Name: production and measurement of organic particulate matter in the harvard environmental chamber
Uploaded: 2018-11-18T12:30:00.000+00:00
Duration: 9 min 46 s
Description: Watch this Scientific Journal Video about Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber at JoVE.com

This material is based upon work supported by the Environmental Chemical Sciences Program in the Division of Chemistry of the U.S. National Science Foundation (NSF) under grant number 1111418, the Atmospheric-GeoSciences Division of the U.S. National Science Foundation (NSF) under grant number 1524731, as well as Harvard Faculty Publication Award. We acknowledge Pengfei Liu, Qi Chen, and Mikinori Kuwata for useful discussions and assistance with the experiments, as well as Eric Etcovitch for being the voiceover of the video.

NOTE: The key measured environmental parameters include ozone (ozone analyzer), NO and NO2 (NOx analyzer), relative humidity (RH sensor), temperature (type K thermocouple), and the differential pressure between the bag and the chamber. The instrument brands are listed in the Table of Materials. The environmental parameters measured by the instruments must be stable and within design requirements prior to the start of the experiments. The environmental chamber uses a feedback system to constantly monitor and adjust controls so that the environmental parameters remain within requirements throughout the course of an experiment.
1. Start Up Procedures
<ol>
	<li>Parameters Setting and oxidants injection
	<ol>
		<li>Set the physical parameters of the environmental chamber by the feedback system (PID). Set the differential pressure to 4 Pa (30 mTorr). When the pressure is too high or too low, the solenoid valve opens or closes to adjust the pressure of the bag within the set range.</li>
		<li>Turn on the ozone generator to generate ozone flow by passing the dry air through an ultraviolet lamp. Set the flow rate to 0.1 sLpm to reach 100 ppb of final ozone concentration inside the HEC. Turn on the ozone monitor and activate the corresponding software.</li>
		<li>Set the relative humidity of the bag to the designated values. In this particular experiment, use 40% RH but the RH level can change from &#60;5% to 80%. The RH sensor and the feedback control system keep the relative humidity in the bag stable by adjusting the ratio of dry and humid air flows. The dry air supply is provided by the pure air generator, which generates zero air free of hydrocarbons, water (RH &#60; 1%), and oxides of nitrogen. A flow of humid air is preparing by bubbling dry air through high purity water (18 M&#937; cm) to create near-saturated air flow.</li>
		<li>Set the temperature of the chamber to 25.0 &#177;0.1 &#176;C. An internal conditioning plenum distributes the air evenly through a stainless-steel ceiling with feedback control systems that keep the temperature within designated values.</li>
		<li>Wait for other environmental parameters to be stable and within the design requirements.</li>
	</ol>
	</li>
	<li>Connect the inlets of instruments to the environmental chamber. Start the self-developed software by clicking the Start button. Check the real-time data displayed on the self-developed software that integrates the feedback control (Figure 2).</li>
	<li>Turn on all the instruments and wait for them to warm up completely.</li>
</ol>
2. Seed Particle Production
NOTE: Prior to seed particle injection, the initial particle concentration is below 1 cm-3.
<ol>
	<li>Production of sulfate seed particles
	<ol>
		<li>Inject quasi-monodisperse dry sulfate particles into the bag, both to better simulate the ambient conditions and also to act as medium for the condensational growth of secondary organic materials. The injection of seed particles is carried out as follows.</li>
		<li>Dissolve ammonium sulfate (0.01 g) in high purity water (18 M&#937;&#8729;cm) in a 100 mL volumetric flask to prepare a (NH4)2SO4 solution (0.1 g&#8729;L-1).</li>
		<li>Use an atomizer to produce (NH4)2SO4 aerosol particles at a flow rate of 3 sLpm.</li>
		<li>Pass the aerosol flow through a diffusion dryer (silica gel) to bring the relative humidity down to 10%.</li>
	</ol>
	</li>
	<li>Seed particle selection and bag injection
	<ol>
		<li>Pass the aerosol flow through a bipolar charger (85Kr) and a Differential Mobility Analyzer (DMA) to size select the particles and prepare a quasi-monodisperse distribution by electric mobility. The transmission function is widened by using a sheath-to-aerosol flow in the DMA of 10:3. The selected electric mobility diameter of the particles exiting the DMA varies from 50 to 150 nm depending on the experiment. The typical number concentration ranges from 4,000 to 8,000 cm-3.</li>
		<li>Feed the quasi-monodisperse aerosol to the bag with a flow rate of 3 sLpm. Monitor the particles exiting the bag by using a Scanning Mobility Particle Sizer (SMPS). Wait for the size distribution of the seed particles to become stable. The particle loss due to the bending tubing as well as static charges on the bag is minimal, especially for particles sizes used in this experiment (larger than 100 nm)29,30.</li>
	</ol>
	</li>
</ol>
3. Injection of Gas Phase Precursors
<ol>
	<li>Injection of the gas-phase precursors
	<ol>
		<li>Use a syringe to withdraw 1.00 mL of the isoprene solution. Rinse the syringe three times with the solution prior to final withdrawal.</li>
		<li>Place the syringe into a syringe injector. Insert the needle tip through a rubber seal into a round-bottom flask (25 mL). Pre-heat the flask to 90 &#177;1 &#176;C by heating tape. Turn on the syringe injection and set it to an appropriate value (1.1 to 4.4 &#956;L&#8729;min-1). The gas-phase concentration of the precursor is adjusted for different experiment by controlling the syringe injection rate. For long experiments, refresh the syringe as needed.</li>
		<li>Introduce a flow of 2 sLpm of purified air to vaporize and carry away isoprene injected in the round-bottom flask. The flow of the air is large enough that the sessile droplet at the tip of the syringe is vaporized instead of dripping into the flask. As a result, the concentration of the gas phase precursor remains stable.</li>
	</ol>
	</li>
	<li>Turn on the switch of the ultraviolet lights inside the HEC.</li>
</ol>
4. Instrument Measurement
NOTE: The combination of isoprene and UV light leads to the production of secondary organic material onto the sulfate seed particles.
<ol>
	<li>Number-diameter distribution of the particle exiting the bag
	<ol>
		<li>Sample the outflow from the bag by using the electrostatic-resistant tubing.</li>
		<li>Start the aerosol measurement software and create a new file by clicking on Create A New File. Each parameter is set as shown in Figure 3. Record the number-diameter distributions of particles exiting the bag by clicking on the OK button.</li>
	</ol>
	</li>
	<li>Chemical characterization of organic particulate matter.
	<ol>
		<li>Open the sampling valve of a stainless steel inside the bag. The sampled aerosol flow in a High-Resolution Time-of-Flight Aerosol Mass Spectrometer (HR-ToF-AMS).</li>
		<li>Start the data acquisition software by pressing the Acquire button on the lower left of the panel (Figure 4). High-resolution mass spectra of the organic PM are recorded during the time course of the experiments. The total organic mass concentration is also obtained.</li>
	</ol>
	</li>
	<li>Characterization of gas-phase species
	<ol>
		<li>Open the sampling valve of a PTFE Teflon tube inside the bag. The sampled flow is guided to a Proton-Transfer-Reaction Time-of-Flight Mass Spectrometer (PTR-TOF-MS). The mass spectra of gaseous species having a higher proton affinity than water are obtained.</li>
		<li>Use the parameter settings of the ion source of the PTR-TOF-MS shown in Figure 5 in the software PTR-Manager. Start the data acquisition by accessing the drop-down menu Acquisition in the TofDAQ Viewer software and then pressing Start. Record the time series of each ion through this software.</li>
	</ol>
	</li>
</ol>
5. End of Experiment and Bag Cleaning
<ol>
	<li>Stop the injection of the gas-phase precursors and the aerosol seed particles.</li>
	<li>For several days continuously inject pure air at 40 L&#8729;min-1 into the bag. Turn on all ultraviolet lights. Set the ozone concentration to 600 ppb and set the temperature to 40 &#176;C. In this way, an aggressive oxidation environment is maintained for several days to scrub the bag. When the number concentration of the particles inside the chamber is less than 0.2 cm-3, the chamber is considered clean and can be used for the next experiment.</li>
</ol>

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The authors declare no competing financial interests.

The increasing significance in understanding the formation and evolution of organic aerosols leads to the urge to construct environmental chambers to simulate such processes in a well-controlled setting. At present, most of the environmental chambers are based on batch reactor mode19,31,32,33,34&#160;while there have been very few chambers that utilize continuously mixing reactor mode15,35. Operating the environmental chamber in continuously flow reactor mode provides the convenience of continuous aerosol sampling for days or even weeks at ambient-like concentrations. It is worth noting that the ambient conditions are much more complex than the well-controlled laboratory settings. For instance, the temperature of ambient fluctuates while in the chamber it is maintained at a constant value. The reaction time of the gases and particles in the chamber will be controlled and limited by the residence of the chamber, rather than reaching days of reaction time in the real world. The use of blacklights, instead of natural solar radiation, can also generate OH radicals and simulate the reactions in the ambient. But blacklight sometimes can lead to elevated concentration of OH radicles compared with those in the ambient environment, which may affect the oxidation state of the organic molecules and needs to be carefully examined. However, by tuning only one or two variables and controlling all other variables through environmental chamber, we can systematically study these chemical/physical processes.
One of the critical steps in operating continuously mixing chambers is to keep the internal pressure of the chamber within an optimal range. A high pressure within the chamber will cause leaking of the gases and particles from the chamber, while a low pressure within the chamber will suck air and particles from the laboratory into the chamber and cause contamination. A pressure gauge is needed to monitor the pressure of the chamber within safe values (&#60;5 Pa) throughout the course of the experiments. Another common observed issue for the environmental chamber is unexpected organic particle self-nucleation. Either a lower VOC/oxidant injection rate or a higher seed particle concentration is needed to avoid this phenomenon. Depending on the purpose of the experiments, the concentrations of ozone, VOC, and seed particles can vary by one order of magnitude. The following equation can be used to calculate the flow rate,&#160;finjection, of each species injected into the chamber.
<img alt="Equation 1" src="/files/ftp_upload/55685/55685eq1.jpg" style="opacity: 0.9; font-size: 14px;" />&#160;(1)
where&#160;ctarget&#160;and&#160;cinitial&#160;each represent the final target concentration of the reactant inside the chamber and the initial concentration of the reactant that is generated from the source. The symbol&#160;ftotal&#160;represent the total flow of all species that were injected into the chamber.
The third critical step for successfully operating the environmental chamber and obtaining the results is to calibrate each instrument before the experiments. The SMPS system can be calibrated by injecting known size of the PSL particles36. The NOx&#160;and ozone analyzer are calibrated by using a 5 ppm NO cylinder diluted by N2, and 10 ppm of ozone diluted by N2, respectively26. The calibration procedures for AMS and PTR-MS are complicated and can be found in instrument manuals or previous literatures27,37.
The environmental chamber setup described above is not only suitable for studying the production and evolution of organic aerosols, but also applicable in coating various particles with organic coating as well as examining gas phase reactions by injecting gas precursors only. These multiple directions provide environmental chamber the flexibility in studying a variety of research fields related to the air quality, climate change, and human health topics.

Atmospheric organic particulate matter (PM) is produced from the oxidation of volatile organic compounds (VOCs) emitted by the biosphere and anthropogenic activities1,2. Despite the important effects of these aerosol particles on climate, human health, and visibility3, the production mechanisms remain incompletely understood and characterized, both qualitatively and quantitatively. One challenge for laboratory studies, which are necessarily of limited scope and time, is to simulate the atmospheric evolution of gas and particle phase species. Residence times must be long enough that compounds in both gas and particles phases can undergo oxidation and multiphase reaction as they would in ambient environments4,5,6,7,8. Another challenge is to work in the laboratory at concentrations sufficiently low that represent the ambient environment9,10,11. Many important processes scale with concentrations. For instance, excessively high mass concentration of organic PM in a laboratory experiment can erroneously shift the partitioning of semivolatile species from the gas phase to the particle phase. The composition of the gas and particle phases can become non-representative of atmospheric conditions. The Harvard Environmental Chamber was designed to respond to these challenges, principally by using the approach of a continuous flow configuration operated under an indefinite timescale, thereby allowing low concentrations and long integration times for signal detection. The chamber celebrates a milestone anniversary of twelve years of scientific discovery in 2018.
Environmental chambers vary based on the light source, the flow mixing system, size, and the number of chambers operating together. There are outdoor chambers that receive natural sunlight12,13&#160;as well as indoor chamber that operate with artificial light14,15,16,17,18,19,20,21. Outdoor chambers can also be built relatively large, minimizing artifacts that can be introduced by wall effects, although challenges include the variation of illumination because of clouds as well as variance in temperature. Although indoor chambers can carefully control temperature and relative humidity, the intensity and the spectrum from the artificial light are generally different from the natural sunlight, which may affect certain photochemical reactions14. Chambers can also be operated as batch reactors or completely mixed flow reactors (CMFR)22. Batch reactors are generally easier to operate and maintain but CMFR can be operated for weeks, as needed, to allow for signal integration and thereby work at low, atmospherically relevant concentrations.
Herein, the hardware and the operation of the Harvard Environmental Chamber (HEC)7,23,24,25 are described in detail. The HEC consists of a 4.7 m3 PFA Teflon bag housed inside a constant-temperature chamber (2.5 &#215; 2.5 &#215; 2.75 m3)26. Reflective aluminum sheets cover the inner walls of the chamber to allow multipath illumination through the bag and thereby increase the rate of photochemistry. The HEC is operated as a CMFR, using a total flow rate of 21 sLpm and corresponding to a mean residence time of 3.4 h27. Temperature, humidity, and ozone concentration are maintained by feedback controls. Ammonium sulfate particles are used as seed particles to mimic the condensation of organic components onto inorganic particles in the ambient environment. The mode diameter of the inorganic sulfate particles is selected to be 100-200 nm to simulate the particle sizes measured in the field28. Operation procedures are described in the protocol section herein, including a visual presentation, followed by a brief discussion of applications and research results of the HEC.

<table><tbody><tr><td>(-)-&amp;alpha;-pinene&amp;nbsp;</td><td>Sigma-Aldrich</td><td>305715</td><td></td></tr><tr><td>2-butanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>294810</td><td></td></tr><tr><td>5.00 mL syringe&amp;nbsp;</td><td>Hamilton</td><td>201300</td><td></td></tr><tr><td>Aerosol particle mass analyzer</td><td>Kanomax</td><td>3600</td><td></td></tr><tr><td>Condensational particle counter</td><td>TSI</td><td>3022</td><td></td></tr><tr><td>Differential mobility analyzer</td><td>TSI</td><td>3081</td><td></td></tr><tr><td>Heating mantle</td><td>Cole-parmer</td><td>WU-36225-10</td><td></td></tr><tr><td>Mass flow controller</td><td>MKS</td><td>M100B</td><td></td></tr><tr><td>Nafion tube</td><td>Perma Pure</td><td>MD-700-24F-1</td><td></td></tr><tr><td>Nanometer aerosol sampler</td><td>TSI</td><td>3089</td><td></td></tr><tr><td>Ozone generator</td><td>Jelight</td><td>600</td><td></td></tr><tr><td>Ozone monitor</td><td>Ecosensors</td><td>UV-100</td><td></td></tr><tr><td>Pressure sensor</td><td>Omega</td><td>PX409</td><td></td></tr><tr><td>RH sensor</td><td>Rotronic</td><td>60587161</td><td></td></tr><tr><td>Round-bottom, three neck flask</td><td>Aceglass</td><td>6944-04</td><td></td></tr><tr><td>Scanning electron microscope</td><td>Zeiss</td><td>N/A</td><td>Ultra plus FESEM</td></tr><tr><td>Scanning mobility particle sizer</td><td>TSI</td><td>3071A+3772</td><td>electrostatic classifier is model 3071A and the condensational particle ocunter is 3772</td></tr><tr><td>Silicon substrate</td><td>University Wafer</td><td>1707</td><td></td></tr><tr><td>Syringe Needle</td><td>Hamilton</td><td>90025</td><td>25 G, 2 inch</td></tr><tr><td>Syringe pump</td><td>Chemyx</td><td>Fusion Touch 200</td><td></td></tr><tr><td>Temperature sensor</td><td>National Instrument</td><td>USB-TC01</td><td></td></tr><tr><td>water circulator</td><td>Brinkmann</td><td>RC6</td><td></td></tr></tbody></table>

production and measurement of organic particulate matter in the harvard environmental chamber

The production and the evolution of atmospheric organic particulate matter (PM) are insufficiently understood for accurate simulations of atmospheric chemistry and climate. The complex production mechanisms and reaction pathways make this a challenging research topic. To address these issues, an environmental chamber, providing enough residence time and close-to-ambient concentrations of precursors for secondary organic materials, is needed. The Harvard Environmental Chamber (HEC) was built to serve this need, simulating the production of gas and particle phase species from volatile organic compounds (VOCs). The HEC has a volume of 4.7 m3 and a mean residence time of 3.4 h under typical operating conditions. It is operated as a completely mixed flow reactor (CMFR), providing the possibility of indefinite steady-state operation across days for sample collection and data analysis. The operation procedures are described in detail in this article. Several types of instrumentation are used to characterize the produced gas and particles. A High-Resolution Time-of-Fight Aerosol Mass Spectrometer (HR-ToF-AMS) is used to characterize particles. A Proton-Transfer-Reaction Mass Spectrometer (PTR-MS) is used for gaseous analysis. Example results are presented to show the use of the environmental chamber in a wide variety of applications related to the physicochemical properties and reaction mechanisms of organic atmospheric particulate matter.

An example of the time series of organic mass concentration recorded by the HR-ToF-AMS during one experiment is shown in Figure 6. Experimental conditions were 490 ppb of isoprene with UV lights turning on to provide OH radical as oxidant. The mass concentration of organic PM steadily increased after the beginning of the experiment until after about 4 h steady state was reached. The evolution of gas-phase organic compounds was simultaneously studied using the PTR-TOF-MS. Figure 7 shows the time series of the C4H6O+ signal intensity under the same experiment, which arises from major isoprene oxidation products (e.g., methyl vinyl ketone, methacrolein, and several organic hydroperoxides). After illumination was begun, the C4H6O+ signal intensity increased and continued doing so until steady state was reached after 50 min.
The figures illustrate the time evolution of secondary organic material in the HEC. After injection, reaction, and spin up to steady state, the AMS data indicate that the particles are composed of organic compounds, and the concentration of the organic components increases with time. The PTR-MS data show that the parent precursor is lost from the gas phase and first-generation product species appear after the reaction is initiated. Data analysis of online and offline measurements typically focus on the steady-state period. There is the opportunity to conduct experiments that require days to finish because the concentrations of the gas and particle phase species remain stable indefinitely by use of the CMFR operation with feedback controls on important chamber parameters. In summary, the HEC is used to simulate atmospheric chemistry and thereby test hypotheses and understanding on important topics related to understanding air pollution, the climate influences of particles, and even health effects of aerosols.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55685/55685fig1.jpg" /> 
Figure 1. A schematic flow diagram of the Harvard Environmental Chamber (HEC). The lines represent the aerosol flow. The left panels show the instruments used to monitor reaction conditions. The right panels list those instruments used to characterize the particle and gas phase species. This figure is adapted from King et al.26&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55685/55685fig1large.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/55685/55685fig2.jpg" /> 
Figure 2. Graphical user interface for the self-developed program used to monitor temperature, ozone, relative humidity, and pressure, among other species. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55685/55685fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55685/55685fig3.jpg" /> 
Figure 3. Graphical user interface of the software used to record the number-diameter distribution using a SMPS. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55685/55685fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55685/55685fig4.jpg" /> 
Figure 4. Graphical user interface of the software to operate the Aerosol Mass Spectrometer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55685/55685fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55685/55685fig5.jpg" /> 
Figure 5. Graphical user interface of the software to control the Proton-Transfer-Reaction Mass Spectrometer (PTR-MS) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55685/55685fig5large.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/55685/55685fig6v2.jpg" /> 
Figure 6. Example measurements for organic PM, as characterized by the Aerosol Mass Spectrometer. The red line represents the total signal intensity for organic ions. The shown data correspond to shortly after the isoprene was injected and the ultraviolet illumination commenced. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55685/55685fig6large.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/55685/55685fig7v2.jpg" /> 
Figure 7. An example of the signal intensity of C 4 H 6 O+ ion, a major product of isoprene photo-oxidation measurement is by Proton-Transfer-Reaction Mass Spectrometry. Signal intensity began to increase 8 min after illumination and reached steady state at 50 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55685/55685fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber at JoVE.com

Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber

This paper describes operation procedures for the Harvard Environmental Chamber (HEC) and related instrumentation for measuring gaseous and particle species. The environmental chamber is used to produce and study secondary organic species produced from the organic precursors, especially related to atmospheric organic particulate matter.

The production and the evolution of atmospheric organic particulate matter (PM) are insufficiently understood for accurate simulations of atmospheric ...

production-measurement-organic-particulate-matter-harvard

Nanyang Technological University

Research

JoVE Journal

Environment

7.2K Views.  Harvard University. Het algemene doel van dit project is om organisch materie in de milieukamer te produceren en de chemische en fysische eigenschappen ervan te karakteriseren. De Harvard Environmental Chamber werd gebouwd om de vorming van organische deeltjes en de reactie van gasfase componenten te bestuderen in de buurt van omgevingsomstandigheden. Dit is het tienjarig bestaan van de kameroperaties.Het unieke deel van deze kamer is het wordt bediend als een volledig gemengde stroom reactor die de kans ...

Video: Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher 
spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis 
and treatment of neurovascular diseases such as stroke, Alzheimer's disease, and head injury.
Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. 
Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.
Click here to view the related article <a href="https://www-jove-com.remotexs.ntu.edu.sg/Details.php?ID=1731">Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.</a>

We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

Neuroscience

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface  

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

Medicine

Making Record-efficiency SnS Solar Cells by Thermal Evaporation and Atomic Layer Deposition

Tin sulfide (SnS) is a candidate absorber material for Earth-abundant, non-toxic solar cells. SnS offers easy phase control and rapid growth by congruent thermal evaporation, and it absorbs visible light strongly. However, for a long time the record power conversion efficiency of SnS solar cells remained below 2%. Recently we demonstrated new certified record efficiencies of 4.36% using SnS deposited by atomic layer deposition, and 3.88% using thermal evaporation. Here the fabrication procedure for these record solar cells is described, and the statistical distribution of the fabrication process is reported. The standard deviation of efficiency measured on a single substrate is typically over 0.5%. All steps including substrate selection and cleaning, Mo sputtering for the rear contact (cathode), SnS deposition, annealing, surface passivation, Zn(O,S) buffer layer selection and deposition, transparent conductor (anode) deposition, and metallization are described. On each substrate we fabricate 11 individual devices, each with active area 0.25 cm2. Further, a system for high throughput measurements of current-voltage curves under simulated solar light, and external quantum efficiency measurement with variable light bias is described. With this system we are able to measure full data sets on all 11 devices in an automated manner and in minimal time. These results illustrate the value of studying large sample sets, rather than focusing narrowly on the highest performing devices. Large data sets help us to distinguish and remedy individual loss mechanisms affecting our devices.

Tin sulfide (SnS) is a candidate material for Earth-abundant, non-toxic solar cells. Here, we demonstrate the fabrication procedure of the SnS solar cells employing atomic layer deposition, which yields 4.36% certified power conversion efficiency, and thermal evaporation which yields 3.88%.

Tin sulfide (SnS) is a candidate absorber material for Earth-abundant, non-toxic solar cells. SnS offers easy phase control and rapid growth by congruent ...

Engineering

Production and Measurement of Organic Particulate Matter in a Flow Tube Reactor

Organic particulate matter (PM) is increasingly recognized as important to the Earth's climate system as well as public health in urban regions, and the production of synthetic PM for laboratory studies have become a widespread necessity. Herein, experimental protocols demonstrate approaches to produce aerosolized organic PM by &#945;-pinene ozonolysis in a flow tube reactor. Methods are described for measuring the size distributions and morphology of the aerosol particles. The video demonstrates basic operations of the flow tube reactor and related instrumentation. The first part of the video shows the procedure for preparing gas-phase reactants, ozonolysis, and production of organic PM. The second part of the video shows the procedures for determining the properties of the produced particle population. The particle number-diameter distributions show different stages of particle growth, namely condensation, coagulation, or a combination of both, depending on reaction conditions. The particle morphology is characterized by an aerosol particle mass analyzer (APM) and a scanning electron microscope (SEM). The results confirm the existence of non-spherical particles that have grown from coagulation for specific reaction conditions. The experimental results also indicate that the flow tube reactor can be used to study the physical and chemical properties of organic PM for relatively high concentrations and short time frames.

This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols.

Organic particulate matter (PM) is increasingly recognized as important to the Earth's climate system as well as public health in urban regions, and the ...

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation.

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

Bioengineering

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Biology

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Fabricating Highly Open Porous Microspheres (HOPMs) via Microfluidic Technology

Compared to bulk scaffolds and direct injection of cells alone, the injectable modular units have garnered enormous interest in repairing malfunctioned tissues due to convenience in the packaging of cells, improved cell retention, and minimal invasiveness. Moreover, the porous conformation of these microscale carriers could enhance the medium exchange and improve the level of nutrients and oxygen supplies. The present study illustrates the convenient fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (PLGA-HOPMs) by the facile microfluidic technology for cell delivery applications. The resultant monodispersed PLGA-HOPMs possessed particle sizes of ~400 &#181;m and open pores of ~50 &#181;m with interconnecting windows. Briefly, the emulsified oil droplets (PLGA solution in dichloromethane, DCM), wrapped with the 7.5% (w/v) gelatin aqueous phase, were introduced into the 1% (w/v) continuous flowing poly(vinyl alcohol) (PVA) aqueous solution through the coaxial nozzle in the customized microfluidic setup. Subsequently, the microspheres were subjected to solvent extraction and lyophilization procedures, resulting in the production of HOPMs. Notably, various formulations (concentrations of PLGA and porogen) and processing parameters (emulsifying power, needle gauge, and flow rate of dispersed phase) play crucial roles in the qualities and characteristics of the resulting PLGA HOPMs. Moreover, these architectures might potentially encapsulate various other biochemical cues, such as growth factors, for extended drug discovery and tissue regeneration applications.

Fabricating Highly Open Porous Microspheres HOPMs via Microfluidic Technology

The present protocol describes the fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (HOPMs) via the single-emulsion formulation based facile microfluidic technology. These microspheres have potential applications in tissue engineering and drug screening.

Compared to bulk scaffolds and direct injection of cells alone, the injectable modular units have garnered enormous interest in repairing malfunctioned ...

Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the effects of nitrosated compounds. Measuring NO metabolites is essential to studying NO activity both at vascular levels and in other tissues, especially in the experimental settings where exogenous NO is administered. Ozone-based chemiluminescence assays allow precise measurements of NO and NO metabolites in both fluids (including plasma, tissue homogenates, cell cultures) and gas mixtures (e.g., exhaled breath). NO reacts with ozone to generate nitrogen dioxide in an excited state. The consequent light emission allows photodetection and the generation of an electric signal reflecting the NO content of the sample. Aliquots from the same sample can be used to measure specific NO metabolites, such as nitrate, nitrite, S-nitrosothiols, and iron-nitrosyl complexes. In addition, NO consumed by cell-free hemoglobin is also quantified with chemiluminescence analysis. An illustration of all these techniques is provided.

Here, we present protocols for detecting nitric oxide and its biologically relevant derivatives using chemiluminescence-based assays with high sensitivity.

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the ...

Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber

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