This project is based on research that was partially supported by the Nebraska Agricultural Experiment Station with funding from the Hatch Act (Accession Number 0223605) through the USDA National Institute of Food and Agriculture.

All human subject research was performed in accordance with the Institutional Review Board of the University of Nebraska-Lincoln, and informed consent was obtained from all subjects.
1. Locating the MCA signal by freehand TCD
NOTE: &#8220;Freehand&#8221; TCD refers to operation of TCD with a handheld transducer to find a CBFV signal before beginning an fTCD experiment.
<ol>
	<li>Setting TCD parameters
	<ol>
		<li>Keep the power at a reasonably high value (e.g., 400 mW) during the initial search for the MCA. Once the MCA signal is located, reduce the power as much as possible while still maintaining a &#8220;good&#8221; signal (see step 2.2.7). 
		NOTE: Using a reasonably high power during the initial search does not violate the &#8220;As Low As Reasonably Achievable&#8221; (ALARA) principle of exposure to acoustic radiation because higher power will allow the MCA signal to be discovered more quickly10.</li>
		<li>Set the sample volume to 8&#8211;12 mm during the initial search for the MCA signal. If the signal is difficult to find, increase the gate size to increase the intensity of the signal, but note that this may incorporate the signal from one or more nearby arteries into the signal from the MCA.</li>
		<li>Set the gain at a medium level, with the goal of &#8220;keeping background noise at a minimum, but present&#8221;10.&#160;</li>
		<li>Set the high-pass filter cutoff (normally termed &#8220;threshold&#8221;) to 50&#8211;150 Hz.&#160;</li>
		<li>If the subject is an adult, set the depth to 50 mm, which is the average mid-point depth of the M1 segment of the MCA10 (Figure 1).&#160; 
		&#8203;NOTE: This setting will be discussed in more detail in subsequent steps. Depth settings for children are given in Table 1.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62048/62048fig1.jpg" /> 
Figure 1: Representation of the circle of Willis and the major arteries of the cerebral circulatory system. The bifurcation of the ICA into the ACA and MCA is marked with a black circle. The M1 segment of the MCA is shown. This figure has been modified from24. Abbreviations: ACA = anterior cerebral artery; Bif. = bifurcation; ICA = internal carotid artery; MCA = middle cerebral artery. <a href="https://www.jove.com/files/ftp_upload/62048/62048fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Locating the temporal window 
	NOTE: The temporal window, also called the transtemporal acoustic window, is a part of the skull where the bone is thinnest11, thus allowing transmission of low-frequency ultrasound energy through the cranium (Figure 2).
	<ol>
		<li>For infants and small children, locate the temporal window just in front of the ear (the &#8220;intertragal space&#8221;) and above the rostral edge of the zygomatic arch, which can be easily felt under the skin.&#160;</li>
		<li>For teenagers and young adults, locate the temporal window via any of the subwindows.&#160; 
		NOTE: The posterior subwindow usually provides the best signal (Figure 2).</li>
		<li>For adults aged 30 years or older, locate the temporal window just in front of the ear. 
		NOTE: The acoustic window decreases in size as people age due to increasing porosity of the bone of the cranium, causing some older people to have a very limited temporal window12. In such individuals, bilateral insonation of the MCA is sometimes impossible.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62048/62048fig2.jpg" /> 
Figure 2: The transtemporal window (marked by the dashed ellipse), zygomatic arch (arrow), and subwindows11. (A) Frontal subwindow. (B) Anterior subwindow. (C) Middle subwindow. (D) Posterior subwindow.&#160;<a href="https://www.jove.com/files/ftp_upload/62048/62048fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Applying the transducer
	<ol>
		<li>Apply enough ultrasound gel to cover the surface of the transducer.&#160; 
		&#8203;NOTE: When placed on the head, the gel should cover sufficient space to maintain a seal between the scalp and the Doppler probe&#8217;s surface, thus preventing signal interruption from air coupling underneath the probe&#8217;s surface.</li>
		<li>Alert the subject that the gel may feel cold (if at room temperature).</li>
		<li>Place the transducer on the temporal window, which was located in section 1.2.</li>
	</ol>
	</li>
	<li>Searching for the MCA
	<ol>
		<li>After placing the transducer on the scalp, search for the MCA signal, which will generally be located slightly anterior (forwards) and rostral (towards the head) from the location of the initial transducer scalp placement10.</li>
		<li>If the TCD spectral signal is not immediately obvious, adjust the angle of the transducer while keeping it in the same location relative to the scalp. Slowly angle the probe from rostral to caudal (towards feet) and posterior to anterior.&#160; 
		NOTE: Figure 3 shows two spectra taken from the same position, but at different angles.</li>
		<li>If a signal is still absent after performing step 1.4.2, check the color M-mode display for flow in the MCA at different depths (indicated by red coloring). Increment or decrement the signal depth in 5 mm steps and search as described in step 1.4.2. If flow is visible in M-mode but not in the Doppler spectrum, increase or decrease the depth until the flow signal is visible in the Doppler spectrum.</li>
		<li>If a satisfactory signal is still not obtained, move the transducer to a nearby position on the scalp, which is slightly more anterior, and repeat steps 1.4.1&#8211;1.4.3.&#160;</li>
		<li>When an optimal MCA signal is obtained, note the depth and maximum velocity.</li>
		<li>Using a washable makeup pen, place a mark on the scalp (trace part of the transducer edge) where the optimal signal was found.&#160;</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62048/62048fig3.jpg" /> 
Figure 3: Sample Doppler spectra and M-mode images from midpoint of M1 segment of the MCA. (A) Spectrum taken right after applying transducer to the temporal window, just in front of the ear. (B) Sample Doppler spectrum at same location and depth as (A). The only change is that the transducer has been angled upwards (superiorly) slightly. In both (A) and (B), depth = 50 mm, gain = 50, sample volume = 12 mm, power = 420 mW/cm2, and filter = 100 Hz. <a href="https://www.jove.com/files/ftp_upload/62048/62048fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Searching for the bifurcation 
	NOTE: Finding the bifurcation of the internal carotid artery (ICA) is important to help confirm that the MCA is the artery being monitored. This step should be performed on both sides if bilateral monitoring will be performed, as the bifurcation may not be at the same depth on both sides.
	<ol>
		<li>Increase the depth until the signal from the bifurcation of the ICA into the MCA and ACA is noted (Figure 4), typically at a depth of 51&#8211;65 mm10.</li>
		<li>Search for the optimum bifurcation spectral signal using the procedure described in step 1.4.2. Always strive for the highest-velocity spectral signal possible10.&#160;</li>
		<li>When an optimal bifurcation signal is obtained, note the depth of the bifurcation.</li>
		<li>For bilateral monitoring, repeat sections 1.1&#8211;1.4 and steps 1.5.1&#8211;1.5.3 on the other side of the head.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62048/62048fig4.jpg" /> 
Figure 4: Spectral Doppler (top) and M-mode (bottom) image of bifurcation of the ICA into the MCA and ACA. Depth = 65 mm, gain = 50, sample volume = 12 mm, power = 420 mW/cm2, and filter = 100 Hz.&#160;<a href="https://www.jove.com/files/ftp_upload/62048/62048fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Relocating the MCA after placing a fixation device
NOTE: For fTCD experiments, it is necessary to monitor CBFV for 10&#8211;90 min or longer. Therefore, a fixation device (Figure 5) is crucial to provide stability.
<ol>
	<li>Placing the fixation device
	<ol>
		<li>By visual inspection, adjust the fixation device (Figure 5) to the subject&#8217;s approximate head size.</li>
		<li>Alert the subject before placing the headset on his or her head. Place the headset on the subject&#8217;s head. 
		NOTE: If the subject has long or thick hair, it may be necessary to tie the subject&#8217;s hair back, depending on the fixation device being used.</li>
		<li>Adjust the fixation device&#8217;s fit, and ask the subject if the device is too tight. 
		NOTE: The device should be tight enough that it does not move when bumped slightly, but loose enough that the subject is not uncomfortable.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62048/62048fig5.jpg" /> 
Figure 5: Subject wearing custom fixation device. <a href="https://www.jove.com/files/ftp_upload/62048/62048fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Locating the MCA signal
	<ol>
		<li>Loosen the mechanism of the fixation device holding the transducer in place (e.g., loosen the mechanism, shown in in Figure 5, by turning a knob counterclockwise) so that the transducer can move freely.</li>
		<li>Alert the subject before applying gel to the transducers (which should already be in place from section 2.1), and that the gel may be cold (if it has been stored at room temperature).</li>
		<li>Apply enough ultrasound gel to the transducer to cover the face of the transducer.&#160;</li>
		<li>Adjust the fixation device so that the transducer is located over the top of the mark made in step 1.4.6.</li>
		<li>Search for the optimal MCA spectral signal using the procedure described in steps 1.4.1&#8211;1.4.3. Always strive for the highest-velocity spectral signal possible10.&#160; 
		NOTE: When compared to freehand TCD, the optimal depth at which the MCA is located using the fixation device may differ slightly (at most 1&#8211;2 mm) from the depth for the freehand device. This is because the fixation device may hold the transducer slightly further away from the scalp while still maintaining a coupling gel seal.</li>
		<li>When the optimal MCA spectral signal is found, tighten the mechanism of the fixation device to lock the transducer in place. Note the depth and all other settings.&#160;</li>
		<li>Decrease the power (see step 1.1.1) as much as possible while still maintaining a spectral envelope that traces the maximal velocity accurately.</li>
		<li>For bilateral monitoring, repeat steps 2.2.1&#8211;2.2.7 on the other side.</li>
	</ol>
	</li>
</ol>
3. Performing a breath-hold maneuver
NOTE: This section is given as an example of a functional experiment that may be performed using the experimental setup described in section 1 and section 2.
<ol>
	<li>Perform all steps described in section 1 and section 2.</li>
	<li>Begin recording on the TCD software.</li>
	<li>Breathe normally for 3 min to achieve a good baseline recording, and allow CBFV to stabilize from any previous experiments or stimuli.</li>
	<li>Count down slowly from three. On the count of one, ask the subject to begin breath-holding following a normal inspiration13.&#160; 
	NOTE: The subject should not inhale deeply, as this would decrease carbon dioxide in the lungs and decrease the likelihood of observing the increase in CBFV due to cerebrovascular reactivity. The subject should also avoid performing a Valsalva maneuver, in which intrathoracic pressure is substantially increased against a held inspiration14.&#160;</li>
	<li>Place a marker in the TCD recording to signify the start of breath-holding.</li>
	<li>Have the subject hold their breath for 30 s, or until they are no longer comfortable holding their breath.&#160;</li>
	<li>When the subject inhales, place a marker in the TCD recording to signify the end of breath-holding.</li>
	<li>Continue monitoring CBFV using TCD and recording for at least 30 s following the end of breath-holding to ensure that CBFV returns to baseline values.</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physics+of+functional+magnetic+resonance+imaging+(fMRI).">The physics of functional magnetic resonance imaging (fMRI).</a> Reports on Progress in Physics. 76 (9), 096601 (2013).</li><li>Lohmann, H., Dr&#228;ger, B., M&#252;ller-Ehrenberg, S., Deppe, M., Knecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Language+lateralization+in+young+children+assessed+by+functional+transcranial+Doppler+sonography.">Language lateralization in young children assessed by functional transcranial Doppler sonography.</a> NeuroImage. 24 (3), 780-790 (2005).</li><li>Knecht, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+determination+of+language+lateralization+by+functional+transcranial+Doppler+sonography:+a+comparison+with+the+Wada+test.">Noninvasive determination of language lateralization by functional transcranial Doppler sonography: a comparison with the Wada test.</a> Stroke. 29 (1), 82-86 (1998).</li><li>Knecht, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successive+activation+of+both+cerebral+hemispheres+during+cued+word+generation.">Successive activation of both cerebral hemispheres during cued word generation.</a> Neuroreport. 7 (3), 820-824 (1996).</li><li>Hage, B., Way, E., Barlow, S. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+Doppler+physics+and+techniques,+lecture+notes.">Transcranial Doppler physics and techniques, lecture notes.</a> American Society of Neuroimaging Conference. , (2020).</li><li>Alwatban, M., Truemper, E. J., Al-rethaia, A., Murman, D. L., Bashford, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+breath-hold+acceleration+index:+a+new+method+to+evaluate+cerebrovascular+reactivity+using+transcranial+Doppler.">The breath-hold acceleration index: a new method to evaluate cerebrovascular reactivity using transcranial Doppler.</a> Journal of Neuroimaging. 28 (4), 429-435 (2018).</li><li>Tiecks, F. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+fMRI+in+cognitive+neuroscience.">The future of fMRI in cognitive neuroscience.</a> NeuroImage. 62 (2), 1216-1220 (2012).</li><li>Oh, H., Custead, R., Wang, Y., Barlow, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+encoding+of+saltatory+pneumotactile+velocity+in+human+glabrous+hand.">Neural encoding of saltatory pneumotactile velocity in human glabrous hand.</a> PLoS ONE. 12 (8), 0183532 (2017).</li><li>Rosner, A. O., Barlow, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+changes+in+cortical+sensorimotor+systems+following+hand+and+orofacial+motor+tasks+and+pulsed+pneumotactile+stimulation.">Hemodynamic changes in cortical sensorimotor systems following hand and orofacial motor tasks and pulsed pneumotactile stimulation.</a> Somatosensory &amp; Motor Research. 33 (3-4), 145-155 (2016).</li><li>Alexandrov, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+rate+of+complete+recanalization+and+dramatic+clinical+recovery+during+tPA+infusion+when+continuously+monitored+with+2-MHz+transcranial+doppler+monitoring.">High rate of complete recanalization and dramatic clinical recovery during tPA infusion when continuously monitored with 2-MHz transcranial doppler monitoring.</a> Stroke. 31 (3), 610-614 (2000).</li><li>Watt, B. P., Burnfield, J. M., Truemper, E. J., Buster, T. W., Bashford, G. 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The authors declare no conflicts of interest.

Critical steps in the protocol include 1) finding the MCA, 2) placing the headband, and 3) performing the breath-holding maneuver.
Modifications may be necessary depending on the subjects in the study. For example, subjects with Alzheimer&#8217;s disease may have difficulty following instructions, necessitating the use of a capnograph to ensure compliance with breath-holding instructions15. Young children may have difficulty following i...

In neuroscience research, it is often desirable to monitor real-time brain activity noninvasively in a variety of environments. However, conventional functional neuroimaging modalities have limitations that impede the ability to capture localized and/or rapid activity changes. The true (non-jittered, non-retrospective) temporal resolution of functional magnetic resonance imaging (fMRI) is currently of the order of a few seconds1, which may not capture transient hemodynamic changes linked to transient neural activation. In another example, although functional near-infrared spectroscopy (fNIRS) has high temporal resolution (milliseconds) and reasonable spatial resolution, it can only probe hemodynamic changes within the cerebral cortex and cannot provide information about changes taking place in the larger arteries supplying the brain.
In contrast, fTCD&#8212;classified as a neuroimaging modality&#8212;&#8220;imaging&#8221; refers to the dimensions of time and space, rather than two orthogonal spatial directions that are more familiar in an &#8220;image&#8221;. fTCD provides complementary information to other neuroimaging modalities by measuring high temporal resolution (typically 10 ms) hemodynamic changes at precise locations within vessels of the basal cerebral circulation.&#160;As with other neuroimaging modalities, fTCD may be used for a variety of experiments such as studying lateralization of cerebral activation during language-related tasks2,3,4, studying neural activation in response to various somatosensory stimuli5, and exploring neural activation in various cognitive stimuli such as visual tasks6, mental tasks7, and even tool production8.
Although fTCD offers several advantages for use in functional imaging, including low cost of equipment, portability, and enhanced safety (compared to Wada test3 or positron emission tomography [PET] scans), operation of a TCD machine requires skills obtained by practice. Some of these skills, which must be learned by a TCD operator, include the ability to identify various cerebral arteries and the motor skills necessary to precisely manipulate the ultrasound probe during the search for the relevant artery. The goal of this Methods paper is to present a technique for using fTCD to perform a functional imaging experiment. First, the basic steps for identifying and optimizing the signal from the MCA, which perfuses 80% of the cerebral hemisphere9, will be listed. Next, placement of a fixation device for holding the TCD probe in place during the experiment will be described. Finally, the breath-holding experiment, which is one example of a functional imaging experiment using fTCD, will be described, and representative results will be shown.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aquasonic</td>
			<td>Parker Laboratories, Inc., Fairfield, NJ, USA</td>
			<td>01-50</td>
			<td>Ultrasound Gel</td>
		</tr>
		<tr>
			<td>Doppler Box X</td>
			<td>DWL Compumedics Gmbh, Singen, Germany</td>
			<td>Model &#34;BoxX&#34;</td>
			<td>Transcranial Doppler with 2-MHz monitoring probes</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark Professional</td>
			<td>34256</td>
			<td>Delicate Task Wipers</td>
		</tr>
		<tr>
			<td>Transeptic&#160;</td>
			<td>Parker Laboratories, Inc., Fairfield, NJ, USA</td>
			<td>09-25</td>
			<td>Cleaning Spray</td>
		</tr>
	</tbody>
</table>

functional transcranial doppler ultrasound for monitoring cerebral blood flow

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli such as physical movement, activation of tactile sensors in the skin, and viewing images. Neural activation is inferred from an increase in the cerebral blood flow velocity (CBFV) supplying the region of the brain involved in processing sensory input. For example, viewing bright light causes increased neural activity in the occipital lobe of the cerebral cortex, leading to increased blood flow in the posterior cerebral artery, which supplies the occipital lobe. In fTCD, changes in CBFV are used to estimate changes in cerebral blood flow (CBF).
With its high temporal resolution measurement of blood flow velocities in the major cerebral arteries, fTCD complements other established functional imaging techniques. The goal of this Methods paper is to give step-by-step instructions for using fTCD to perform a functional imaging experiment. First, the basic steps for identifying the middle cerebral artery (MCA) and optimizing the signal will be described. Next, placement of a fixation device for holding the TCD probe in place during the experiment will be described. Finally, the breath-holding experiment, which is a specific example of a functional imaging experiment using fTCD, will be demonstrated.

Figure 3 shows sample Doppler spectra and color M-modes from the midpoint of the M1 segment of the MCA. Figure 3A,B were taken at the same position on the scalp, but at different angles. Note how a very small change in angle, without changing the contact position on the scalp, can greatly improve Doppler signal strength, as shown by the higher-intensity yellow coloring of the spectrogram in Figure 3B. Note ...

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Functional Transcranial Doppler Ultrasound for Monitoring Cerebral Blood Flow

Functional transcranial Doppler ultrasound complements other functional imaging modalities, with its high temporal resolution measurement of stimulus-induced changes in cerebral blood flow within the basal cerebral arteries. This Methods paper gives step-by-step instructions for using functional transcranial Doppler ultrasound to perform a functional imaging experiment.&#160;

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli ...

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7.3K Views.  University of Nebraska-Lincoln. Functional transcranial Doppler ultrasound complements other functional imaging modalities, with its high temporal resolution measurement of stimulus-induced changes in cerebral blood flow within the basal cerebral arteries. This Methods paper gives step-by-step instructions for using functional transcranial Doppler ultrasound to perform a functional imaging experiment. 

Video: Functional Transcranial Doppler Ultrasound for Monitoring Cerebral Blood Flow

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence (e.g. ferricrocin or N',N",N'''-triacetylfusarinine C)1. The reaction catalyzed by SidA can be divided into reductive and oxidative half-reactions (Scheme 1). In the reductive half-reaction, the oxidized FAD bound to Af SidA, is reduced by NADPH2,3. In the oxidative half-reaction, the reduced cofactor reacts with molecular oxygen to form a C4a-hydroperoxyflavin intermediate, which transfers an oxygen atom to ornithine. Here, we describe a procedure to measure the rates and detect the different spectral forms of SidA using a stopped-flow instrument installed in an anaerobic glove box. In the stopped-flow instrument, small volumes of reactants are rapidly mixed, and after the flow is stopped by the stop syringe (Figure 1), the spectral changes of the solution placed in the observation cell are recorded over time. In the first part of the experiment, we show how we can use the stopped-flow instrument in single mode, where the anaerobic reduction of the flavin in Af SidA by NADPH is directly measured. We then use double mixing settings where Af SidA is first anaerobically reduced by NADPH for a designated period of time in an aging loop, and then reacted with molecular oxygen in the observation cell (Figure 1). In order to perform this experiment, anaerobic buffers are necessary because when only the reductive half-reaction is monitored, any oxygen in the solutions will react with the reduced flavin cofactor and form a C4a-hydroperoxyflavin intermediate that will ultimately decay back into the oxidized flavin. This would not allow the user to accurately measure rates of reduction since there would be complete turnover of the enzyme. When the oxidative half-reaction is being studied the enzyme must be reduced in the absence of oxygen so that just the steps between reduction and oxidation are observed. One of the buffers used in this experiment is oxygen saturated so that we can study the oxidative half-reaction at higher concentrations of oxygen. These are often the procedures carried out when studying either the reductive or oxidative half-reactions with flavin-containing monooxygenases. The time scale of the pre-steady-state experiments performed with the stopped-flow is milliseconds to seconds, which allow the determination of intrinsic rate constants and the detection and identification of intermediates in the reaction4. The procedures described here can be applied to other flavin-dependent monooxygenases.5,6

We describe the use of a stopped-flow instrument to investigate both the reductive and oxidative half-reactions of Aspergillus fumigatus siderophore A (SidA), a flavin-dependent monooxygenase. We then show the spectra corresponding to the species in the reaction of SidA and we calculate the rate constants for their formation.

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...