates the pressure in the humidifier canister for both safety and consistency. Without regulation canister pressure could reach that of the input line pressure (25+ psi), creating a potentially dangerous situation. The sensor attached to the top of the canister (C40) provides pressure feedback for the software loop. When the pressure is low, the software increases a multiplicative factor for the flowrates of MFC6 and MFC7. That is, when the pressure is low MFC6 and MFC7 increase flow so that more air is going into the canister than leaving until the setpoint pressure is achieved. Once the desired pressure is achieved, the PID loop compensates by reducing MFC6 and MFC7. Although only MFC7 directly affects canister pressure, MFC6 is equally adjusted so as to not disrupt the humidity PID loop control. 2.1.1.6. Temperature. A constant temperature is maintained throughout the olfactometer, and is typically either set to room temperature or 37 ◦ C. Entering air is heated with an inline air heater (C22) (Sylvania OSRAM #038823 12.5 ). The heater is capable of operating up to 220 V ac but was chosen to run at 24 V dc for safety purposes. According to manufacture specifications, 24 V dc is sufficient power to heat 30 LPM to 90 ◦ C. The heater’s 12.5  heating element draws (24/12.5 =) 1.92 A of current at 24 Vdc. An OGDEN ETR-9090 PID temperature controller with an internal 3 A maximum relay is used to control the inline air heater. A K-type thermocouple is placed approximately 10 cm downstream of the inline air heater to provide temperature feedback to the controller. A 3 A fuse is placed between the controller relay and the heater for over current protection of the controller’s internal relay. The water within the humidifier canister is heated by a screw plug immersion heater (C43) (Omega Engineering Inc., Part No. RIO-300/120v). The heater is threaded through the bottom cap of the humidifier canister (C26). The heater is supplied with 110 V ac through a solid-state-relay (Carlo Gavazzi RS1A23D25). The SSR is controlled by an OGDEN ETR-9090 PID temperature controller with relay output. A K-type thermocouple sealed within a 0.25 in. compression fitting (C48) provides the water temperature feedback for the controller. The olfactometer cabinet is heated to be slightly warmer than the air temperature setting. This minimizes electrical circuit and odorant diffusion temperature dependencies, and prevents condensation within the valves, meters, and electronics. The heating and recirculation of the air within the cabinet is with a modified 1600 W space heater (Kaz/Honeywell HZ-315, http://www.kaz.com). The bulky plastic housing of the heater was removed and the heater-fan assembly mounted to a 10 in. L × 8 in. H piece of 0.25 in. PVC sheet. There is a 5 in. diameter hole in the center of the sheet to allow for airflow by the heater’s fan. The assembly is situated so the airflow is directed forward through the long direction of the cabinet. The original wiring

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utilized a single ac power cord with the fan and heater operating together. The fan and heater power were separated so the fan can be left on while the heater can be switched on and off. The fan is plugged into the power-strip on the top of the cart and can be turned on or off via the strip’s switch. The heater is switched on and off by a SSR relayed (Carlo Gavazzi RS1A23D25) controlled by an OMRON E5GN temperature controller. A K-type thermistor is placed in the free-air of the middle of the cabinet for temperature feedback to the controller. 2.1.1.7. Olfactometer control. The olfactometer relies on electrical valves and solenoids to regulate its behavior. These electromechanical devices are interfaced to computer software through multifunction acquisition boards within a computer. Two additional computers operate the external physiological and stimulus design software and hardware. All three computers on the cart are Apple Macintosh (Apple Computers, Cupertino, CA, http://www.apple.com). The three computers are connected to an Ethernet hub for local data transfer. The first computer, DRIVER, contains the software and data acquisition hardware necessary for controlling the operation of the olfactometer. DRIVER has two National Instrument (http://www.ni.com) PCI I/O boards, the NI 6011E for data input and the NI 6703 for data output. DRIVER also contains the LabVIEW software package from National Instruments. LabVIEW is a graphical programming language that is particularly suited for interfacing with external hardware for data acquisition and providing feedback control. The second computer, PSYSCOPE, is dedicated to running the software package “PsyScope” (Cohen et al., 1993). The PsyScope package is a shareware/freeware block-design visual programming language orientated towards running experiments (http://psy.ck.sissa.it/). It is simpler and more robust than LabVIEW for this task. The software can be used to present questions (e.g., “please estimate concentration”) and commands (e.g., “sniff now”) either visually (on a monitor or projected to a screen) or by sound (speakers or headphones). Psyscope can record user responses indicated either verbally or by keyboard or button press. Psyscope also provide triggers for external devices. The PSYSCOPE computer is linked to DRIVER via the PsyScope button-box (http://newmicros.com/) to set the content of odor-events and to trigger their onset. The digital out lines of the Psyscope button box are wired to the digital I/O lines of the NI-6011E, see Appendix 3 in supplementary data for specific wiring between subsystems. The third computer, PHYSIO is a laptop (Macintosh iBook) mounted to the front panel of the cart. PHYSIO is the computer end of the PowerLab physiological recording equipment (ADInstruments, http://www.adinstruments.com) used to monitor airflow in the nose throughout tasks. PHYSIO can also be used to trigger odor-events based on nasal respiration data. For example, odor onset can be initiated at any time point within a sniff. Additionally, the PowerLab recording equipment can record the state of the three-way vacuum valve (e.g. clean air or stimulus air to the subject) via a BNC cable that is connected across the relay that drives the valve (see Appendix 2 in supplementary data). This combination provides information on odor onset and offset, together with sniff charts, all in the

same file. In the MRI scanner, individual TRs can be logged onto the same file, providing ideal timing between stimulus, subject behavior, and scanner. Between the NI boards in DRIVER and the various electromechanical devices are conditioning circuits. The circuitry is fairly simple and primarily required because the NI boards cannot supply the power the devices require. Example circuits and wiring diagrams can be found in the supplemental materials online. These circuit designs are not the only approaches available; for an experienced circuit designer it will likely be more efficient to use his or her own familiar techniques in interfacing electromechanical devices for computer control. 2.1.1.8. Power supply. All electricity used on the olfactometer cart is provided through a single 110 V ac patch cord. The patch cord connects between a panel on the cart and a ground-faultcircuit-interrupter (GFCI) wall-outlet. There are inline GFCI units than can be used to promote portability. The GFCI is essential for operator safety in case any ac wires become loose and potentially lethal. The patch cord connects to the switched receptacle and immediately adjacent are additional switches for internal ac power (computer, dc power, etc.), ac heaters, and ac fans. Immediately below the galvanized steel electrical boxes that protect the ac wiring, there is a 3 in. length of (1/8) in. allthread secured perpendicular to the bottom shelf. A (3/16) in. hole was drilled through the bottom shelf, the all-thread placed through the hole and secured to the shelf by double nuts. The all-thread was situated so the long end protrudes up into the cart. The cart’s paint around the hole was removed to ensure a low-resistance electrical connection. The ac patch panel plug is grounded to the all thread post. The post is also the converging point for all dc signal grounds to help eliminate ground loops. Devices are grounded to the post in a hierarchical fashion; large current carrying devices are grounded directly to the post. The initial ac entry point splits three ways: into a two-outlet receptacle controlled by an external switch, into the ac heater system controlled by the second external switch, and into the ac fan system controlled by a third external switch. The two switched receptacles provide power to an uninterruptible power supply (UPS) and a surge-protector outlet strip. The UPS provides uninterrupted ac power to the two desktop Macintosh computers and gas analyzer. It also provides surge protected power to the two flat-panel monitors mounted on the front of the cart. The surge-protected outlet strip provides ac power to the computer network hub, the two dc power supplies, the OMRON temperature controller, and a second surge-protector strip located on the top shelf of the cart. This second strip provides ac power to the Macintosh iBook, the PsyScope button box, the four-channel PowerLab amplifier, and the cabinet heater fan. The strip can also be used for extra external devices. Many of the devices on the cart require dc power. A medicalgrade dc power supply (Condor DC Power Supplies Inc., Model GLD 140E, http://condorpower.com) is on the cart to provide the different voltage levels required: −15, +5, +15, +24. The following is a list of devices on the cart that require dc power: OGDEN temperature control (+24 V dc), HydroFlex

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temperature and humidity sensor (+15 V dc), MFCs (+15, −15), Teqcom valves (+24 V dc), Humphrey three-way valves (24 V dc), Redhat three-way valves (24 V dc), and various circuits (5 V dc). 2.1.1.9. Olfactometer software. The electronic mass-flowcontrollers and solenoid valves that regulate the airflows through the olfactometer are computer controlled. The control software provides a simple event-driven interface that only requires the user to pre-program desired delivery flowrate, odorant identity (e.g. SOC channel), concentration and event duration. These pre-programmed events are saved in ASCII formatted files to ensure each subsequent run is identical. The user, or external experiment control software (psyscope), then select the preprogrammed event number and time the switch between clean and stimulus air (‘triggering’). The operation of the olfactometer is completely reliant on software written in LabVIEW, Version 6. LabVIEW programs are called “Virtual Instruments” or VIs. Like LabVIEW’s internal organization, the olfactometer software is written in a hierarchical structure. At the lowest level, many small and specialized programs control the hardware within the olfactometer. Above these small programs are a handful of rudimentary programs that operate the individual subsystems. Finally there is a single master program that allows the user to access all the important experimental parameters and to run experiments. This organization allows rapid development of additional VIs for experiment paradigms that are not supported by the current software. There are four main groups of bottom-level programs: Analog In, Analog Out, Digital In, and Digital Out. These four groups handle the data exchange between the LabVIEW software and the olfactometer’s electrical components, and handle the conversion of the data and preparing it for use in the rest of the olfactometer software. The two ‘OUT’ groups take commands from the higher software, formats it and then relays the commands to the hardware. The two ‘IN’ groups collect data received from the hardware and make it available for use by the higher programs. There are three intermediate VIs that maintain the airflow rates through the olfactometer: two proportional-integralderivative (PID) control loops and one logic controller. The PID VIs are: one for humidity and one for humidifier canister pressure (to help regulate humidity, and provide a consistent overpressure to compensate for slight MFC calibration inconsistencies). The humidity is controlled, as previously described, by adjusting the ratio of flow through the ‘wet’ and ‘dry’ air supply lines. The canister pressure is regulated by adjusting the flowrate through the MFCs supplying the humidifier plumbing. The logic controller VI determines which odorant valves should be open and how much total air flows through the olfactometer. The top-level software relies on odor events. These events prescribe the stimulus parameters: odor(s), concentration, and duration. During an experiment, the olfactometer sits in its normal (non-triggered) mode where it is constantly flowing a set volumetric rate of clean air to the subject. When given the trigger command, the olfactometer switches states and the subject is given the odor event.

Fig. 3. The proposed standard olfactory canister. Two units are shown to illustrate how they can be joined in series. Each street tee is shown with the hex-cap “thimble” that holds the odorant. The minimal mass of the hex cap (∼40 g) allows it to be massed with a high precision balance.

We have made the olfactometer software available on the Internet at http://socrates.berkeley.edu/∼borp/supp.htm. 2.1.2. Odorant source—standard olfactory canister (SOC) As standard odor canister (SOC) we used the combination of two standard stainless steel pipe fittings: the (1/2) in. street tee (T) and (1/2) in. hollow cap (http://www.ham-let.com). Two milliliters of odorant liquid are placed within the cap, and the cap is then screwed into the branch of the T (Fig. 3). The carrier (we use bottled medical grade breathable air) is then flown through the run of the T. Several of these functional units can be connected in series when higher end-concentrations are desired. In our olfactometer we typically use four SOCs in series. Depending on properties of the odorant in question, this setup produces odorant concentrations ranging from marginally discernible from background odor up to an intense perceptual experience. In choosing the odor canister we were primarily concerned with selecting a discrete, functional unit that could be inserted into a wide variety of olfactometer-devices and still provide comparable odorant concentrations. As such, such a functional unit produces the same concentration if it is attached to computer controlled mass-flow-controllers, solenoids, etc, or if attached to manually operated needle-valve flowmeters and hand-valves. 2.1.3. Delivery apparatus An overview and expanded views of the three components of the delivery apparatus are shown in Fig. 4. The following section is again best viewed with the figure in hand. From the olfactometer the delivery-line (C58) extends to the railroad manifold (C60) which in turns connects to the subject mask (C51). 2.1.3.1. Delivery-line. The delivery-line (C58) is an aggregate of 15 tubes going from the olfactometer to the subject. Several delivery-lines can be made at different lengths to enable use of the olfactometer in different situations. For example, we use a 10 m delivery-line in the imaging environment such that the olfactometer is in the control room and the subject is in the center of the magnet bore, and a 3 m delivery-line in the psychophysical lab such that the olfactometer and the subject are in adjacent rooms sharing a wall. Delivery-line length affects

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(pneumatic control lines for the three-way Teflon valves in monorhinal experiments) are made of 0.25 in. Tygon tubing. Tubes 12–15 (pneumotachometer lines) are made of thickwalled 0.125 in. nylon tubing. At the olfactometer, the deliveryline connects to the olfactometer with a series of quick-connects. The four vacuum tubes (1–4) together with the odor tube (5) connect with a five-port Twintec connector (C41, TWINTEC1) (http://www.twintecinc.com). The four pneumatic control tubes (8–11) and the four pneumotachometer tubes (12–15) each connect with a four-port Twintec connector (C42 and C61, respectively). The clean air tube (6) and the opposing nostril clean air tube (7) are connected with individual quick-connects (C31 and C33, respectively). The entire bundle of tubes is then enwound by a tube (C59) from a heat recirculator (RCB300, Hoefer Scientific Instruments, San Francisco, CA) and then ensheathed in a thermal insulating sleeve (C58) (Thermwell Pipe Insulation, P12X 3 ft × 1 in.). It is not necessary, but often convenient, to include the pneumatic control lines (8–11) or the pneumotachometer tubes (12–15) in the thermal insulating sleeve. At the subject end of the bundle each tube connects to its respective target.

Fig. 4. Delivery apparatus. The delivery-line connects to the external quickconnects of the olfactometer cart and then to the railroad manifold. The manifold in turn connects to the subject mask. See text for callout descriptions.

odorant concentration (see results) and determines the shortest possible inter-stimulus-interval (as a function of washout time), but does not affect the temporal resolution of odor onset and removal. Tubes 1 (railroad switch vacuum 1), 2 (railroad switch vacuum 2), 3 (mask vacuum 1), and 4 (mask vacuum 2) are made of 0.3125 braided nylon hose. Tubes 5 (odorized air), 6 (clean air), and 7 (clean air for opposing nostril in monorhinal experiments only) are made of 0.3125 in. Teflon tubing. Tubes 8–11

2.1.3.2. Railroad manifold: switching between the two prepared gas streams. The railroad manifold design described by Kobal (Kobal, 1985) is a crafty olfactometer design feature that enables moving the point of temporal resolution from the olfactometer itself to the far end of the delivery-line. This is especially critical in the imaging environment where the olfactometer cannot be placed in the magnet room due to its ferrous components. Our version of the railroad manifold is shown in Fig. 4 (C60) (MAN—5W5T-CV TEQCOM, http://www.teqcom.com). The unit was machined so that all wetted parts were Teflon. The manifold has two positive pressure inlets (pC and pS), two vacuum outlets (vC and vS), and a final positive pressure outlet (O) that is directed to the subject. Tube 1 connects to vC, tube 2 connects to vS, tube 5 connects to pS, and tube 6 connects to pC. In practice, the subject receives air from one positive pressure inlet while the other positive pressure supply is removed through its vacuum port. One positive port (pC) is always supplied with clean air while the other (pS) is supplied with the stimulus (either odorized air or non-odorized air for “blank” trials). The two vacuum ports are connected to a three-way valve of which the third port is attached to a regulated vacuum supply. The railroad switch functions as follows. Tube 5 delivers the odorized air at flowrate x and tube 6 delivers clean air also at flowrate x. A vacuum, also at flowrate x, is diverted through three-way valve (Asco/Redhat 8320G178Q) to either tube 2 or 3. When vacuum is at tube 2, the odorized air is vacuumed away at the railroad switch, and clean air enters the nostrils. When vacuum is switched to tube 1, the clean air is vacuumed away at the railroad switch, and odorized air enters the nostrils. Oneway flow valves immediately between the vacuum tees and the center tee prevent any possibility of contamination. This design allows the three-way valve to be located tens of meters from the manifold, maintaining MR compatibility. The vacuum removes either the clean air or the stimulus air while the remainder goes to the subject. Switching the valve changes

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which air the subject receives—from clean to stimulus, or from stimulus to clean. This arrangement moves the point of odorant temporal control into the railroad manifold. The new air after switching reaches the subject at time T = V/Q. V is the volume of air that must be replaced, that is, the amount of air between the vacuum inlet and the subject mask and Q is the volumetric flowrate of air. Based on tube diameter and length, we estimated the relevant volume of our manifold to be approximately 6 ml. With a typical flowrate of 10 liters-per-minute (LPM) it takes approximately 30 ms for the new air to reach the subject (see Section 3 for an actual measurement). Remote placement of the olfactometer (say ∼10 m from the subject) does incur a penalty however. To change the identity or concentration of the stimulus, the new air must be generated at the olfactometer and then traverse the delivery-line (at ∼1.3 m/s for 10 LPM through 0.25 in. tubing) to be readied in the railroad manifold. Considering such speed (∼1.3 m/s) it would take approximately 8 s to change the stimulus that is ‘at the manifold’ if a 10 m delivery line is used. However this lag in changing stimulus properties is generally acceptable, as most olfactory experiments require long inter-stimulus-intervals to minimize sensory habituation. 2.1.3.3. Mask. The mask (C51) (SleepNet Corp., Phantom Nasal Mask, www.sleep-net.com) can be used for both monorhinal (both nostrils exposed to same olfactory environment) or birhinal (each nostril exposed to a different olfactory environment). Several holes must be drilled and hose-barb fittings secured into the mask (see C52, C53, C54, and C55). The mask receives air from the railroad manifold (C60). The output of the manifold (C49) connects to Y-connector (C50) that diverts air to both the right (C52) and left (C53) sides of the mask. The airflows into the mask and then exits via the two delivery-line vacuum tubes (C54 and C55). When the subject is receiving stimulus air (e.g. odor laden air), the vacuum helps prevent this odorized air from escaping the mask and accumulating near the subject. The pneumotachometers (not shown) are attached to C56 and C57 to monitor the subject’s sniffing. It is certain that both the position of the fittings for vacuum tubes (C54 and C55) and the nasal geometry of individual subjects will affect the odor flow profiles within the mask. We recommend choosing locations for these fittings that (a) can be easily replicated between masks and (b) do not cause inference with the other tubing. However, additional consideration for this positioning must be given if one is interested in how the flow profiles influence perception. 2.1.3.4. Birhinal experiments. In a birhinal experiment, delivery-line tubes 12–15, along with the railroad switch output (O), connect to the two pneumatic three-way Teflon valves (M443WPFS-T TEQCOM, http://www.teqcom.com). Lines 12–15 control the valves, which are configured to switch the opposing-nostril clean air, tube 7, and railroad output (O) between the two nostrils. Although one could use two nasal canulas, we typically put a septum in the mask, thus creating two separate spaces within the mask. These two spaces are independently served by an inlet and outlet. The Y-connector (C50)

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is not used in birhinal experiments. The inlets (C52 and C53) are connected to the outputs of the pneumatic three-way Teflon valves while the outlets (C54 and C55) are connected to the two mask vacuum tubes in the delivery-line. Similarly, a pair of MRsafe pneumotachometers (http://www.gm-instruments.com) are attached to the left and right chambers to measure sniffing in each nostril separately. 2.2. Olfactometer characterization 2.2.1. Measuring concentration—gas analyzer The olfactometer is equipped with an on-board gas-analyzer (photo-ionization method, 8800 PID, Baseline-Mocon Inc., Lyons, CO) to measure the concentration of odorant in the delivered gas stream. The GA sampling port is connected to a tee immediately prior to the odor line exiting the olfactometer. When active, the GA draws a small amount of air from the odor line (0.1 LPM). The GA purge line is connected to the air line immediately after the on-cart pressure regulator. An LCD on the device displays concentration; the external current output is connected to NI-6011E to allow continuous recoding of device concentration. This particular GA is well suited for tracking long-term stability and relative concentrations—it can measure concentration differences as low as 10 parts per billion (ppb). The device, however, is not accurate in absolute terms or for measuring rapidly changing concentration. The GA reading takes only a few seconds for the reading to reach equilibrium if the sample concentration is increasing or very slowly decreasing. However, when the sample concentration is decreased more than a few percent, it takes a much longer time to obtain an accurate reading. The GA is slow to purge the sample volume, so that previous high concentration samples effectively contaminate the sampling chamber. This contamination has persisted for over an hour in several of the tests performed. 2.2.2. Measuring concentration—mass-loss An additional way to measure concentration delivered from the olfactometer is by measuring the rate of depletion of the odorant source (Allison and Katz, 1919; Johnston, 1967). Once a popular technique when reporting stimulus concentration, massloss measurement are rarely seen in current literature. This is unfortunate for this method is very accurate regardless of stimulus identity, unlike almost any analytical device. Furthermore, a sensitive electronic laboratory balance is much cheaper than a gas-analyzer. The liquid that is depleted from the standard olfactory canisters is the same that is produced at the output of the olfactometer (provided no leaks and substantial adhesion). Liquid loss from the canisters can be measured gravimetrically. The individual hex caps are massed and the liquid loss recorded. We measure the amount of odorant mass depleted from the SOC in relative long time period (15–30 min intervals) all the while integrating the volume passed through the SOC. Various measures of concentration (volume, molar or mass) can be calculated with knowledge of the odorants chemical formula, liquid density, assumed air pressure and temperature, etc.

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2.2.3. Measuring concentration—UV-absorption To characterize the temporal dynamics of the olfactometer we used the principles of ultraviolet light spectroscopy (method by personal communication, Briglin S, Lewis N, 2002). This technique has excellent temporal resolution (sub millisecond) but requires the use of a UV absorbing liquid and thus is only practical in characterizing the stimulus timing. Acetone (99.9+% HPLC grade; Sigma–Aldrich, 27,072-5) has a strong absorption peak in the UV wavelengths of the energy spectrum, and a high vapor pressure so it is excellent choice. Instead of the typical subject mask, the odorant flow is directed into a quartz chamber with an Hg(Ar) lamp (6035 Spectral Calibration Lamp, Oriel Instruments, Stratford, CT) on one side and a UV photodiode detector (Model #UV-50, UDT Sensors Inc., Hawthorne, CA) on the other (Fig. 10A). The passage of acetone through the tube attenuates the amount of energy received by the photodiode detector. The lamp’s 253.7 nm Hg peak was isolated with a low-pass filter (Oriel Instruments, 6041 Short Wave Filter) that attenuated the higher, visible frequencies. With the filter, the lamp’s output was largely limited to the 253.7 nm Hg peak. At the Hg peak, the molar absorptivity is 1.6 × 104 l/mol cm for acetone, providing a strong attenuation of the lamp’s radiant energy if acetone is present in the path between the detector and lamp. The attenuation can be calculated with the Beer–Lambert law, %T = 100 × [exp(−ελCsolute )], where ε is the molar absorptivity (1.6 × 104 l/mol cm), λ the pathlength (approximately 6 mm) and Csolute concentration of the solute in the solution. This attenuation is ONLY to the light passing through the odorant airflow. It is thus crucial to minimize the UV light reaching the photodetector that does not pass through the airflow. The stray light biases the photodetector and consequentially small changes in light intensity are difficult to detect. We built an enclosure for the apparatus and used internal structures to limit the paths to the photodetector to those passing through the quartz tube. The photodiode detector was connected to a transimpedence current-to-voltage pre-amplifier (AD8015 Wideband/Differential Transimpedance Amplifier, Analog Devices, Norwood, MA) and then to an instrumentation amplifier and digital acquisition system (ADInstruments, Powerlab 4sp). The quartz tube is connected to the subject side of the railroad manifold. This connection is with a piece of Teflon tube (1/4 in. i.d.). The tube length is selected to approximate the volume between the railroad manifold and the subject’s mask that is used in human experiments. 2.3. Olfactometer maintenance Olfactometer maintenance is an unavoidable issue. Although there is occasional failure of MFCs, solenoids, etc., the primary maintenance task is removal of the inevitable buildup of odorant contamination. Maintenance should be performed according to the contamination tolerance of individual experiments. With adequate design consideration, a rigorous olfactometer maintenance schedule is very manageable. Components that are exposed to odorants should be located and installed in a manner that allows for easy removal. Olfactometer maintenance can

be minimized by avoiding odorants that have higher adhesion properties (e.g. Citral). Considering the odorant flow, all parts downstream of the odorant-selection-manifolds (C38 and C39) are susceptible to contamination; this hardware includes the street tees (e.g. SOCs), one-way flow valves, the mixing manifold, the plumbing to the TWINCTEC connector, the delivery line, and finally, the railroad manifold. The one-way valves can be disassembled and the individual parts cleaned and then reassembled. It is a good idea to replace any o-rings in the one-ways as they often deteriorate after prolonged odorant exposure. Any method for cleaning the one-way valve parts is dictated by their construction. Most of the stainless steel hardware (e.g. street-tees, plumbing, some valve parts, etc.) can either be cleaned in an autoclave or placed on hot plate (∼60 ◦ C) until the contaminants evaporate. The Teflon odorant tube within the delivery line must be replaced unless a suitable method of cleaning the tubing is available (e.g. large autoclave). The Teflon railroad manifold can be cleaned with copious amounts of hot water and soap or autoclaved according to material specifications. The hot plate method does not work with Teflon parts because, in contrast to metal, the heat will not transfer through the plastic. Finally, the SOC thimbles can be reused even for different odorants by rinsing and washing with soap and then autoclaving or placing them on a hot plate as described above. We have found that the hot plate method works very well for metal parts but care must be taken to not get the parts “too hot” or they will discolor and possible warp, making assembly difficult. 3. Results Using the three methods described above, we characterized the key properties of the olfactometer. 3.1. Concentration stability over time We measured concentration stability by continuously sampling the concentration produced by the olfactometer over a 60 min period. A 2 ml of amyl acetate (Aldrich Chemical Company Inc.) was placed in each of four inline SOCs, a total of 8 ml. Five LPM of dry unheated air (28 ◦ C, 28% RH) was flown through the SOCs while the LabVIEW software sampled the photoionization detector reading every second. The odorant concentration stability is shown in Fig. 5. The unadjusted concentration was 41.5 ± 0.38 ppm for a 1 h period. This was a total air volume of approximately 307 l. Psychophysical research suggests, that although humans have superb odorant detection from background capabilities (Nagata and Takeuchi, 1990; Whisman et al., 1978), they lack equally superb abilities in detecting minute concentration deviations (Cain, 1977; Gamble, 1899; Slotnick and Ptak, 1977) that we measured here. 3.2. Control of odorant concentration without ‘air dilution’ A primary function of the olfactometer is producing perceptually different stimuli. We measured the output concentration as we increased the volumetric rate of air passing through the

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Fig. 5. (A) Time-series of odor concentrations recorded over a period of 60 min. The carrier flowrate was 5 LPM giving an average concentration of 41.5 ppm as determined by the on-board PID gas analyzer. During 60 min of continued odorant generation the system drifted less than 3 ppm. (B) Distribution of concentrations recorded over the same 60 min period. The histogram shows the distribution frequency of normalized concentration whereas the line shows the cumulative percentage of the normalized concentration distribution. This data suggest the odorant stream was highly consistent over time. Used with permission from (Johnson et al., 2003).

SOCs. In practice, the subject receives a constant rate of air, so that increasing odorant flow is compensated with decreasing dilution air. However, we were interested in first examining the undiluted characteristics of the odorant canisters. For each experiment, a number of inline SOCs were filled with 2 ml of amyl acetate. The flowrate through the inline SOCs was increased in steps and the gas-analyzer value recorded once the reading stabilized. This procedure was repeated until the odorant flowrate reached 10 LPM. This experiment was performed with one, two, three, and four inline SOCs, the results are shown in Fig. 6. Fig. 6A shows that the odorant concentration in the carrier gas stream was not a simple function of carrier flowrate. As flowrate increased the concentration actually decreased (see 0–3 LPM), suggesting that the odorant evaporation was near its maximum rate and the decreasing measured concentration was due to the increased air. Fig. 6B shows that odorant delivery rate (e.g. concentration multiplied by flow

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Fig. 6. Odorant output vs. flowrate and number of inline SOCs, same data but presented differently between A and B. Concentration was measured using the PID as carrier gas flowrate was increased for 1, 2, 3, or 4 SOCs (“thimbles”) in series. (A) Direct concentration provided by PID. The graph shows that concentration is not a simple function of flowrate. (B) Data from (A) multiplied by respective carrier flowrate to give an estimate of molar rate of odorant delivery.

rate of the carrier gas) increases monotonically with flow rate. However, the odorant delivery rate (e.g. the amount of odorant evaporating from the SOCs) was indeed rather constant for 1, 2, or 3 SOCs up to 4 LPM. 3.3. Control of odorant concentration with ‘air dilution’ In a second experiment we repeated the previous measurements but also included the dilution air that would normally be used in a behavioral experiment. A 2 ml of amyl acetate was placed in each of four inline SOCs. The ratio of flowrates through the SOCs and dilution vessel was varied between 0% (no odor, all dilution air) and 100% (all odor, no dilution air) for a total flowrate of 10 LPM. Several ascending and descending experiments were ran, to provide six data points for each of the shown concentrations. All data was collected over a period of 11 days. The gas analyzer was only calibrated immediately before the first experiment and not before any other experiment providing data

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Fig. 7. Control of odorant concentration using air-dilution. This data suggest a simple relation between dilution ratio and output concentration. Here the olfactometer produced a total flow of 10 LPM, and a set percentage (x-axis) of that flow was odorized while the on-board PID gas analyzer measured the output concentration of the output flow (e.g. the full 10 LPM airstream). Each point represents six experiments repeated over a period of 11 days. The first data point is at 2% (0.2 LPM), the lowest flowrate the mass-flow-controllers can produce reliably. To obtain lower concentrations we could use fewer SOCs (4 used here) or dilute the liquid odorant with an appropriate solution (water or mineral oil). Mean variance across all points was less than 5%. This data suggest that a given odor event was highly repeatable over experiments performed many days apart. Used with permission from (Johnson et al., 2003).

for the graph. The measured concentrations are shown in Fig. 7. Unlike the undiluted concentration measurements, the diluted concentration measurements are nearly linear when using a fixed final flow rate (10 LPM here). These data suggest a simple relation between dilution ratio and output concentration. 3.4. Measuring absolute odorant concentration To accurately measure absolute output concentration we used the aforementioned mass-loss technique (see Section 2). We also used these more precise measurements to verify the simple relationship between dilution ratio and output concentration shown with the PID data of Fig. 7. This finding suggests that the airdilution output concentration at any given dilution percentage can be accurately predicted with knowledge of the concentration when the olfactometer output is not air-diluted (e.g. the ‘undiluted concentration’). This undiluted concentration is the concentration obtained when the entire airflow is directed through the SOCs and no additional dilution air is added. With an estimate of the undiluted concentration (Cundiluted ), the output concentration is reduced according to the equation Cdiluted = Cundiluted × X/100, where X is the dilution percentage. To verify this relationship, we first measured undiluted concentration at 10 LPM and then calculated the dilution concentration at 25%, 50%, and 75%. Next, we directly measured the concentration at the flows represented by these three dilutions (2.5, 5.0, and 7.5 LPM, respectively). The results of the mass-loss characterization, and verification of the simple relationship between dilution percentage and concentration are shown in Fig. 8. Specifically, amyl acetate was loaded into the olfactometer after measuring the initial mass with an analytical balance (L-Series, 0.1 mg precision, Acculab, Bradford, MA). Odorized air at 10 LPM carrier gas flowrate was produced by the olfactometer for a 20–30 min period, after which the remaining amyl acetate mass was measured. The total volume of air was measured by integrating the flow measurement by

Fig. 8. Estimates of output concentration using the mass-loss measurement technique. Amyl acetate was produced at different flowrates by the olfactometer for a duration sufficient to measurable reduce the mass of liquid in the SOC. For each flowrate, concentration was calculated for a total ‘to-subject’ flowrate of 10 LPM (diamonds). Additionally, we predicted the concentration based on the measured mass loss at 10 LPM using [Cdiluted = Cundiluted × X/100] (circles).

the olfactometer. This procedure was repeated at additional flowrates (2.5, 5.0, and 7.5 LPM). The mass-loss was converted to moles (mass/molecular-weight) and then to molar concentration (moles of odor vapor per million moles of carrier gas at 1 atm and 37 ◦ C). The obtained values were then used to assess the diluted concentration when using a total airflow of 10 LPM to a subject. The diamonds in Fig. 8 represent our most precise estimate of the concentration at each of the four dilution steps. Next, we verified the Cdiluted = Cundiluted × X/100 prediction by calculating the concentration at each dilution based on the mass-loss measured at 10 LPM. The open circles in Fig. 8 thus represent our estimate of the four dilution steps based on our single measurement at 10 LPM. The predictions were very similar to the actual measurements. In comparing the concentrations measured by the PID and mass-loss techniques, the PID was systematically lower by a factor of approximately 20, well within the range of human discriminability. That is, if two labs used “10” ppm stimuli but one measured with the PID and the other with mass loss they would be delivering perceptually different stimuli. We repeated the mass-loss measurement procedure with seven additional odorants, citral, 1-butanol, l-carvone, acetophenone, propionic acid, valeric acid, and octane, and the results are presented in Table 1. Data is presented as such to simplify concentration estimation for different combinations of carrier and dilution flows (see table legend for calculations). 3.5. Output concentration versus exit resistance In designing the olfactometer we wanted to generate identical stimuli in either our psychophysics lab or in the MR scanner. Since we use different length delivery-lines in these two envi-

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Table 1 Estimates of output concentration using the mass-loss measurement technique for the odorants citral, 1-butanol, l-carvone, acetophenone, propionic acid, valeric acid, octane, and amyl acetate (rows) Undiluted concentration (ppm)

Citral 1-Butanol l-Carvone Acetophenone Propionic acid Valeric acid Octane Amyl acetate

2.5 LPM

5 LPM

7.5 LPM

10 LPM

0.051 3.453 0.079 0.216 2.136 0.214 4.056 2.059

0.067 3.583 0.075 0.174 2.202 0.193 5.277 2.245

0.057 2.521 0.085 0.220 2.539 0.281 5.309 2.844

0.047 2.104 0.069 0.232 2.389 0.289 5.244 2.746

Values shown are the moles of liquid odorant per million moles of carrier gas (e.g. ppm) when 2.5, 5.0, 7.5, and 10 LPM are passed through four SOCs and no dilution air is added. Data is presented in this manner to estimate concentration for different combinations of carrier and dilution. To estimate concentration, first choose the amount of odorized air and the amount of final to-subject flowrate. Interpolate the table data to obtain the value for the desired odorant flowrate if the exact flowrate is not on the table. Finally, divide this value by the percentage of odorized air. For example, 5 LPM through amyl acetate combined with 5 LPM of dilution air to give a final flow of 10 LPM has a concentration of (2.245 × 5/10 =) 1.1225 ppm. Or, 2.5 LPM of amyl acetate combined with 7.5 LPM to give a final flow of 10 LPM has a concentration of (2.059 × 2.5/10 =) 0.515 ppm.

ronments we were interested in measuring the effect of external resistance (delivery-line length) on concentration. The evaporation of the odor molecules into the air-stream depends on a variety of factors, one being headspace pressure. Unlike flowrate, which depends on pressure differential, the absolute pressure within the olfactometer lines affects evaporation. As the resistance to flow is increased, a larger upstream pressure is required to overcome the resistance. Thus as external resistance increases the pressure over the headspace of the odorant increases. This results in a relationship between odor concentration and resistance. From fluid dynamics theory we hypothesized that the concentration of the olfactometer would linearly vary with the length of the delivery-line. Lengthening the delivery-line increases the pressure differential required to maintain the airflow rate (Ph − Patm = QR). Ph is the pressure maintained by the MFC immediately upstream from the SOCs and is very close to the pressure above the headspace. Since the evaporation rate of the odorant is proportional to Ph − Pvp , the evaporation rate is proportional to delivery-line length. To test this we varied the length of the delivery-line while using the PID to measure relative concentration. The collected data is shown in Fig. 9. The points indicate the mean PID signal over 60 s. The graph shows that end-point concentration is linear to tube length, as expected. Using this graph, we can alter dilution ratios between our imaging and psychophysics labs, compensating for different tube lengths, to get equal concentrations. 3.6. Temporal resolution of railroad switch We measured the temporal resolution of the olfactometer using the UV spectroscopy method. The olfactometer’s delay for switching between no-odor and odor conditions is due to

Fig. 9. PID concentration measurement for various lengths of the delivery-line. The delivery-line connects the olfactometer cart, and its associated MFCs, SOCs, etc., to the switching manifold that is immediately before the subject’s mask. As predicted from potential flow (dP = resistance × flow) increasing external resistance resulted in higher pressure above the odorant headspace. Consequentially, odorant evaporation flux (i.e. concentration) decreased proportional to the decrease in pressure differentially between the odorant’s saturated vapor pressure and actual headspace pressure.

the volume of air that must be replaced within the railroad manifold, and the speed at which the air is replaced. We measured the time it took the output of the manifold to switch from clean-air to acetone-laden odorant air. We were forced to sparge acetone through a custom designed apparatus, similar to the humidifier apparatus, to achieve an end concentration that was high enough to influence the UV signal. We used a LabView VI to automatically control the olfactometer so we could average multiple trials. The VI was programmed to complete twenty cycles: 3 s of clean-air, 3 s of acetone, and finally 3 s of clean-air. This routine allowed the concentration to stabilize within each cycle and allow measuring both odor on-set and odor removal delays. The routine was ran once at each of 5, 7.5, and 10 LPM. The acetone source was replenished between each routine. The 20 blocks in each series were zero and first-order corrected to accommodate for decreases in acetone due to decreases in liquid volume in the sparger and shift of photodiode temperature during the experiment. The first three corrected blocks of the 5 LPM experiment are shown in Fig. 10B. Fig. 10C is an enlarged view of the first block. A switching delay was then determined for each of the 20 blocks, based on the time to reach a given percentage of the average signal during the odorant period. The average signal during the odorant period can be referenced to the pre-odor signal and used to calculate concentration ratios using the Beer–Lambert law (Levine, 1995). The delays for 50%, 75%, and 95% C/Cmax are shown in Fig. 10D. The average delay represents the time elapsed from activation of the three-way vacuum solenoid valve until the odorant was present in the mask/quartz chamber at the specified percentage. The error bars at each point represent the variance in this time, or in other words, the temporal resolution of the olfactometer. For example, at 5 LPM the time to reach 95% C/Cmax was 84.6 ± 1.8 ms. Thus, if we want an odorant to

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of interest is predicted to occur in the hundreds-of-milliseconds range. 4. Discussion In this manuscript we had two goals. One was to provide detailed information on olfactometer construction, and the other was to propose and characterize a standardized olfactory canister, or SOC. 4.1. Olfactometer construction

Fig. 10. (A) Schematic of the apparatus used to determine the temporal resolution of the olfactometer used in this study. The apparatus was connected to the olfactometer with the same diameter and length of tubing between the railroad switch and nasal mask as that used in the study. The UV energy generated by the light source passed through the quartz tube window and then activated the UV-sensitive silicon photodiode. As olfactometer air flowed through the quartz tube, any acetone in the air stream attenuated the UV energy measured at the photodiode. The output from the photodiode was amplified, digitized, and stored on computer for analysis (components not shown). Used with permission from (Johnson et al., 2003). (B) Raw UV blocks. Silicon photodiode signal data recorded on the computer. The gray line indicates the state of the three-way valve that controlled the vacuum drawn away from the railroad switch: “low” indicates clean air was delivered to the apparatus (odorized air pulled away), “high” indicates odorized (acetone) air was delivered to the apparatus (clean air pulled away). The black trace represents the photodiode signal from an experiment where 5 LPM was sparged through an acetone vessel. (C) Zoomed UV block. The inset is an expanded view of the first acetone event. (D) Switching delays from UV experiments. The UV experiment was conducted three times (at 5, 7.5, and 10 LPM). At each flowrate, the switching delay was determined by three separate criteria: 50% C/Cmax , 75% C/Cmax , and 95% C/Cmax . The mean values represent the systematic delay, whereas the error bars represent the uncertainty (temporal resolution). Reprinted with permission from (Johnson et al., 2003).

reach the nose at time “X,” we should have the olfactometer trigger 84.6 ms earlier, and it is assured that the odorant will indeed be present at time “X” ± 1.8 ms. This temporal resolution of less than 2 ms is more than sufficient considering that the process

The pioneers of olfactometry have published several manuscripts describing the conceptual framework of olfactometer design (Benignus and Prah, 1980; deWijk et al., 1996; Kobal, 1985; Lorig et al., 1999; Prah et al., 1995; Slotnick, 1990; Vigouroux et al., 1988). Here we made no claims to innovate beyond these concepts. In turn, we aimed to provide a detailed account in nearly “manual format” regarding various considerations in olfactometer construction. The purpose of this section of the manuscript was to serve as a helpful tool to those entering into the field. It is our hope that this effort will spare new practitioners costly excursions in their path to a functional olfactometer. A point in case can be taken from our path to finding an appropriate in-line air heater. Ideally, the entire airline of an olfactometer should consist of Teflon, glass, or stainless steel components only. With this criteria in mind, we failed to find an appropriate off-the-shelf in-line air heater, so set off to build one from scratch. After lengthy research and design, component acquisition, custom modifying of components, and construction, we had spent several thousand dollars on this inline air heater. Although an impressive artifact, it failed to heat the air at a sufficient rate. We then went looking again for off-the-shelf alternatives, and this time found the ∼US$ 100 stainless steel in-line Sylvania air heater, that performed exceptionally well. If new practitioners are spared the above arduous route by reading this manuscript, then it has served its purpose. The particular olfactometer design we have described here is not without drawbacks. In that it relies on MFCs, it is expensive, and constructing it calls for a fair amount of skills. Furthermore, once constructed, it has to be occasionally calibrated, and often cleaned. Also, in that it relies on a single flushable odor line from olfactometer to subject (rather than multiple lines, one for each odor used in a given experiment), it is prone to slight contamination, and is therefore inappropriate for applications such as detection threshold testing. Furthermore, as described the olfactometer provided a dynamic range of approximately 20-fold in concentration. The output concentration adjustment range is not constrained by the SOC themselves, but by the characteristics of the MFCs controlling the air-dilution. To increase the dilution factor, an additional MFC with lower calibration range (e.g. 0–1 LPM instead of 0–30 LPM) need be used in parallel with the odorant airflow control MFC. Finally, preventing a tactile puff upon alteration from odor to no-odor and back calls for particularly careful calibration of flows. All that said, this design also has several advantages. Foremost of these is robustness.

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Two such olfactometers in our lab have each ran for several hundred hours over the past few years, essentially without incident. Also, this particular design offers significant flexibility in experimental design, and a high level of control over stimulus characteristics. 4.2. Standardized olfactory canister In addition to providing detailed information on building an olfactometer, in this manuscript we proposed what can be used as a standardized olfactory canister. As noted in the introduction, for a canister to become a standard it would ideally satisfy the following criteria: 1, safe; 2, cheap; 3, readily available; 4, have minimal odor adherence; 5, be easily integrated into olfactometers of various kinds; 6, liquid and solid odorant compatible; 7, durable; 8, easily calibrated. It is our view that the canister we have described fulfils all of these criteria. (1) In that it is made of stainless steel (rather than glass), it is safe. (2) It costs a few tens of dollars at most. (3) It can be obtained from countless manufacturers that all construct to the same specifications given that the item is a plumbing standard. (4) It is made of stainless steel which has minimal odor adherence. (5) In that it is threaded with NPT threads, it can be easily incorporated in the flow line of any olfactometer. (6) The cap can be filled with either liquid or solid (e.g., decanoic acid). (7) The stainless steel construction is particularly durable. (8) The output can be measured easily as detailed in Section 3. A particularly appealing aspect of using this canister, and one we see as the key aspect of this manuscript, is the consistency of the output given a consistent airflow input. In other words, if one flows an airflow at a particular flow velocity, temperature, and humidity, through the SOC, one always obtains the identical odorant output. Considering that nearly all olfaction experiments are conducted within a limited range of airflow values (typically 2–10 LPM over the odorant source), we have generated a table of flow/concentration graphs for several odorants typically used in olfaction experiments. Thus, any practitioner can now use the SOC, and as long as he/she uses up to 10 LPM of airflow at room temperature and humidity, they can now know exactly what output they are getting. They do not need to build a complex olfactometer, and they do not need to invest in expensive analytical measurements as we have here. They can use the information in this manuscript to generate a well-characterized stimulus. For example, if one flows 5 LPM of air through four SOCs in series that contain Amyl Acetate, one will obtain a stimulus flow of 1.12 ppm (Table 1). Thus, in addition to the possibility of sparing someone a lot of work charging down the wrong path in olfactometer construction, we see Table 1 as a central practical contribution of this manuscript. If additional practitioners take on the proposed SOC, the list of odorants in Table 1 will grow over time, and allow distant practitioners to generate nearly identical stimuli, thus enabling better comparison of results across labs and across time.

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