Supported by NIH awards RO1 HL073087, GM 077185, and GM 069589 to CKS. The work is also supported by a grant from the Regenerative Medicine program of the DHLRI. The authors are grateful to the technical support and clinical inputs from the following personnel at the Ohio State University: Dr. Sabyasachi.Biswas (Comprehensive Wound Center), Dr. Allison Spiwak (Circulation Technology Division), Joseph Agoston (Circulation Division), Thoma Shives (Circulation Division), Joseph Ewing (Mechanical Engineering), Scott Killinger (Mechanical Engineering), and Xiaoyin Ge (Electrical Engineering).

1. Mapping tissue oxygenation by multispectral imaging
A Comprehensive Wound Center (CWC) dual-mode imaging system, henceforth referred to as the CWC system, was developed at the Ohio State University for multispectral imaging of cutaneous tissue oxygenation and thermographic imaging of vascular function. The wide gap second derivative spectroscopic technique was used to reconstruct tissue oxygenation maps based on multispectral images 1. In this study, a healthy subject sat with the left forearm resting on a countertop. A portion of the forearm within the field of view of the CWC system was painted with India ink (1% dissolved in ethanol) to mimic different skin colors. Multispectral images were acquired and an oxygenation map was reconstructed based on the wide gap second derivative spectrum. The reconstructed oxygenation map was compared with that acquired by a commercial Hypermed OxyVu hyperspectral tissue oxygenation measurement system.
Tissue oxygen responses to vascular occlusion were studied on the same subject following a protocol of post-occlusive reactive hyperemia (PORH) 2. Before the PORH test, the subject's systolic and diastolic blood pressures were recorded by a pressure cuff placed on the left upper arm. The PORH protocol consisted of a pre-occlusive baseline period of two minutes, a suprasystolic occlusion (systolic + 50 mm Hg) period of two minutes, and a reactive hyperemia period of two minutes. Multispectral images were acquired at four wavelengths (i.e., 530nm, 550nm, 570nm, and 590nm) during the PORH test at the sampling rate of 0.75 seconds per wavelength. Deep tissue oxygen saturation and cutaneous tissue oxygen tension on the same arm were simultaneously recorded by an OxiplexTS tissue spectrophotometer (ISS Inc., Urbana Champaign, IL) and a TCM transcutaneous oxygen monitor (Radiometer, Denmark) respectively.
2. Mapping vascular function by dynamic thermographic imaging
Dynamic thermographic imaging was demonstrated on a healthy subject using the same CWC system. The subject comfortably lied on a table in supine position, with the left arm resting on a countertop and the dorsum of the left hand facing up toward the infrared camera unit of the CWC system. A laser Doppler probe was placed on the finger tip of the same hand for continuous monitoring of finger skin perfusion. A pressure cuff was placed on the left upper arm to produce different levels of occlusion. Before the experiment, the subject was asked to rest for at least 10 minutes, with the systolic and diastolic pressures recorded by the pressure cuff. Dynamic thermographic images were captured at the following cuff pressure levels: no occlusion, 0.5 x diastolic blood pressure, 0.5 x (diastolic blood pressure + systolic blood pressure), and 1.5 x systolic blood pressure. At each cuff pressure level, a thermal stimulation was introduced by placing a water bag of room temperature (25&deg;C) on the left hand for 30 seconds. Immediately after the removal of the thermal stimulation, the left hand thermographic images were acquired at a rate of 2 frames/second and the finger skin perfusion was recorded by the laser Doppler probe at the sampling rate of 10 Hz. The time interval between tests was 10 min. The position of the left hand was marked in advance so that the subsequent measurements were at the same position. Tissue vascular functions at different occlusion levels were evaluated by calculating both the temperature response and the vascular function index. The vascular function index was defined as the ratio between the square root of the time after thermal stimulation and the corresponding temperature change.
3. Co-registration between tissue oxygenation and vascular function maps
The map of cutaneous tissue vascular function acquired by dynamic thermographic imaging and the map of cutaneous tissue oxygenation acquired by multispectral imaging were co-registered by an advanced imaging algorithm. Four fiduciary markers, with simultaneous thermal and optical contrasts, were placed on the biological tissue to facilitate image co-registration. Currently the protocol for co-registering the optical image (and the oxygenation map) with the thermal image (and the vascular function map) includes the following key steps: 1) transform all the images to gray scale and normalize the pixel intensity values between 0 and 1; 2) for the optical photo, identify the foreground (skin) region which has high pixel intensity values than the an empirical global threshold (0.8 times the mean intensity value of the whole image); the small holes in the foreground region were filled using the morphological close operation; 3) identify the fiduciary marker regions as darker regions in the foreground whose intensity values are below both the global threshold and the adaptive local threshold defined by the mean pixel values in the 20-by-20 pixel neighborhood. The adaptive local threshold allowed us to accommodate the illumination variation; 4) refine the fiduciary marker regions using morphological operations to remove noise and spikes; the centroids of the four regions were selected as control points in the optical photo; 5) repeat similar steps to identify marker regions in the thermal image; 6) match the two sets of control points based on proximity (given that the two cameras were closely positioned); 7) compute an affine transformation between the two images with the thermal image as the reference and transform the optical photo and the oxygen map (which is obtained from the same camera as the optical photo) correspondingly; 8) finally generate the overlaid images for visualization.
4. Representative Results:
The representative results for the oxygenation protocol (i.e., #1) are cutaneous tissue oxygenation maps reconstructed based on wide gap second derivative spectroscopy. The method of wide gap second derivative spectroscopy effectively reduced the measurement artifacts caused by tissue background absorption so that the cutaneous oxygenation measurement was less affected by the simulated skin color changes. We also demonstrated a PORH protocol where the cutaneous tissue oxygenation map, the deep tissue oxygenation, and the cutaneous tissue oxygen tension were simultaneously recorded.
The representative results for the vascular function protocol (i.e., #2) include the cutaneous tissue temperature distributions, the cutaneous tissue temperature changes in response to external thermal stimulation, and the cutaneous tissue vascular index maps derived from dynamic thermographic imaging. Correlations were observed between the thermographic imaging of cutaneous tissue vascular index and the laser Doppler measurement of skin tissue perfusion.
The representative results for the co-registration protocol (i.e., #3) include the tissue oxygenation map, the vascular index map, and the image fusion among photographic, oxygenation, and vascular index images co-registered by multi-contrast fiduciary markers.
<img src="/files/ftp_upload/2095/2095fig1.jpg" alt="Figure 1" /> 
 Figure 1. CWC dual-mode system setup for non-contact imaging of tissue oxygenation and vascular function. The system was installed on a mobile cart. It consisted of an infrared thermal camera, a CCD camera, a liquid crystal tunable filter, and a broadband light source. A computer was used to synchronize the tasks of data collection, analysis, and display.
<img src="/files/ftp_upload/2095/2095fig2.jpg" alt="Figure 2" /> 
 Figure 2. Software interface for the CWC system. The software interface was programmed in the Labview environment. On the top left of the interface is the control panel for hardware configuration and system calibration. On the right of the interface is the real-time display of the tissue temperature map acquired by the infrared camera. On the bottom of the interface are laser Doppler measurements of skin tissue perfusion.
<img src="/files/ftp_upload/2095/2095fig3.jpg" alt="Figure 3" /> 
 Figure 3. Software interface for the CWC system (Cont.). This is a pop-up window to show tissue oxygenation parameters. On the left is the tissue oxygenation map reconstructed from the multispectral images. The right side of the interface displays the following tissue oxygenation parameters from top down: (1) cutaneous tissue oxygenation averaged from a selected region of interest (ROI) in the tissue oxygenation map; (2) deep tissue oxygenation monitored by an OxplexTS tissue oximeter; (3) cutaneous tissue oxygen tension monitored by a TCM transcutaneous oxygen monitor.
<img src="/files/ftp_upload/2095/2095fig4.jpg" alt="Figure 4" /> 
 Figure 4. Cutaneous tissue oxygenation imaging by an OxyVu imaging system. The left figure is a grayscale image of the skin tissue. A layer of ink was painted on the skin to simulate the skin color. The map of cutaneous tissue oxygenation acquired by the OxyVu system is shown on the right. Two square-shaped regions of interest (ROIs) were selected within and outside the ink-painted area respectively. For the ROI within the ink-painted area, the averaged cutaneous tissue oxygenation was 62.8&plusmn;11.0%. For the ROI outside the ink-painted area, the averaged cutaneous tissue oxygenation was 44.0&plusmn;11.0%. A difference of 18.8% was observed for the cutaneous tissue oxygenation measurements within and outside the ink-painted skin area.
<img src="/files/ftp_upload/2095/2095fig5.jpg" alt="Figure 5" /> 
 Figure 5. Cutaneous tissue oxygenation imaging by the CWC system. The multispectral images were captured by the CWC system at the same skin location on the same subject as that in Figure 4. The left figure is the single wavelength image of the skin tissue and the right figure is the reconstructed cutaneous tissue oxygenation map. A layer of ink was painted on the skin with the ink concentration the same as that in Figure 4. . Two square-shaped regions of interest (ROIs) were selected within and outside the ink-painted area respectively. For the ROI within the ink-painted area, the averaged cutaneous tissue oxygenation was 60.9&plusmn;6.9%. For the ROI outside the ink-painted area, the averaged cutaneous tissue oxygenation was 65.8&plusmn;5.5%. A difference of 4.9% was observed for the cutaneous tissue oxygenation measurements within and outside the ink-painted skin area.
<img src="/files/ftp_upload/2095/2095fig6.jpg" alt="Figure 6" /> 
 Figure 6. The effect of simulated skin color changes on the reliability of cutaneous tissue oxygenation measurements. Statistic analysis was carried out to determine the significance of the simulated skin color changes to oxygenation measurements for both the OxyVu and the CWC imaging systems. For each imaging system, the averaged cutaneous tissue oxygenations were calculated within and outside the ink-painted skin areas by randomly selecting 10 regions of interest (ROIs) in each area. Our null hypothesis is that the change of skin color will not affect the cutaneous tissue oxygenation measurement. This null hypothesis was tested using oxygenation maps in Figure 4 (i.e., the OxyVu measurements) and Figure 5 (i.e., the CWC measurements) respectively. Student's t-tests show that the p value for the OxyVu measurements is much less than 0.001, implying that the null hypothesis is rejected. Therefore, the change of skin color does affect the cutaneous tissue oxygenation measurements in an OxyVu imaging system. In contrast, the p value for the CWC measurements is 0.728, implying the likelihood that the change of skin color does not affect the cutaneous tissue oxygenation measurements in a CWC imaging system.
<img src="/files/ftp_upload/2095/2095fig7.jpg" alt="Figure 7" /> 
 Figure 7. Cutaneous tissue oxygenation images obtained from the CWC system during post-occlusive reactive hyperemia (PORH). The PORH test was carried out on the forearm of a healthy subject following #1. (a). Single wavelength gray scale image of the arm with four fiduciary markers placed for image co-registration. (b) The baseline cutaneous tissue oxygenation map acquired before vascular occlusion. (c) The cutaneous tissue oxygenation map after vascular occlusion (systolic pressure + 50mmHg) for 2 minutes. (d) The cutaneous tissue oxygenation map after reactive hyperemia. Significant changes in cutaneous tissue oxygenation were observed before, during, and after vascular occlusion.
<img src="/files/ftp_upload/2095/2095fig8.jpg" alt="Figure 8" /> 
 Figure 8. Tissue oxygen parameters continuously monitored during a PORH test. The test protocol in (a) shows a period of pre-occlusion baseline, followed by a suprasystolic occlusion period, and end with a reactive hyperemia period. The history of cutaneous tissue oxygenation duration the PORH procedure is plotted in (b). It was obtained by averaging a selected region of interest (ROI) in the CWC cutaneous oxygenation map. We also monitored the deep tissue oxygenation by an OxiplexTS tissue oximeter, as plotted in (c). The transcutaneous tissue oxygen tension was also monitored by a TCM device and plotted in (d). The CWC measurements of cutaneous tissue oxygenation coincide well with other oxygen parameters during the PORH procedure.
<img src="/files/ftp_upload/2095/2095fig9.jpg" alt="Figure 9" /> 
 Figure 9. Cutaneous tissue responses at different vascular occlusion pressures. Thermographic images were acquired immediately after the thermal stimulation (i.e., a water bag at room temperature 25&deg;C) was removed from the subject's left hand. The horizontal axis corresponds to the different time spots after the thermal stimulation was removed. The vertical axis corresponds to the following 4 levels of vascular occlusion pressures: 0 (No occlusion), 0.5DBP, 0.5(DBP+SBP), 1.5SBP, where DBP is the diastolic blood pressure and SBP is the systolic blood pressure. For this specific subject, the DBP is 69mmHg and the SBP is 123mmHg. The test results indicate that the tissue temperature response to the external thermal stimulation is correlated with the level of vascular occlusion. Increasing the occlusion pressure reduces the thermal response rate.
<img src="/files/ftp_upload/2095/2095fig10.jpg" alt="Figure 10" /> 
 Figure 10. Reconstructed vascular index maps at different vascular occlusion pressures. Vascular function index (v) at each pixel of the thermographic images was derived by regressing the cutaneous tissue temperature change (&Delta;T) against the square root of the time after the thermal stimulation (&radic;t): &Delta; &radic;t + K + &#949; , where &#949; is the random error and K is a constant. Vascular function index maps from left to right correspond to the following occlusion conditions: (a) no occlusion, (b) 0.5DBP, (c) 0.5 (DBP+SBP), (d) 1.5SBP. For this specific subject, the diastolic blood pressure (DBP) is 69mmHg and the systolic blood pressure (SBP) is 123mmHg.
<img src="/files/ftp_upload/2095/2095fig11.jpg" alt="Figure 11" /> 
 Figure 11. Correlation between the vascular function index and the skin tissue perfusion. The finger tip vascular function index at each occlusion pressure in #2 was calculated by averaging five regions of interest (ROIs) in the tissue vascular index map. The skin tissue perfusion was measured by a laser Doppler device at one of the finger tip. The finger tip vascular index correlated with the laser Doppler measurement, indicating the potential of using the dynamic thermographic method for quantitative assessment of tissue vascular function.
<img src="/files/ftp_upload/2095/2095fig12.jpg" alt="Figure 12" /> 
 Figure 12. The image co-registration process. In the first row, we show: (a) the normalized photo, (b) the segmented foreground region, and (c) the segmented markers in the photo image. In the second row, we show: (d) the normalized perfusion map, (e) the segmented foreground region, and (f) the segmented markers in the perfusion map. The affine transformation between the two sets of marker positions was calculated. The co-registered images (g and h) were obtained by transforming the photo image and the oxygen map using this recovered affine transformation.
<img src="/files/ftp_upload/2095/2095fig13.jpg" alt="Figure 13" /> 
 Figure 13. The co-registration results. The transformed photo and oxygen map, together with the perfusion map were co-registered and displayed in as a heat map. The heat map in (a) was presented with 100% of transformed photo image in the red channel, 100% of transformed oxygen image in the green channel and 50% of the perfusion map in the blue channel. In order to visualize the vessels better, we present another version of the co-registration result as in (b). The heat map was composed of 100% of transformed photo image in the red channel, 50% of transformed oxygen image in green channel, and 50% of changed perfusion map in the blue channel, where the vascular index map was inverted and only the information within the foreground region was kept.

<ol>
	<li>Myers, D. E., Anderson, L. D., Seifert, R. P., Ortner, J. P., Cooper, C. E., Beilman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+method+for+measuring+local+hemoglobin+oxygen+saturation+in+tissue+using+wide+gap+second+derivative+near-infrared+spectroscopy.">Noninvasive method for measuring local hemoglobin oxygen saturation in tissue using wide gap second derivative near-infrared spectroscopy.</a> J Biomed Opt. 10 (3), 034017-034017 (2005).</li><li>Jarm, T., Kragelj, R., Liebert, A., Lukasiewitz, P., Erjavec, T., Preseren-Strukelj, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postocclusive+reactive+hyperemia+in+healthy+volunteers+and+patients+with+peripheral+vascular+disease+measured+by+three+noninvasive+methods.">Postocclusive reactive hyperemia in healthy volunteers and patients with peripheral vascular disease measured by three noninvasive methods.</a> Adv Exp Med Biol. 530, 661-669 (2003).</li><li>Treacher, D. F., Leach, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+transport-1.+Basic+principles.">Oxygen transport-1. Basic principles.</a> Bmj. 317 (7168), 1302-1306 (1998).</li><li>Leach, R. M., Treacher, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+transport-2.+Tissue+hypoxia.">Oxygen transport-2. Tissue hypoxia.</a> BMJ. 317 (7169), 1370-1373 (1998).</li><li>Sen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+essentials:+let+there+be+oxygen.">Wound healing essentials: let there be oxygen.</a> Wound Repair Regen. 17 (1), 1-18 (2009).</li><li>RL, G. r. e. e. n. m. a. n., Panasyuk, S., Wang, X., TE, L. y. o. n. s., Dinh, T., Longoria, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+changes+in+the+skin+microcirculation+and+muscle+metabolism+of+the+diabetic+foot.">Early changes in the skin microcirculation and muscle metabolism of the diabetic foot.</a> Lancet. 366 (9498), 1711-1717 (2005).</li><li>Valvano, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The use of thermal diffusivity to quantify tissue perfusion [dissertation]. , (1981).</li><li>Hassan, M., Togawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+skin+thermal+inertia+distribution+during+reactive+hyperaemia+using+a+single-hood+measurement+system.">Observation of skin thermal inertia distribution during reactive hyperaemia using a single-hood measurement system.</a> Physiol Meas. 22 (1), 187-200 (2001).</li><li>Ley, O., Deshpande, C., Prapamcham, B., Naghavi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumped+parameter+thermal+model+for+the+study+of+vascular+reactivity+in+the+fingertip.">Lumped parameter thermal model for the study of vascular reactivity in the fingertip.</a> J Biomech Eng. 130 (3), 031012-031012 (2008).</li><li>Wilson, S. B., Spence, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+thermographic+imaging+method+for+quantifying+dermal+perfusion:+potential+and+limitations.">Dynamic thermographic imaging method for quantifying dermal perfusion: potential and limitations.</a> Med Biol Eng Comput. 27 (5), 496-501 (1989).</li></ol>

No conflicts of interest declared.

Oxygen exists in biological tissue in multiple forms such as hemoglobin or myoglobin bound oxygen, dissolved oxygen, and reactive oxygen species. Oxygen transport plays a critical role in maintaining tissue viability and normal metabolic processes 3. Acute mild to moderate hypoxia will initiate metabolic adaptation, vascular regulation, and angiogenic responses 4. Extreme hypoxia and anoxia will lead to insufficient angiogenesis and cell death. The imbalance between limited oxygen supply and increased oxygen demand is one of major causative factors for many disorders such as chronic wounds 5. In the case of ischemic wounds, oxygen supply is limited by lack of perfusion and cannot meet the metabolic need of the healing process and compromising respiratory burst function aimed at fighting infection. Simultaneous assessment of cutaneous tissue oxygenation and vascular function has clinical significance in chronic wound management. The hyperspectral imaging technique estimates cutaneous tissue oxygenation by illuminating tissue and detect tissue reflectance at different wavelengths 6. One major advantage of hyperspectral imaging is non-invasive and non-contact detection of tissue functional properties. However, the oxygen measurement reliability for many hyperspectral imaging systems is affected by skin color and other background absorption variations. Wide gap second derivative spectroscopy was developed previously for tissue oxygenation measurement with minimal effect of scattering and skin pigmentation 1. We applied the principle of the second derivative spectroscopy to multispectral imaging and demonstrated the consistent measurement of cutaneous tissue oxygenation independent with the simulated skin color changes. Tissue perfusion was previously studied by measuring tissue thermal diffusivity 7. A single hood method was developed to introduce thermal stimulation and image tissue dynamic responses in order to estimate the skin tissue thermal inertia distribution 8. Experiments on human forearm skin subjected to arterial cuff occlusion demonstrated linear relationship between thermal inertia and blood perfusion measured by a laser Doppler imager before and during blood flow occlusion 8. A lumped bioheat model was also used to estimate the finger tip vascular reactivity during venous occlusion plethysmography 9. Despite the above efforts, quantifying skin blood perfusion from skin temperature measurements is challenging because of the lack of sensitivity, the dependence on subcutaneous fat thickness, and other contributing factors such as vasoconstriction, vasodilation, and motion artifacts 10. In this protocol, we used an infrared camera to capture tissue temperature dynamics in response to a thermal stimulation at room temperature. The thermal stimulation was selected so that the effect of vasoconstriction and vasodilation was minimized. Further modeling and measurement efforts are necessary in order to delineate a reliable quantitative correlation between the vascular function index and the skin tissue perfusion. In #3, we used the affine transformation for the co-registration tasks. However, given that the two cameras are positioned with different angles in the 3D space, it is potentially more accurate to apply a transformation related to the 3D transformation between the two cameras. Currently we are exploring in that direction which involve extrinsic calibration of the cameras in 3D space using epipolar geometry.

<table><tbody><tr><td>Higgins Calligraphy waterproof black ink</td><td>Sanford</td><td>44314</td><td>diluted to 1%</td></tr><tr><td>Hamamatsu ORCA ER deep cooling CCD camera</td><td>Hamamatsu Corp.</td><td>C4742-80-12AG</td><td></td></tr><tr><td>Varispec SNIR liquid crystal tunable filter</td><td>Cambridge Research Systems</td><td>VIS-10-HC-20</td><td></td></tr><tr><td>ThermoVision A40 infrared camera</td><td>FLIR Systems Inc.</td><td>A40</td><td></td></tr><tr><td>Thorlabs OSL1 high intensity fiber light source</td><td>Thorlabs Inc.</td><td>OSL1</td><td></td></tr></tbody></table>

dual-mode imaging of cutaneous tissue oxygenation and vascular function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

Watch this Scientific Journal Video about Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function at JoVE.com

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

dual-mode-imaging-cutaneous-tissue-oxygenation-vascular

Research

JoVE Journal

Medicine

16.5K Views.  The Ohio State University. Een dual-mode imaging-systeem is ontwikkeld voor non-contact beoordeling van cutane zuurstofvoorziening van de weefsels en vasculaire functie.

Video: Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

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

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Bioengineering

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Preclinical Imaging of Tumor Oxygen Levels Using EPRI and Ultrasound

Tumor Hypoxia Assessment: In Vivo 3D Oxygen Imaging Through Electron Paramagnetic Resonance

The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 (i.e., hypoxia) is connected to tumor aggressiveness and poor response to therapy. The quantification of tumor pO2 allows the evaluation of treatment effectiveness. Electron Paramagnetic Resonance Imaging (EPRI), particularly Pulse EPRI, has emerged as an advanced three-dimensional (3D) method of assessing tissue oxygenation in vivo. This innovation was enabled by the technological developments in EPR (Electron Paramagnetic Resonance) and the application of the water-soluble oximetric spin probes from the triaryl family, offering fast and sensitive oxygenation data. The relaxation time of the spin probe (T1 and/or T2) provides accurate information about pO2 in selected voxels.
Human glioblastoma LN229 tumors were grown in the interscapular fad pad of BALB/c nude mice. Ultrasound (US) imaging was used as a reference for tumor anatomical information. To image tissue pO2, the animals were placed in a fixed position in the animal bed with fiducials, enabling registration between the imaging modalities. After the OX071 contrast agent was administered, EPRI was performed, followed by US B-mode. Because of the low spin probe toxicity, the procedure can be repeated during tumor growth or treatment. Following imaging, the registration process was carried out using software written in MATLAB. Ultimately, the hypoxic fraction can be calculated for a specific tumor, and the histogram of pO2 tissue distribution can be compared over time. EPRI combined with ultrasound is an excellent tool for oxygen mapping of tumors in the preclinical setting.

Quantitative 3D oxygen maps of murine tumors were imaged noninvasively using Pulse Electron Paramagnetic Resonance. Ultrasound B-mode and Power Doppler were used for anatomy and vascular structure. Images from both modalities were superimposed enabling multi-parametric tumor analysis.

The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 ...

Cancer Research

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 ...

Multispectral Optoacoustic Tomography for Functional Imaging in Vascular Research

Microcirculatory impairment has been recognized in various disease processes, underlying this growing theme within vascular research. In recent years, the development of live imaging systems has set the (analytical) pace in both basic and clinical research, with the objective of creating new instruments capable of providing real-time, quantifiable endpoints with clinical interest and application. Near-infrared spectroscopy (NIRS), positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI) are available, among other techniques, but cost, image resolution, and reduced contrast are recognized as common challenges. Optoacoustic tomography (OT) offers a new perspective on vascular functional imaging, combining state-of-the-art optical absorption and spatial resolution capacities (from micrometer optical to millimeter acoustic resolution) with tissue depth. In this study, we tested the applicability of multispectral optoacoustic tomography (MSOT) for functional imaging. The system uses a tunable optical parametric oscillator (OPO) pumped by an Nd: YAG laser, providing excitation pulses sensed by a 3D probe at wavelengths from 680 nm to 980 nm. Images obtained from the human forearm were reconstructed through a specific algorithm (supplied within the manufacturer&#39;s software) based on the response of specific chromophores. Maximal Oxygenated Hemoglobin (Max HbO2) and Deoxygenated Hemoglobin (Max Hb), Total Hemoglobin (HbT), and mean Oxygen Saturation (mSO2) to vascular density (&#181;Vu), inter-unit average distances (&#950;Ad), and capillary blood volume (mm3) may be measured using this system. The applicability potential found with this OT system is relevant. Ongoing software developments will surely improve the utility of this imaging system.

The present protocol describes the acquisition of multispectral optoacoustic images of in vivo human skin vasculature. These include the quantification of hemoglobin and melanin, regarded as chromophores of interest for functional analysis.

Microcirculatory impairment has been recognized in various disease processes, underlying this growing theme within vascular research. In recent years, the ...

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of the retina. This process, referred to as retinal vascular reactivity (RVR), is mediated by neurovascular coupling, which is impaired very early in retinal vascular diseases such as diabetic retinopathy. Therefore, a clinically feasible method of assessing vascular function may be of significant interest in both research and clinical settings. Recently, in vivo imaging of the retinal vasculature at the capillary level has been made possible by the FDA approval of optical coherence tomography angiography (OCTA), a noninvasive, minimal risk and dyeless angiography method with capillary level resolution. Concurrently, physiological and pathological changes in RVR have been shown by several investigators. The method shown in this manuscript is designed to investigate RVR using OCTA with no need for alterations to the clinical imaging procedures or device. It demonstrates real time imaging of the retina and retinal vasculature during exposure to hypercapnic or hyperoxic conditions. The exam is easily performed with two personnel in under 30 min with minimal subject discomfort or risk. This method is adaptable to other ophthalmic imaging devices and the applications may vary based on the composition of the gas mixture and patient population. A strength of this method is that it allows for an investigation of retinal vascular function at the capillary level in human subjects in vivo. Limitations of this method are largely those of OCTA and other retinal imaging methods including imaging artifacts and a restricted dynamic range. The results obtained from the method are OCT and OCTA images of the retina. These images are amenable to any analysis that is possible on commercially available OCT or OCTA devices. The general method, however, can be adapted to any form of ophthalmic imaging.

This article describes a method for measuring retinal vasculature reactivity in vivo with human subjects using a gas breathing provocation technique to deliver vasoactive stimuli while acquiring retinal images.

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of ...

Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction

Oxygenation-sensitive cardiac magnetic resonance imaging (OS-CMR) is a diagnostic technique that uses the inherent paramagnetic properties of deoxyhemoglobin as an endogenous source of tissue contrast. Used in combination with standardized vasoactive breathing maneuvers (hyperventilation and apnea) as a potent non-pharmacologic vasomotor stimulus, OS-CMR can monitor changes in myocardial oxygenation. Quantifying such changes during the cardiac cycle and throughout vasoactive maneuvers can provide markers for coronary macro- and microvascular function and thereby circumvent the need for any extrinsic, intravenous contrast or pharmacologic stress agents.
OS-CMR uses the well-known sensitivity of T2*-weighted images to blood oxygenation. Oxygenation-sensitive images can be acquired on any cardiac MRI scanner using a modified standard clinical steady-state free precession (SSFP) cine sequence, making this technique vendor-agnostic and easily implemented. As a vasoactive breathing maneuver, we apply a 4-min breathing protocol of 120 s of free breathing, 60 s of paced hyperventilation, followed by an expiratory breath-hold of at least 30 s. The regional and global response of myocardial tissue oxygenation to this maneuver can be assessed by tracking the signal intensity change. The change over the initial 30 s of the post-hyperventilation breath-hold, referred to as the breathing-induced myocardial oxygenation reserve (B-MORE) has been studied in healthy people and various pathologies. A detailed protocol for performing oxygen-sensitive CMR scans with vasoactive maneuvers is provided.
As demonstrated in patients with microvascular dysfunction in yet incompletely understood conditions, such as inducible ischemia with no obstructive coronary artery stenosis (INOCA), heart failure with preserved ejection fraction (HFpEF), or microvascular dysfunction after heart transplantation, this approach provides unique, clinically important, and complementary information on coronary vascular function.

The assessment of microvascular function by oxygenation-sensitive cardiac magnetic resonance imaging in combination with vasoactive breathing maneuvers is unique in its ability to assess rapid dynamic changes in myocardial oxygenation in vivo and, thus, may serve as a critically important diagnostic technique for coronary vascular function.

Oxygenation-sensitive cardiac magnetic resonance imaging (OS-CMR) is a diagnostic technique that uses the inherent paramagnetic properties of ...

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.

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

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