Name: Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function (Video) | JoVE Journal
Uploaded: 2010-12-08T17:00:00.000+00:00
Duration: 11 min 35 s
Description: Explore dual-mode imaging for non-invasive assessment of cutaneous tissue oxygenation and vascular function using infrared and CCD cameras. This method enables precise monitoring of wound healing and vascular thermal reactivity.

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5 minutes

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°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="Thermal imaging setup diagram with CCD camera, tunable filter; mobile cart for spectroscopic analysis." data-altupdatedat="1788235205000" data-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="Tissue oxygen and vascular function system; thermal imaging, laser Doppler flux/concentration graph." data-altupdatedat="1788235205000" data-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="Tissue oxygenation map; diagram with SO2 graphs, dual-mode system for vascular function analysis." data-altupdatedat="1788235205000" data-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="Tissue oxygenation map, grayscale image, color scale, tissue analysis, oxygen percentage representation." data-altupdatedat="1788235205000" data-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±11.0%. For the ROI outside the ink-painted area, the averaged cutaneous tissue oxygenation was 44.0±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="Tissue oxygenation percentage map, grayscale image comparison of blood oxygen levels, color-coded results." data-altupdatedat="1788235205000" data-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±6.9%. For the ROI outside the ink-painted area, the averaged cutaneous tissue oxygenation was 65.8±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="Tissue oxygenation bar chart; skin ink effect; CWC vs OxyVu; statistical results p=0.728, p&lt;0.001." data-altupdatedat="1788235205000" data-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="Static equilibrium analysis, thermal imaging sequence, heat distribution, data chart, occlusion study." data-altupdatedat="1788235205000" data-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="Occlusion pressure diagram; cutaneous and deep tissue oxygenation, tension graphs showing data trends." data-altupdatedat="1788235205000" data-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="Thermal imaging sequence, vascular occlusion pressure, hand heat map, temperature over time." data-altupdatedat="1788235205000" data-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°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="Thermal imaging, blood perfusion; occlusion effects, hand diagram, color scale analysis." data-altupdatedat="1788235205000" data-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 (ΔT) against the square root of the time after the thermal stimulation (√t): Δ √t + K + ε , where ε 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="Vascular index vs. Doppler measurement graph; linear fit R²=0.8611; data correlation analysis." data-altupdatedat="1788235205000" data-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="Vascular index map process; image segmentation, marker tracking, oxygen mapping, data analysis." data-altupdatedat="1788235205000" data-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="Co-registration result comparison; image analysis of vascular function map; diagram; research method." data-altupdatedat="1788235205000" data-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/pubmed?term=((Noninvasive+method+for+measuring+local+hemoglobin+oxygen+saturation+in+tissue+using+wide+gap+second+derivative+near-infrared+spectroscopy%5BTitle%5D)+AND+%22J+Biomed+Opt%22%5BJournal%5D)">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>
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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

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.

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 ...

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders, including chronic wounds. Here, cutaneous tissue oxygenation and tissue vascular thermal reactivity are imaged non-invasively. Using a novel dual mode imaging system that integrates an infrared camera, A CCD camera, a liquid crystal tuneable filter, and a high intensity fiber light source.Oxygenation changes are induced in the arm tissue of a healthy subject using a blood pressure cuff monitored by the dual mode imaging system, and compared with simultaneous measurements of deep tissue oxygenation and cutaneous tissue, oxygen tension tissue temperature changes in response to external thermal stimulation are also recorded to reconstruct the vascular thermal reactivity map. The cutaneous tissue oxygenation map and the tissue vascular thermal reactivity map are fused through an advanced co-registration algorithm. In this work, we describe the development of a novel imaging system for the non-invasive monitoring of wound tissue oxygen saturation, as well as dynamic thermographic profile, which helps study exothermic inflammation and in tracing warm blood flowing into the wound tissue during the post-inflammatory angiogenic phase.From a clinical perspective, this imaging system has the potential to help answer key questions in the field of acute and chronic wound management, such as using image guided debridement. It'll also help monitor responsiveness of the wound therapeutic interventions non-invasively by way of repeated measurements of tissue oxygenation. This dual model imaging system developed at the Ohio State University's Comprehensive Wound Center is the first step toward the simultaneous assessment of wound tissue ox oxygenation and vascular function.As you will see, the system is now ready for functional test efforts are in progress to improve the engineering design standardized protocol, and a validated system for routine clinical use. In addition to the imaging hardware and the reconstruction algorithms, we have developed a faster image co-registration measures for integrating and visualizing overlay images acquired by the different imaging modalities. The approach is modular living room for future edition of new imaging platforms.Begin by having a healthy subject. Sit with his or her left forearm resting on a countertop. Use a brush to paint a thin layer of India ink on the subject's forearm to mimic different skin colors.Place four fiduciary markers on the subject's forearm to define a region of interest for ROI that consists of both the ink, painted skin and the regular skin. The fiduciary markers have simultaneous thermal and optical contrasts that facilitate image co-registration here, the dual mode imaging system developed at the comprehensive wound center of the Ohio State University is used. The imaging system consists of an infrared camera, a CCD camera, a liquid crystal tunable filter, and a high intensity fiber light source to perform multi-spectral imaging of tissue oxygenation.Turn off all external light sources, then have the subject position his or her arm and capture images. The imaging software automatically calibrates the exposure times at each wavelength. After the sample images are collected from the region of interest, place a whiteboard in the imaging area and acquire reflectance images at different wavelengths.These will be used for calibration. Once the reference images have been acquired. Calculate the reflectance ratio and derive the tissue oxygenation at each pixel based on the wide gap.Second derivative spectrum. For comparison, acquire a cutaneous tissue oxygenation map using a commercial system to determine whether change of skin color affects oxygenation measurements in each system. Use statistical analysis software to perform a student's T-test, comparing the measurements in the regions with and without ink.Next, place a pressure cuff on the subject's left upper arm. Determine and record the subject's systolic and diastolic blood pressures. Place the sensors of an plex TS tissue spectrophotometer and A TCM transcutaneous oxygen monitor on the same arm and turn on the devices to warm them up for 20 minutes while monitoring the cutaneous tissue oxygenation map.Introduce oxygenation changes in the arm tissue by performing a postocclusive reactive hyperemia or PORH. Click the start button to begin imaging. Monitor the pre occlusive baseline for two minutes.Next, inflate the blood pressure cuff to 50 millimeters mercury above the systolic pressure to induce a supra systolic occlusion. Leave the cuff inflated for two minutes, continuing to monitor changes in tissue parameters. Finally rapidly deflate the pressure cuff to induce reactive hyperemia.Record all the tissue oxygen parameters for two minutes. To perform dynamic thermographic imaging. Have a healthy subject comfortably.Sit with his or her left arm resting on a countertop. Position the dorsum of the left hand up toward the infrared camera unit of the imaging system. Next place a laser doppler probe on the fingertip of the left hand to continuously monitor skin perfusion at a sampling rate of 10 hertz.Place a pressure cuff on the subject's left upper arm. Then determine and record the subject systolic and diastolic blood pressures. Generate different levels of vascular occlusion by controlling the inflation of the pressure cuff at the following pressure levels, no occlusion 0.5 times the diastolic blood pressure 0.5 times the diastolic blood pressure and systolic blood pressure combined, and 1.5 times the systolic blood pressure.Next, perform thermographic imaging tests at two vascular occlusion levels, no occlusion and s systolic occlusion at each vascular occlusion level. Introduce a thermal stimulation by placing a room temperature water bag on the left hand for 30 seconds. Immediately after the removal of the water bag, start acquiring thermographic images continuously at an imaging rate of two frames per second.Using software, determine the tissue vascular thermal reactivity function. Calculate the function index at each pixel of the thermographic image by taking the ratio between the square root of the thermal stimulation duration and the corresponding tissue temperature change. Calculate the finger vascular thermal reactivity value at each occlusion pressure by averaging five ROIs in the tissue vascular thermal reactivity Map the finger vascular thermal reactivity values are compared with laser doppler measurements of finger vascular perfusion at different occlusion levels.To coregister the oxygenation map obtained from multispectral imaging and the vascular function map obtained from dynamic thermographic imaging. Use MATLAB to transform all the images to gray scale. Then normalize the pixel intensity values by setting them between zero and one.Next on the oxygenation map or multi-spectral image, identify the foreground or skin region with pixel intensity values greater than the empirical global threshold of 0.8 times the mean intensity value of the whole image. Further improve the image using the morphological operation. Next to accommodate the illumination variation, identify the fiduciary markers.These markers will appear as darker regions in the foreground and will have intensity values. Below the global and adaptive local thresholds. Refine the fiduciary marker regions using morphological operations to remove noise in the image, select the OIDs of these four regions as control points in the optical photo.Repeat similar steps to identify marker regions in the vascular function map or the thermographic image. Match the two sets of control points based on proximity. After normalizing them to the same scale, match a control point in the first image to the closest control point.In the second one, compute in a fine transformation with translation rotation and scaling information between the two images. Using the vascular function map as a reference. Then transform the oxygenation map using the computed transformation.Finally, overlay the coregistered images for visualization. A reconstructed cutaneous tissue oxygenation map acquired from the forearm of a healthy subject using the comprehensive wound centered dual mode imaging system is shown here. Note that significant oxygenation differences between the painted and non-pain tissue regions are not observed.10 ROIs were randomly selected from outside and within the ink painted region to evaluate oxygenation measurements. The results shown here suggest that tissue oxygenation measurement by the comprehensive wound center system is not significantly affected by skin color. In comparison, tissue oxygenation measurements acquired using a commercial system are significantly affected by skin color.The comprehensive wound center imaging system is able to monitor changes in cutaneous tissue oxygenation continuously throughout baseline occlusion and hyperemia phases of the PORH procedure. During the PORH procedure, the change of cutaneous tissue oxygenation coincides with changes in deep tissue oxygenation. The comprehensive wound center imaging system is also able to image the cutaneous tissue vascular thermal reactivity.These figures show representative vascular function maps of the same subject at different vascular occlusion pressures introduced by a pressure cuff. The averaged vascular function index at the fingertips of the subject correlates with the laser doppler measurement of fingertips. Skin tissue perfusion.The oxygenation map and the vascular function map acquired by the imaging system are fused by a co-registration algorithm. This figure shows a representative image fusion. What we have here is a dual model image system for simultaneous assessment of cut continuous tissue ox oxygenation and tissue vascular thermal reactivity.Other platforms such as the fluorescence imaging can be added. System integration of hardware and software components represents a key cornerstone. In this effort, the image co registration algorithm in the system can be easily adapted to accommodate additional imaging modalities making this a versatile product.After watching this video, you should have a good understanding of the promise offered by this new imaging approach. Development of the system is an example of interdisciplinary translational research at the Ohio State University's comprehensive wound Center. Good luck with your experiments.

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Research

JoVE Journal

Medicine

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.

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

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.

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

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

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.

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

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.

Multispectral Optoacoustic Tomography for Functional Imaging in Vascular Research

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

This article presents a multimodal approach that aims to overcome the limitations of traditional methods in detecting mesenteric ischemia and preventing bowel necrosis. The presented technique offers a promising solution by combining state-of-the-art ultrasonography with cutting-edge near-infrared light technologies.

Multimodality Diagnosis of Mesenteric Ischemia

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.

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

Here, we describe a non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia in the lower limb. This protocol provides a standardized assessment of vascular and microvascular responsiveness that may be used to determine the presence of vascular dysfunction as well as the efficacy of therapeutic interventions.

Near-Infrared Spectroscopy During Reactive Hyperemia for the Assessment of Lower Limb Vascular Function

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.

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

Here, we present a protocol for performing Oxygen Sensitive – Dynamic Contrast Enhanced Optoacoustic Imaging (OS-DCE OAI) for evaluating pre-clinical models of cancer and wound healing. OS OAI can image oxy‑ and deoxyhemoglobin, mapping tissue oxygenation. DCE-OAI can image the pharmacokinetics of an exogenous contrast agent, thereby mapping vascular perfusion.

Evaluations of Oxygenation and Vascular Perfusion in Pre-Clinical Models of Cancer and Wound Healing Using OS-DCE OAI

Explore dual-mode imaging for non-invasive assessment of cutaneous tissue oxygenation and vascular function using infrared and CCD cameras. This method enables precise monitoring of wound healing and vascular thermal reactivity.

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

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