We acknowledge the support by the S&#227;o Paulo Research Foundation (FAPESP) through Proc. 2012/02500-8 (RM), 2014/25486-6 (RF) and 2013/07559-3. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The protocol was approved by the local committee of the University of Campinas (protocol number 56602516.2.0000.5404). Written informed consent was obtained from the patient or a legal representative prior to the measurements. We monitored patients that were admitted to the Clinics Hospital at the University of Campinas with a diagnosis of either ischemic stroke or a subarachnoid hemorrhage affecting the anterior circulation. Patients with ischemic strokes affecting the posterior circulation, patients with decompressive craniectomies due to elevated intracranial pressure and patients with other neurodegenerative diseases (dementia, Parkinson&#39;s or any other disease that can be associated with cortical atrophy) were excluded from the study protocol.
1. Preparations before moving the system to the ICU
<ol>
	<li>Connect all the fibers to the relevant lasers and detectors, and make sure they are properly attached to the optical probe (Figure 1B).</li>
	<li>Check that the optical probe is covered with a black cloth to avoid the lasers shining in the room.</li>
	<li>Turn the system power switch to the &#8216;ON&#8217; position. After powering the system, wait 30s and then turn the DCS laser key switch to the &#8216;ON&#8217; position. The FD-DOS lasers are automatically turned on when the system is powered.</li>
	<li>While the system is being prepared, obtain consent from either the participant or a legal representative. After obtaining consent, bring the cart to the patient&#8217;s room. 
	NOTE: Since the hybrid system has a built-in battery that lasts up to 45 min, it does not need to be turned off during transport.</li>
</ol>
2. Calibration and gain settings of the DOS system
<ol>
	<li>Upon arrival at the ICU, turn off the DCS laser by switching the key to the &#8216;OFF&#8217; position.</li>
	<li>Starting with the solid phantom marked &#8216;Calibrate&#8217;, run the calibration process on the FD-DOS software (BOXY, ISS) by following the steps below.
	<ol>
		<li>On the &#8216;File&#8217; menu, load the appropriate settings file for the probe being used by clicking on the &#8216;Load settings file&#8217; option.</li>
		<li>Place the probe on the curved side of the phantom, ensuring a good contact with the surface and then optimize the PMT bias voltage by clicking on the &#8216;Optimize All Detectors&#8217; button in the FD-DOS software.</li>
		<li>Run the calibration for multiple source-detector separations by clicking on the option &#8216;Calc. Waveform Calib. Values for Optical Props. and Multiple Distances&#8217; from the &#8216;Calibrate&#8217; menu.</li>
		<li>Open the &#8216;User-defined Calculations&#8217; option from the &#8216;Text-Mon&#8217; menu to check that the measured optical properties match the prespecified values (written in the solid phantom), and that the fitting R2 is close to one.</li>
	</ol>
	</li>
	<li>Repeat the steps above (except step 2.2.3) to measure the optical properties of the phantom marked as &#8216;Check&#8217; to ensure the calibration was adequate. The measured optical properties should match, within 10%, the values specified in the phantoms. 
	CAUTION: Make sure to turn off PMTs (by clicking on the &#8216;All Detectors OFF&#8217; button) every time the probe is moved to avoid damaging PMTs due to direct illumination from ambient light.</li>
	<li>If the calibration is not adequate, re-run the calibration process (steps 2.2 and 2.3). Ensuring a good calibration of the FD-DOS system is essential to the validity of the FD-DOS measurements.</li>
</ol>
3. Preparation of the participant at the bedside
<ol>
	<li>Use sanitizing wipes to clean both the probe and patient forehead.</li>
	<li>Place the double-sided tape on the probe (Figure 1B), ensuring the tape is not in direct contact with the optical fiber tips.</li>
	<li>Place a laser safety googles on the subject.</li>
	<li>Place the probes over the region-of-interest (ROI) and wrap the elastic straps around the subject&#8217;s head. Although not strictly necessary for FD-DOS and DCS, it is advisable to cover the optical probe with a black cloth or black bandage to reduce noise due to ambient light. 
	NOTE: It is important to assure that the elastic strap is neither too tight&#160;nor too loose. If the strap is too tight it may cause significant discomfort to the patient, and if the strap is too loose it may lead to poor data quality as the double-sided tape is not strong enough to keep the probes in place.</li>
	<li>After the probe is properly secured to the patient&#39;s forehead, turn on the DCS laser by switching the key to the &#8216;ON&#8217; position. 
	CAUTION: The DCS system uses a Class 3B laser which is hazardous for eye exposure. It is very important to only turn on the lasers when the probe is properly attached to the patient&#8217;s forehead.</li>
</ol>
4. Data quality assessment
<ol>
	<li>Before starting to acquire data with the GUI, write the DCS source-detector separations in the &#8216;Settings&#8217; tab of the GUI. 
	NOTE: The DCS system does not require a calibration step, but the proper input of the source-detector separations is necessary for the real-time analysis (see Supplementary Material for details).</li>
	<li>Start the acquisition software by pressing the &#8216;Start&#8217; button in the GUI and check the DOS signal in the FD-DOS software:
	<ol>
		<li>Click on the &#8216;Optimize All Detectors&#8217; button in the FD-DOS software to optimize the PMT bias voltage.</li>
		<li>Check the optical properties and the R2 of the DOS fitting in the &#8216;User Defined Calculation&#8217; option from the &#8216;Text-Mon&#8217; menu. The R2 coefficient should be close to unity and, as a rule of thumb, the absorption coefficient of human patients should be within 0.05 and 0.2 cm-1, while the scattering coefficient should be within 6 and 13 cm-113.</li>
	</ol>
	</li>
	<li>Check the DCS signal in the &#8216;Correlation curves&#8217; tab of the GUI.
	<ol>
		<li>Turn on the DCS detectors by turning the switches to the &#8216;ON&#8217; position.</li>
		<li>Ensure that each DCS detector is measuring an adequate light intensity. As a rule of thumb, more than 10 kHz is required.</li>
		<li>If the measured intensity is higher than 800 kHz, use a neutral density filter to reduce the photon counts to avoid damaging the detectors. This is typically a problem for shorter (&#60; 1 cm) source-detector separations. 
		NOTE: Apart from potentially damaging the DCS detectors, photon counts higher than 800 kHz may also bring errors due to non-linear effects in the detector.</li>
		<li>Check the autocorrelation curves to ensure a good skin coupling (see the Representative Results and Figure 2) and reposition the optical probe if necessary.</li>
		<li>If the repositioning of the probe was necessary in the previous step, repeat Steps 4.2 and 4.3. These steps may need to be repeated multiple times. 
		NOTE: The DCS and the FD-DOS detectors must be turned off each time the probe is moved. To turn the DCS detectors off, manually move the switches to the &#8216;OFF&#8217; position. The FD-DOS detector is turned off by clicking the &#8216;All Detectors OFF&#8217; button in the FD-DOS software.</li>
	</ol>
	</li>
	<li>When a good contact between the probe and the skin is achieved, stop the data collection by clicking the &#8216;Stop&#8217; button in the GUI. Then, set the experiment and patient identifiers in the &#8216;Folder&#8217; textbox and write the ROI name in the &#8216;File name&#8217; textbox.</li>
	<li>Start the data acquisition by pressing the &#8216;Start&#8217; button in the GUI.</li>
	<li>Collect data in the first ROI for as long as required by the protocol. If necessary, move the probe to the other ROIs and repeat the measurement. 
	NOTE: The monitoring period may vary depending on the study goals.</li>
</ol>
5. Considerations for the experimenter during the measurement
<ol>
	<li>After starting the measurement, write in the &#8216;Experiment Info&#8217; tab of the GUI the relevant patient information (e.g., type and location of the injury, drugs being administered, age, sex, etc.).</li>
	<li>Ensure that any relevant event that occurred during the monitoring period is marked by clicking the &#8216;Mark&#8217; button on the GUI. After each mark, make sure to write the event description in the &#8216;Experiment Info&#8217; tab of the GUI.</li>
</ol>
6. Stop data collection
<ol>
	<li>Stop the data collection by pressing the &#8216;Stop&#8217; button in the GUI.</li>
	<li>Stop the FD-DOS software by pressing the stop data acquisition and recording button represented as two red squares in the FD-DOS software.</li>
	<li>Turn off the DCS detectors by flipping the switches to the &#8216;OFF&#8217; position and turn off the DCS laser by turning the key to the &#8216;OFF&#8217; position.</li>
	<li>Turn off the PMTs of the FD-DOS module by clicking the &#8216;All Detectors OFF&#8217; button.</li>
	<li>Remove the probe from the patient&#39;s head and remove the double-sided tape from the probe. Then, clean the probe with sanitizing wipes.</li>
	<li>Repeat the measurement of the optical properties of each solid phantom as soon as possible to ensure the calibration remained adequate throughout the monitoring session (see step 4.2.2). 
	NOTE: Ideally, the calibration step should be done right after removing the optical probes from the patient&#39;s head (step 6.6). However, due to timing issues, in the examples presented in the next section this was done in the storage facility.</li>
	<li>Clean the system and its accessories with sanitizing wipes.</li>
	<li>Wheel the cart back to the storage room.</li>
</ol>

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: RC Mesquita has one pending patent application and two other patents relevant to this work (United States patents 10,342,488 and 10,064,554). No author currently receives royalties or payments from these patents.
&#160; &#160;

This paper presented a hybrid optical system that can provide real-time information about cerebral blood flow, cerebral oxygenation, and cerebral oxygen metabolism of neurocritical patients at the beside. The use of diffuse optical techniques had been previously addressed as a potential marker for non-invasive, bedside monitoring in clinical scenarios. A previous study focused on the clinical aspects and the feasibility of optical monitoring during hospitalization in the neuro-ICU through a case report9...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61608">Real-Time Monitoring of Neurocritical Patients with Diffuse Optical Spectroscopies</a>. The Authors section was updated from:
Rodrigo Menezes Forti1,2 
Marilise Katsurayama2,3 
Lenise Valler2,3 
Andr&#233;s Quiroga1,2 
Luiz Simioni1 
Julien Menko4 
Antonio L. E. Falc&#227;o3 
Li Min Li2,5 
Rickson C. Mesquita1,2 
1Institute of Physics, University of Campinas 
2Brazilian Institute of Neuroscience and Neurotechnology 
3Clinical Hospital, University of Campinas 
4Department of Emergency Medicine, Albert Einstein College of Medicine 
5School of Medical Sciences, University of Campinas
to:
Rodrigo Menezes Forti1,2 
Marilise Katsurayama2,3 
Giovani Grisotti Martins1 
Lenise Valler2,3 
Andr&#233;s Quiroga1,2 
Luiz Simioni1 
Julien Menko4 
Antonio L. E. Falc&#227;o3 
Li Min Li2,5 
Rickson C. Mesquita1,2 
1Institute of Physics, University of Campinas 
2Brazilian Institute of Neuroscience and Neurotechnology 
3Clinical Hospital, University of Campinas 
4Department of Emergency Medicine, Albert Einstein College of Medicine 
5School of Medical Sciences, University of Campinas

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61608">Real-Time Monitoring of Neurocritical Patients with Diffuse Optical Spectroscopies</a>. The Authors section was updated.

Erratum: Real-Time Monitoring of Neurocritical Patients with Diffuse Optical Spectroscopies

Most of the complications that lead to poor outcomes for critically ill neurologic patients are related to secondary injuries caused by cerebral hemodynamic impairments. Therefore, monitoring cerebral physiology of these patients may directly impact morbidity and mortality rates1,2,3,4,5,6,7. Currently, however, there is no established clinical tool for the continuous real-time noninvasive monitoring of cerebral physiology in&#160;neurocritical patients at the bedside. Among the potential candidates, diffuse optical techniques have recently been proposed as a promising tool to fill in this gap8,9,10,11. By measuring the slow changes (i.e., on the order of tens to hundreds of ms) of the diffusively scattered near-infrared light (~650-900 nm) from the scalp, diffuse optical spectroscopy (DOS) can measure concentrations of the main chromophores in the brain, such as cerebral oxy- (HbO) and deoxy-hemoglobin (HbR)12,13. Additionally, it is possible to measure cerebral blood flow (CBF) with diffuse correlation spectroscopy (DCS)10,14,15,16,17 by quantifying the rapid fluctuations in light intensity (i.e., from a few &#181;s to a few ms). When combined, DOS and DCS can also provide an estimate of the cerebral metabolic rate of oxygen (CMRO2)18,19,20.
The combination of DOS and DCS has been explored to monitor patients in several pre-clinical and clinical scenarios. For example, diffuse optics has been shown to provide relevant clinical information for critically-ill neonates21,22,23,24, including during cardiac surgeries to treat heart defects23,25,26,27,28. In addition, several authors have explored the use of diffuse optics to assess cerebral hemodynamics during different cerebrovascular interventions, such as carotid endarterectomy29,30,31, thrombolytic treatments for stroke32, head-of-bed manipulations33,34,35, cardiopulmonary resuscitation36, and others37,38,39. When continuous blood pressure monitoring is also available, diffuse optics can be used to monitor cerebral autoregulation, both in healthy and in critically ill subjects11,40,41,42, as well to assess the critical closing pressure of the cerebral circulation43. Several authors have validated CBF measurements with DCS against different gold standard CBF measures18, while CMRO2 measured with diffuse optics has been shown to be a useful parameter for neurocritical monitoring8,18,23,24,28,43,44,45. In addition, previous studies have validated the optically-derived cerebral hemodynamic parameters for long-term monitoring of neurocritical patients8,9,10,11, including for the prediction of hypoxic46,47,48 and ischemic events8.
The reliability of the diffuse optical techniques to provide valuable real-time information during longitudinal measurements as well as during clinical interventions remains largely unaddressed. The use of a standalone DOS system was previously compared to invasive brain tissue oxygen tension monitors, and DOS was deemed to not have a sufficient sensibility to replace the invasive monitors. However, apart from using relatively small populations, the direct comparison of the invasive and non-invasive monitors may be misguided as each technique probe different volumes containing different parts of the cerebral vasculature. Even though these studies ultimately concluded that diffuse optics is not a replacement for the invasive monitors, in both studies DOS achieved a moderate-to-good accuracy, which may be sufficient for cases and/or places wherein invasive monitors are not available.
Relative to other approaches, the key advantage of diffuse optics is its ability to simultaneously measure blood flow and tissue blood oxygenation non-invasively (and continuously) at the bedside using portable instrumentation. Compared to Transcranial Doppler ultrasound (TCD), DCS has an additional advantage: it measures perfusion at the tissue level, whereas TCD measures cerebral blood flow velocity in large arteries at the base of the brain. This distinction may be particularly important when evaluating steno-occlusive diseases in which both proximal large artery flow and leptomeningeal collaterals contribute to perfusion. Optical techniques also have advantages when compared to other traditional imaging modalities, such as Positron-Emission Tomography (PET) and Magnetic Resonance Imaging (MRI). In addition to simultaneously providing direct measures of both CBF and HbO/HbR concentrations, which is not possible with MRI or PET alone, optical monitoring also provides significantly better temporal resolution, allowing, for example, the assessment of dynamic cerebral autoregulation40,41,42 and the assessment dynamically evolving hemodynamical changes. Moreover, diffuse optical instrumentation is inexpensive and portable in comparison to PET and MRI, which is a critical advantage given the high burden of vascular disease in lower- and middle-income countries.
The protocol proposed here is an environment for real-time bedside neuromonitoring of patients at the intensive care unit (ICU). The protocol uses a hybrid optical device together with a clinical-friendly graphical user interface (GUI) and customized optical sensors to probe the patients (Figure 1). The hybrid system employed for showcasing this protocol combines two diffuse optical spectroscopies from independent modules: a commercial frequency-domain (FD-) DOS module and a homemade DCS module (Figure 1A). The FD-DOS module49,50 consists of 4 photomultiplier tubes (PMTs) and 32 laser diodes emitting at four different wavelengths (690, 704, 750 and 850 nm). The DCS module consists of a long-coherence laser emitting at 785 nm, 16 single-photon counters as detectors and a correlator board. The sampling frequency for the FD-DOS module is 10 Hz, and the maximum sampling frequency for the DCS module is 3 Hz. To integrate the FD-DOS and DCS modules, a microcontroller was programmed inside our control software to automatically switch between each module. The microcontroller is responsible for turning the FD-DOS and DCS lasers on and off, as well as the FD-DOS detectors to allow interleaved measurements of each module. In total, the proposed system can collect one combined FD-DOS and DCS sample every 0.5 to 5s, depending on the signal-to-noise ratio (SNR) requirements (longer collection times leads to better SNR). To couple the light to the forehead, we developed a 3D-printed optical probe that can be customized for each patient (Figure 1B), with source-detector separations varying between 0.8 and 4.0 cm. The standard source-detector separations used in the examples presented here are 2.5 cm for DCS and 1.5, 2.0, 2.5 and 3.0 cm for FD-DOS.
The main feature of the protocol presented in this study is the development of a real-time interface that can both control the hardware with a friendly GUI and display the main cerebral physiology parameters in real-time under different temporal windows (Figure 1C). The real-time analysis pipeline developed within the proposed GUI is fast and takes less than 50 ms to compute the optical parameters (see the Supplementary Material for more details). The GUI was inspired by current clinical instruments already available at the neuro-ICU, and it was adapted through extensive feedback by clinical users during the translation of the system to the neuro-ICU. Consequently, the real-time GUI can facilitate&#160;the adoption of the optical system by regular hospital staff, such as neurointensivists and nurses. The wide adoption of diffuse optics as a clinical research tool has the potential to enhance its ability to monitor physiologically meaningful data and can ultimately demonstrate that diffuse optics is a good option for non-invasively monitoring neurocritical patients in real-time.

<table><tbody><tr><td>3D Printer</td><td>Sethi3D</td><td>S2</td><td>3D-printer used to print the customizable probes</td></tr><tr><td>Arduino UNO</td><td>Arduino</td><td>UNO REV3</td><td>Microcontroller responsible to interleave the DCS and FD-DOS measurements</td></tr><tr><td>DCS Correlator</td><td>Correlator.com</td><td>Flex11-16ch</td><td>Component of the DCS module</td></tr><tr><td>DCS Dectectors IO Boards</td><td>Excelitas Technology</td><td>SPCM-AQ4C-IO</td><td>Component of the DCS module</td></tr><tr><td>DCS Detectors</td><td>Excelitas Technology</td><td>SPCM-AQ4C</td><td>Component of the DCS module</td></tr><tr><td>DCS Laser</td><td>CrystaLaser</td><td>DL785-120-SO</td><td>Component of the DCS module</td></tr><tr><td>DCS Power supply</td><td>Artesyn</td><td>UMP10T-S2A-S2A-S2A-S2A-IES-00-A</td><td>Component of the DCS module (power supply for the DCS detecto; 2, 5 and 30V)</td></tr><tr><td>FD-DOS fibers</td><td>ISS</td><td>Imagent supplies</td><td>The fibers used for FD-DOS detection and illumination are provived by ISS</td></tr><tr><td>Flexible 3D printer material</td><td>Sethi3D</td><td>NinjaFlex</td><td>Material used to print the flexible customizable probes</td></tr><tr><td>Imagent</td><td>ISS</td><td>Imagent</td><td>FD-DOS module</td></tr><tr><td>Laser safety googles</td><td>Thorlabs</td><td>LG9</td><td></td></tr><tr><td>Multi-mode fiber</td><td>Thorlabs</td><td>FT400EMT</td><td>Multi-mode fiber used for DCS illumination</td></tr><tr><td>Neutral density filter 1.0 OD</td><td>Edmund Optics</td><td>53-705</td><td>Neutral density filter for the short source detector separations</td></tr><tr><td>Single-mode optical fiber</td><td>Thorlabs</td><td>780HP</td><td>Single-mode optical fiber used for the DCS detectors</td></tr><tr><td>System battery</td><td>SMS</td><td>NET4</td><td>System battery used for transportation</td></tr></tbody></table>

real-time monitoring of neurocritical patients with diffuse optical spectroscopies

Neurophysiological monitoring is an important goal in the treatment of neurocritical patients, as it may prevent secondary damage and directly impact morbidity and mortality rates. However, there is currently a lack of suitable non-invasive, real-time technologies for continuous monitoring of cerebral physiology at the bedside. Diffuse optical techniques have been proposed as a potential tool for bedside measurements of cerebral blood flow and cerebral oxygenation in case of neurocritical patients. Diffuse optical spectroscopies have been previously explored to monitor patients in several clinical scenarios ranging from neonatal monitoring to cerebrovascular interventions in adults. However, the feasibility of the technique to aid clinicians by providing real-time information at the bedside remains largely unaddressed. Here, we report the translation of a diffuse optical system for continuous real-time monitoring of cerebral blood flow, cerebral oxygenation, and cerebral oxygen metabolism during intensive care. The real-time feature of the instrument could enable treatment strategies based on patient-specific cerebral physiology rather than relying on surrogate metrics, such as arterial blood pressure. By providing real-time information on the cerebral circulation at different time scales with relatively cheap and portable instrumentation, this approach may be especially useful in low-budget hospitals, in remote areas and for&#160;monitoring in open fields (e.g., defense and sports).

Ideally, the normalized autocorrelation curves obtained with the DCS module should be approximately 1.5 at the zero delay-time extrapolation (when using single-mode fibers14), and the curves should decay to 1 at longer delay times. The curve should be smooth, and it should have a faster decay for the longer source-detector separations. An example of a good autocorrelation is shown in Figure 2A. Figure 2B shows an example of a bad auto-cor...

Watch this Scientific Journal Video about Real-Time Monitoring of Neurocritical Patients with Diffuse Optical Spectroscopies at JoVE.com

Real-Time Monitoring of Neurocritical Patients with Diffuse Optical Spectroscopies

Presented here is a protocol for non-invasively monitoring cerebral hemodynamics of neurocritical patients in real-time and at the bedside using diffuse optics. Specifically, the proposed protocol uses a hybrid diffuse optical systems to detect and display real-time information on cerebral oxygenation, cerebral blood flow and cerebral metabolism.

Neurophysiological monitoring is an important goal in the treatment of neurocritical patients, as it may prevent secondary damage and directly impact ...

real-time-monitoring-neurocritical-patients-with-diffuse-optical

Research

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Medicine

2.1K Views.  University of Campinas. Hier wordt een protocol gepresenteerd voor het niet-invasief monitoren van cerebrale hemodynamiek van neurokritische patiënten in realtime en aan het bed met behulp van diffuse optica. In het bijzonder maakt het voorgestelde protocol gebruik van een hybride diffuus optisch systeem om real-time informatie over cerebrale oxygenatie, cerebrale bloedstroom en cerebrale metabolisme te detecteren en weer te geven.

Video: Real-Time Monitoring of Neurocritical Patients with Diffuse Optical Spectroscopies

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="http://www.jove.com/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

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

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 ...

Bioengineering

A Model to Simulate Clinically Relevant Hypoxia in Humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...

A Detailed Protocol for Physiological Parameters Acquisition and Analysis in Neurosurgical Critical Patients

Intracranial pressure (ICP) monitoring is now widely used in neurosurgical critical patients. Besides mean ICP value, the ICP derived parameters such as ICP waveform, amplitude of pulse (AMP), the correlation of ICP amplitude and ICP mean (RAP), pressure reactivity index (PRx), ICP and arterial blood pressure (ABP) wave amplitude correlation (IAAC), and so on, can reflect intracranial status, predict prognosis, and can also be used as guidance of proper treatment. However, most of the clinicians focus only on the mean ICP value while ignoring these parameters because of the limitations of the current devices. We have recently developed a multimodality monitoring system to address these drawbacks. This portable, user-friendly system will use a data collecting and storing device to continuously acquire patients&#39; physiological parameters first, i.e., ABP, ICP, and oxygen saturation, and then analyze these physiological parameters. We hope that the multimodality monitoring system will be accepted as a key measure to monitor physiological parameters, to analyze the current clinical status, and to predict the prognosis of the neurosurgical critical patients.

Recently, we have developed a portable multimodality monitoring system for the monitoring of various physiological parameters in neurosurgical critical patients. Detailed protocols on how to use this multimodality monitoring system are presented here.

Intracranial pressure (ICP) monitoring is now widely used in neurosurgical critical patients. Besides mean ICP value, the ICP derived parameters such as ...

Standardized Hemorrhagic Shock Induction Guided by Cerebral Oximetry and Extended Hemodynamic Monitoring in Pigs

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an insufficient oxygen supply and irreversible organ failure. The brain exerts only limited compensation capacities and is particularly at high risk of severe hypoxic damage.This article demonstrates the reproducible induction of life-threatening hemorrhagic shock in a porcine model by means of calculated blood withdrawal. We titrate shock induction guided by near-infrared spectroscopy&#160;and extended hemodynamic monitoring to display systemic circulatory failure, as well as cerebral microcirculatory oxygen depletion. In comparison to similar models that primarily focus on predefined removal volumes for shock induction, this approach highlights a titration by means of the resulting failure of macro- and microcirculation.

Hemorrhagic shock is a severe complication in seriously injured patients, which leads to life-threatening oxygen undersupply. We present a standardized method to induce hemorrhagic shock via blood withdrawal in pigs that is guided by hemodynamics and microcirculatory cerebral oxygenation.

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an ...

How to Administer Near-Infrared Spectroscopy in Critically ill Neonates, Infants, and Children

Near infrared spectroscopy (NIRS) calculates regional tissue oxygenation (rSO2) using the different absorption spectra of oxygenated and deoxygenated hemoglobin molecules. A probe placed on the skin emits light that is absorbed, scattered, and reflected by the underlying tissue. Detectors in the probe sense the amount of reflected light: this reflects the organ-specific ratio of oxygen supply and consumption - independent of pulsatile flow. Modern devices enable the simultaneous monitoring at different body sites. A rise or dip in the rSO2 curve visualizes changes in oxygen supply or demand before vital signs indicate them. The evolution of rSO2 values in relation to the starting point is more important for interpretation than are absolute values.
A routine clinical application of NIRS is the surveillance of somatic and cerebral oxygenation during and after cardiac surgery. It is also administered in preterm infants at risk for necrotizing enterocolitis, newborns with hypoxic ischemic encephalopathy and a potential risk of impaired tissue oxygenation. In the future, NIRS could be increasingly used in multimodal neuromonitoring, or applied to monitor patients with other conditions (e.g., after resuscitation or traumatic brain injury).

This protocol is designed to assist clinicians to measure regional tissue oxygenation at different body sites in infants and children. It can be used in situations where tissue oxygenation is potentially compromised, particularly during cardiopulmonary bypass, when using non-pulsatile cardiac-assist devices, and in critically ill neonates, infants and children.

Near infrared spectroscopy (NIRS) calculates regional tissue oxygenation (rSO2) using the different absorption spectra of oxygenated and deoxygenated ...

Simultaneous Data Collection of fMRI and fNIRS Measurements Using a Whole-Head Optode Array and Short-Distance Channels

Functional near-infrared spectroscopy (fNIRS) is a portable neuroimaging methodology, more robust to motion and more cost-effective than functional magnetic resonance imaging (fMRI), which makes it highly suitable for conducting naturalistic studies of brain function and for use with developmental and clinical populations. Both fNIRS and fMRI methodologies detect changes in cerebral blood oxygenation during functional brain activation, and prior studies have shown high spatial and temporal correspondence between the two signals. There is, however, no quantitative comparison of the two signals collected simultaneously from the same subjects with whole-head fNIRS coverage. This comparison is necessary to comprehensively validate area-level activations and functional connectivity against the fMRI gold standard, which in turn has the potential to facilitate comparisons of the two signals across the lifespan. We address this gap by describing a protocol for simultaneous data collection of fMRI&#160;and fNIRS signals that: i) provides whole-head fNIRS coverage; ii) includes short-distance measurements for regression of the non-cortical, systemic physiological signal; and iii) implements two different methods for optode-to-scalp co-registration of fNIRS measurements. fMRI and fNIRS data from three subjects are presented, and recommendations for adapting the protocol to test developmental and clinical populations are discussed. The current setup with adults allows scanning sessions for an average of approximately 40 min, which includes both functional and structural scans. The protocol outlines the steps required to adapt the fNIRS equipment for use in the magnetic resonance (MR) environment, provides recommendations for both data recording and optode-to-scalp co-registration, and discusses potential modifications of the protocol to fit the specifics of the available MR-safe fNIRS system. Representative subject-specific responses from a flashing-checkerboard task illustrate the feasibility of the protocol to measure whole-head fNIRS signals in the MR environment. This protocol will be particularly relevant for researchers interested in validating fNIRS signals against fMRI across the lifespan.

We present a method for simultaneously collecting fMRI and fNIRS signals from the same subjects with whole-head fNIRS coverage. The protocol has been tested with three young adults and can be adapted for data collection for developmental studies and clinical populations.

Functional near-infrared spectroscopy (fNIRS) is a portable neuroimaging methodology, more robust to motion and more cost-effective than functional ...

Non-Invasive Monitoring of Microvascular Oxygenation and Reactive Hyperemia using Hybrid, Near-Infrared Diffuse Optical Spectroscopy for Critical Care

The detection of levels of impairment in microvascular oxygen consumption and reactive hyperemia is vital in critical care. However, there are no practical means for a robust and quantitative evaluation. This paper describes a protocol to evaluate these impairments using a hybrid near-infrared diffuse optical device. The device contains modules for near-infrared time-resolved and diffuse correlation spectroscopies and pulse-oximetry. These modules allow the non-invasive, continuous, and real-time measurement of the absolute, microvascular blood/tissue oxygen saturation (StO2) and the blood flow index (BFI) along with the peripheral arterial oxygen saturation (SpO2). This device uses an integrated, computer-controlled tourniquet system to execute a standardized protocol with optical data acquisition from the brachioradialis muscle. The standardized vascular occlusion test (VOT) takes care of the variations in the occlusion duration and pressure reported in the literature, while the automation minimizes inter-operator differences. The protocol we describe focuses on a 3-min occlusion period but the details described in this paper can readily be adapted to other durations and cuff pressures, as well as other muscles. The inclusion of an extended baseline and post-occlusion recovery period measurement allows the quantification of the baseline values for all the parameters and the blood/tissue deoxygenation rate that corresponds to the metabolic rate of oxygen consumption. Once the cuff is released, we characterize the tissue reoxygenation rate, magnitude, and duration of the hyperemic response in BFI and StO2. These latter parameters correspond to the quantification of the reactive hyperemia, which provides information about the endothelial function. Furthermore, the above-mentioned measurements of the absolute concentration of oxygenated and deoxygenated hemoglobin, BFI, the derived metabolic rate of oxygen consumption, StO2, and SpO2 provide a yet-to-be-explored rich data set that can exhibit disease severity, personalized therapeutics, and management interventions.

We describe a protocol to non-invasively and continuously measure absolute microvascular blood flow index and blood oxygen saturation using a multi-modal device based on near-infrared diffuse optics. We then evaluate the metabolic rate of oxygen consumption and reactive hyperemia utilizing a vascular occlusion test.

Real-Time Monitoring of Neurocritical Patients with Diffuse Optical Spectroscopies

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