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Spatial frequency domain imaging (SFDI) is a wide-field diffuse optical imaging technique for separately quantifying tissue reduced scattering (μs ' ) and absorption (μa) coefficients at multiple wavelengths, providing wide potential utility for clinical applications such as burn wound characterization and cancer detection. However, measured μs ' and μa can be confounded by absorption from melanin in patients with highly pigmented skin. This issue arises because epidermal melanin is highly absorbing for visible wavelengths and standard homogeneous light-tissue interaction models do not properly account for this complexity. Tristimulus colorimetry (which quantifies pigmentation using the L * "lightness" parameter) can provide a point of comparison between μa, μs ' , and skin pigmentation.We systematically compare SFDI and colorimetry parameters to quantify confounding effects of pigmentation on measured skin μs ' and μa. We assess the correlation between SFDI and colorimetry parameters as a function of wavelength.μs ' and μa from the palm and ventral forearm were measured for 15 healthy subjects with a wide range of skin pigmentation levels (Fitzpatrick types I to VI) using a Reflect RS® (Modulim, Inc., Irvine, California) SFDI instrument (eight wavelengths, 471 to 851 nm). L * was measured using a Chroma Meter CR-400 (Konica Minolta Sensing, Inc., Tokyo). Linear correlation coefficients were calculated between L * and μs ' and between L * and μa at all wavelengths.For the ventral forearm, strong linear correlations between measured L * and μs ' values were observed at shorter wavelengths (R 0.92 at ≤659 nm), where absorption from melanin confounded the measured μs ' . These correlations were weaker for the palm (R 0.59 at ≤659 nm), which has less melanin than the forearm. Similar relationships were observed between L * and μa.We quantified the effects of epidermal melanin on skin μs ' and μa measured with SFDI. This information may help characterize and correct pigmentation-related inaccuracies in SFDI skin measurements.

Background Burns are devastating injuries, carry significant morbidity, and require long-term treatment or multiple reconstructive procedures. Wound healing and secondary insults caused by burn wound conversion are amendable to therapeutic intervention, where ischemia has been cited as one of the major factors (Dermatol Surg. 2008;34:1159-1169). Halting injury progression in the zone of stasis is crucial as conversion creates increased burn surface area and depth, leading to local and systemic consequences (J Burns Wounds. 2006;5:e2). Oxygen-carrying micro/nanobubbles, MNB(O2), offer a novel technology that can be used to effectively deliver oxygen to burn wounds and potentially counteract burn wound ischemia. Methods Topical irrigation with MNB(O2) of full-thickness burn wounds on a rodent model (n = 3) was compared against saline-treated controls (n = 3). Tissue structure (reduced scattering coefficient, μs'), oxyhemoglobin concentration (cHbO2), and tissue perfusion were quantified over the course of 28 days through spatial frequency domain imaging and laser speckle imaging. Histological samples taken at the end of the experiment were examined for evidence of wound healing. Results Findings in this preliminary study showed hastened healing with significant differences in spatial frequency domain imaging-measured μs' during wound healing (days 11-28) in MNB(O2) group. The healing "tipping point" seemed to occur at days 9 to 11 with increased collagen organization and increased cHbO2 occurring around that period confirming the gross healing improvements observed. In addition, histological evidence indicated that only the MNB(O2) burns had reached the remodeling phase by the end of 28-day study period. Conclusions These preliminary findings propose the potential of MNB(O2) as a topical method for improving burn wound healing.

Significance: Spatial frequency domain imaging (SFDI) is a wide-field imaging technique that provides quantitative maps of tissue optical properties. We describe a compact SFDI imager that employs a multispectral compound-eye camera. This design enables simultaneous image acquisition at multiple wavelengths. Such a device has potential for application for quantitative evaluation of superficial tissues by nonspecialists in low-resource settings. Aim: The aim of this work was to develop a compact SFDI imager for widefield imaging of in-vivo tissue optical properties and verify its ability to measure optical properties of tissue-simulating phantoms and in a preclinical model of burn wounds. Approach: This compound-eye imager was constructed using a CMOS sensor subdivided into multiple regions, each having a bandpass filter and objective lens. The ability of the instrument to image optical properties was compared with (1) a commercial SFDI imager and (2) a laboratory-based system. Initial validation of ability to accurately characterize optical properties was performed using a tissue-simulating optical phantom. It was then applied to an established murine model of thermal contact burn severity. In-vivo measurements of the optical properties of rat skin were performed before and after the application of burns. Histology was used to verify burn severity. Results: Measurements of the tissue-simulating phantom optical properties made using the compound-eye imager agree with measurements made using the two comparison SFDI devices. For the murine burn model, the burns showed a decrease in the reduced scattering coefficient at all measurement wavelengths compared with preburn measurements at the same locations. This is consistent with previously reported changes in scattering that occur in full-thickness burns. Conclusion: We demonstrate the potential for SFDI to be translated into compact form factor using a compound-eye camera that is capable of obtaining multiple wavelengths channels simultaneously.

While clinical examination is needed for burn severity diagnosis, several emerging technologies aim to quantify this process for added objectivity. Accurate assessments become easier after burn progression, but earlier assessments of partial thickness burn depth could lead to earlier excision and grafting and subsequent improved healing times, reduced rates of scarring/infection, and shorter hospital stays. Spatial Frequency Domain Imaging (SFDI), Laser Speckle Imaging (LSI) and thermal imaging are three non-invasive imaging modalities that have some diagnostic ability for noninvasive assessment of burn severity, but have not been compared in a controlled experiment. Here we tested the ability of these imaging techniques to assess the severity of histologically confirmed graded burns in a swine model. Controlled, graded burn wounds, 3 cm in diameter were created on the dorsum of Yorkshire pigs (n=3, 45–55kg) using a custom-made burn tool that ensures consistent pressure has been employed by various burn research groups. For each pig, a total of 16 burn wounds were created on the dorsal side. Biopsies were taken for histological analysis to verify the severity of the burn. Clinical analysis, SFDI, LSI and thermal imaging were performed at 24 and 72 hours after burn to assess the accuracy of each imaging technique. In terms of diagnostic accuracy, using histology as a reference, SFDI (85%) and clinical analysis (83%) performed significantly better that LSI (75%) and thermography (73%) 24 hours after the burn. There was no statistically significant improvement from 24 to 72 hours across the different imaging modalities. These data indicate that these imaging modalities, and specifically SFDI, can be added to the burn clinicians’ toolbox to aid in early assessment of burn severity.

We propose a new image sensor based on multi-aperture optics and multi-tap charge modulators for simultaneous multi-band spatial frequency domain imaging (SFDI) and blood flow mapping by multi-exposure laser speckle contrast imaging (ME-LSCI). Wavelength-multiplexed imaging implemented by attaching different bandpass filters to each lens enables to perform multi-band SFDI and ME-LSCI simultaneously. A prototype camera for 4-band SFDI (568, 668, 734, 846nm) and single-band LSCI (780nm) was built based on a commercial CMOS camera with enhanced NIR sensitivity to demonstrate the concept and tested using a rat model of graded burns.

While visual clinical impression is the standard of care for burn severity assessment, accuracy improves after the injury progresses, leading to patients waiting days for a diagnosis. Spatial Frequency Domain Imaging (SFDI) has shown the potential to assess burn grade as early as 24 hours post-burn. Here, we compared SFDI and Laser Speckle Imaging (LSI) at 24 and 72 hours after burn injury to clinical burn severity diagnosis. Three patients with burns ranging in severity were imaged. Both techniques showed an ability to categorize burn severity 24 hours after the initial injury, well before the outcome was determined by a clinician at 72 hours or later.

Spatial frequency domain imaging (SFDI) is a wide-field imaging technique that provides quantitative tissue optical property maps. We describe a compact, multi-aperture SFDI imager for burn severity evaluation. This was constructed using a CMOS sensor subdivided into multiple regions, each having a bandpass filter and objective lens. This compound-eye design enables simultaneous image acquisition at multiple wavelengths. The imager was evaluated using a rat burn model and compared to conventional SFDI devices that acquire wavelength data sequentially. The burns showed a decrease in the reduced scattering coefficient that is consistent with previously reported changes in full thickness burns.

Adequate resuscitation is critical for burn wound patients where the affected total body surface area (TBSA) exceeds 20%, as fluid loss leads to organ failure, shock, and patient mortality. Protocols have been developed to combat fluid loss, however excess fluids can also lead to compartment syndromes. Here, we used spatial frequency domain imaging (SFDI), a non-contact wide field imaging technique to measure in-vivo water changes in a 40 % TBSA porcine burn model. In this pilot study one pig received intravenous fluids according to the Parkland formula on top of enteral fluid resuscitation, while a second pig received no fluids during the experiment. Unburned regions of skin were imaged with SFDI from 3 to 22 hours post-burn. This imaging technique uses structured illumination, projected at multiple spatial frequencies and wavelengths, to measure tissue reflectance, which is used to obtain the reduced scattering and absorption coefficients. Water fraction is based upon absorption in the near-infrared spectrum at 971 nm, where water has a high extinction coefficient. SFDI measurements of superficial tissue hydration showed increased water fraction for the pig that received fluid resuscitation (+17%), and a decrease for the pig that received no fluids (-5%). Analysis showed decreased scattering measured in the pig that received fluids (+13%), suggesting increased interstitial water pressure. These preliminary results are consistent with systemic hydration, as estimated based on CT-measured subcutaneous fat thickness. SFDI may, therefore offer the possibility of non-invasively monitoring fluid resuscitation of large TBSA patients.

Significance: Spatial frequency domain imaging (SFDI), a noncontact wide-field imaging technique using patterned illumination with multiple wavelengths, has been used to quantitatively measure structural and functional parameters of in vivo tissue. Using SFDI in a porcine model, we previously found that scattering changes in skin could potentially be used to noninvasively assess burn severity and monitor wound healing. Translating these findings to human subjects necessitates a better understanding of the variation in “baseline” human skin scattering properties across skin types and anatomical locations. Aim: Using SFDI, we aim to characterize the variation in the reduced scattering coefficient (μs′) for skin across a range of pigmentation and anatomic sites (including common burn locations) for normal human subjects. These measurements are expected to characterize baseline human skin properties to inform our use of SFDI for clinical burn severity and wound healing assessments. Approach: SFDI was used to measure μs′ in the visible- and near-infrared regime (471 to 851 nm) in 15 subjects at 10 anatomical locations. Subjects varied in age, gender, and Fitzpatrick skin type. Results: For all anatomical locations, the coefficient of variation in measured μs′ decreased with increasing wavelength. High intersubject variation in μs′ at visible wavelengths coincided with large values of the melanin extinction coefficient at those wavelengths. At 851 nm, where intersubject variation in μs′ was smallest for all anatomical locations and absorption from melanin is minimal, significant intrasubject differences in μs′ were observed at the different anatomical locations. Conclusions: Our study is the first report of wide-field mapping of human skin scattering properties across multiple skin types and anatomical locations using SFDI. Measured μs′ values varied notably between skin types at wavelengths where absorption from melanin was prominent. Additionally, μs′ varied considerably across different anatomical locations at 851 nm, where the confounding effects from melanin absorption are minimized.

While visual assessment by a clinician is the standard of care for burn severity evaluations, new technologies at various stages of development are attempting to add objectivity to this practice by quantifying burn severity. Assessment accuracy generally improves after the burn injury has progressed, but early assessments that correctly identify superficial partial and deep partial burns have the potential to lead to more prompt treatments and shorter recovery times. To date, Spatial Frequency Domain Imaging (SFDI) has only been used in animal models of burns, but has shown the potential to categorize burns accurately at earlier time points. Here we examine the potential for SFDI to assess burn severity in clinical patients. We also utilize Laser Speckle Imaging (LSI), an FDA cleared non-invasive imaging technology that typically measures blood perfusion in order to evaluate burns in clinical patients. We present a case series of two patients, both with partial thickness burns of varying severity. Partial thickness burns are often difficult for clinicians to categorize based on visual appearance alone. SFDI and LSI were both performed on each patient at approximately 24 and 72 h after their respective burn incidents. Each technique was able to render spatially resolved information that enabled improved assessment accuracy for each burn. This represents the first publication of SFDI applied to clinical burn patients after being successfully utilized in animal models, and highlights the potential for SFDI as a feasible tool for the timely categorization of burn severity. Keywords: Burns, Clinical, Humans, Spatial Frequency Domain Imaging, Laser Speckle Imaging

Delayed graft function occurs in 40% of kidney transplantation cases, and is not apparent until after surgery has concluded. To understand changes in donor kidney tissue before transplantation, spatial frequency domain imaging (SFDI) was used to measure absorption and reduced scattering coefficients in excised porcine kidneys as they underwent a standard period of cold ischemic time (CIT). SFDI indicated an increase in reduced scattering and absorption. The scattering b parameter increased until 24 hours, then returned towards baseline. These results suggest that continued investigation may lead to the development of a noninvasive assessment for graft viability.

Spatial frequency domain imaging (SFDI), a noncontact imaging technique using structured illumination with multiple wavelengths, has proved feasible for characterizing burns of different severities induced in porcine dorsum. However, translation to human clinical subjects may be subject to variation in tissue optical properties due to different skin types or body parts. To interrogate this potential difference in optical properties, SFDI was used to measure the reduced scattering coefficient (μs’ ) in patients of varying Fitzpatrick skin type groups at 10 anatomical locations on the body. These scattering values were compared against previously measured average μs’ values for burns in a porcine model. At 851nm, superficial partialthickness burns had a 12.6% higher μs’ than unburned porcine dorsum. Deep partial-thickness and had 12.9% higher average μs’ than unburned porcine dorsum, while full-thickness burns had 2.5% higher average μs’ than unburned porcine dorsum. The variation seen in scattering values at 851nm at one anatomical location (dorsal forearm) for the different skin type groups showed a 6.3% lower average μs’ in group 3 (skin types VVI) compared to group 2 (skin types III-IV). Average scattering values were 3.8% lower in group 1 (skin types I-II) compared to group 2 (skin types III-IV). The variation of μs’ at 851nm across multiple anatomical locations for the same skin type group (Group 1) was a 23% difference from the anatomical location with the highest average μs’ at 851nm (forehead) to the lowest (shin). These findings indicate that further consideration of skin type and location will be integral to the translation of classification techniques based μs’ measurements of porcine dorsum to human patients.

Introduction While visual assessment is the standard for burn severity evaluation, new technologies are attempting to increase objectivity. Burn depth assessment accuracy improves over time as the appearance changes, but earlier diagnosis potentially leads to prompt treatment and shorter recovery. Laser Speckle Imaging (LSI), FDA approved for grading burns, is typically done 72hrs after injury. Spatial Frequency Domain Imaging (SFDI), a novel technology only tested in animal burn models, uses multiple wavelengths of patterned light to quantify tissue absorption (hemodynamics) and scattering (structure). SFDI has shown the potential to differentiate burns as early as 24hrs postburn by measuring scattering (µs’) changes due to collagen damage. Here, we examine SFDI burn severity assessment in clinical patients, and compare against LSI and clinical assessment. Methods 5 burn cases were imaged with LSI and SFDI approximately 24 and 72hrs postburn. The clinician was blinded to imaging results and made diagnoses on their standard of care. Results One patient received no surgical intervention, one received excision and xenograft, and two received excision and autograft. An autograft was recommended for one patient, but not performed due to preexisting conditions. SFDI measurements at 24hrs showed low µs’ values in regions that eventually required autografts, and high µs’ in burns that did not. While clinical decisions were made no earlier than 72hrs postburn, µs’ maps at 24hrs postburn were able to illustrate severity in these regions. In 2 of 3 autograft cases, LSI was unable to determine the severity until 72hrs postburn. Conclusions Here, we compared SFDI to LSI in a clinical setting to quantify burn wound severity. Similar to animal models, SFDI was demonstrated to accurately characterize severity earlier than the perfusion-based LSI. This is likely due to sensitivity of µs’ measured by SFDI to the structural changes in damaged collagen (i.e., zone of coagulation) that occur immediately postburn.

Author(s): Ponticorvo, Adrien; Burmeister, David M; Rowland, Rebecca; Baldado, Melissa; Kennedy, Gordon T; Saager, Rolf; Bernal, Nicole; Choi, Bernard; Durkin, Anthony J | Abstract: The current standard for diagnosis of burn severity and subsequent wound healing is through clinical examination, which is highly subjective. Several new technologies are shifting focus to burn care in an attempt to help quantify not only burn depth but also the progress of healing. While accurate early assessment of partial thickness burns is critical for dictating the course of treatment, the ability to quantitatively monitor wound status over time is critical for understanding treatment efficacy. SFDI and LSI are both non-invasive imaging modalities that have been shown to have great diagnostic value for burn severity, but have yet to be tested over the course of wound healing.In this study, a hairless rat model (n = 6, 300-450 g) was used with a four pronged comb to create four identical partial thickness burns (superficial n = 3 and deep n = 3) that were used to monitor wound healing over a 28 days period. Weekly biopsies were taken for histological analysis to verify wound progression. Both SFDI and LSI were performed weekly to track the evolution of hemodynamic (blood flow and oxygen saturation) and structural (reduced scattering coefficient) properties for the burns.LSI showed significant changes in blood flow from baseline to 220% in superficial and 165% in deep burns by day 7. In superficial burns, blood flow returned to baseline levels by day 28, but not for deep burns where blood flow remained elevated. Smaller increases in blood flow were also observed in the surrounding tissue over the same time period. Oxygen saturation values measured with SFDI showed a progressive increase from baseline values of 66-74% in superficial burns and 72% in deep burns by day 28. Additionally, SFDI showed significant decreases in the reduced scattering coefficient shortly after the burns were created. The scattering coefficient progressively decreased in the wound area, but returned towards baseline conditions at the end of the 28 days period. Scattering changes in the surrounding tissue remained constant despite the presence of hemodynamic changes.Here, we show that LSI and SFDI are capable of monitoring changes in hemodynamic and scattering properties in burn wounds over a 28 days period. These results highlight the potential insights that can be gained by using non-invasive imaging technologies to study wound healing. Further development of these technologies could be revolutionary for wound monitoring and studying the efficacy of different treatments. Lasers Surg. Med. 49:293-304, 2017. © 2017 Wiley Periodicals, Inc.

There is a need for noninvasive, quantitative methods to characterize wound healing in the context of longitudinal investigations related to regenerative medicine. Such tools have the potential to inform the assessment of wound status and healing progression and aid the development of new treatments. We employed spatial frequency domain imaging (SFDI) to characterize the changes in optical properties of tissue during wound healing progression in a porcine model of split-thickness skin grafts and also in a model of burn wound healing with no graft intervention. Changes in the reduced scattering coefficient measured using SFDI correlated with structural changes reported by histology of biopsies taken concurrently. SFDI was able to measure spatial inhomogeneity in the wounds and predicted heterogeneous healing. In addition, we were able to visualize differences in healing rate, depending on whether a wound was debrided and grafted, versus not debrided and left to heal without intervention apart from topical burn wound care. Changes in the concentration of oxy- and deoxyhemoglobin were also quantified, giving insight into hemodynamic changes during healing.

Accurate assessment of burn severity is critical for wound care and the course of treatment. Delays in classification translate to delays in burn management, increasing the risk of scarring and infection. To this end, numerous imaging techniques have been used to examine tissue properties to infer burn severity. Spatial frequency-domain imaging (SFDI) has also been used to characterize burns based on the relationships between histologic observations and changes in tissue properties. Recently, machine learning has been used to classify burns by combining optical features from multispectral or hyperspectral imaging. Rather than employ models of light propagation to deduce tissue optical properties, we investigated the feasibility of using SFDI reflectance data at multiple spatial frequencies, with a support vector machine (SVM) classifier, to predict severity in a porcine model of graded burns. Calibrated reflectance images were collected using SFDI at eight wavelengths (471 to 851 nm) and five spatial frequencies (0 to 0.2 mm−1). Three models were built from subsets of this initial dataset. The first subset included data taken at all wavelengths with the planar (0 mm−1) spatial frequency, the second comprised data at all wavelengths and spatial frequencies, and the third used all collected data at values relative to unburned tissue. These data subsets were used to train and test cubic SVM models, and compared against burn status 28 days after injury. Model accuracy was established through leave-one-out cross-validation testing. The model based on images obtained at all wavelengths and spatial frequencies predicted burn severity at 24 h with 92.5% accuracy. The model composed of all values relative to unburned skin was 94.4% accurate. By comparison, the model that employed only planar illumination was 88.8% accurate. This investigation suggests that the combination of SFDI with machine learning has potential for accurately predicting burn severity.

While most superficial (first degree) burns and full thickness (third degree) burns are easily diagnosed through standard clinical inspection, burns that fall between these extremes are challenging to accurately classify based on clinical appearance alone. Accurate early assessments have the potential to enable earlier debridement and grafting, thereby reducing complication rates, healing times, and chances for infection. To this end, several emerging technologies aim to objectively and non-invasively quantify burn severity, including Spatial Frequency Domain Imaging (SFDI), Laser Speckle Imaging (LSI), and infrared thermal imaging (IR). While these imaging modalities have some diagnostic ability for in-vivo assessment of burn severity, they have not been directly compared in a controlled experiment. Here we compare the ability of these techniques to assess the severity of graded burns in a porcine model (n=96 burns, 6 pigs). Biopsies were taken for histological analysis to verify burn severity. Clinical assessment, SFDI, LSI and thermal imaging were performed for all graded burns at 24 and 72 hours post-burn. In terms of accuracy of burn severity assessment, using histology as a reference, SFDI (85%) and clinical analysis (83%) performed significantly better that LSI (75%) and thermography (73%) at the 24 hour post-burn time point. There was no statistically significant improvement in assessment accuracy at the 72 hour post-burn time point, across the imaging modalities. These data indicate that SFDI may provide valuable information to clinicians in future clinical trials. This information can be obtained early in the process, which may hasten surgical intervention and reduce complications.

Assessment of burn severity is critical for wound treatment. Spatial frequency domain imaging (SFDI) has been previously used to characterize burns based on the relationships between histology and tissue optical properties. Recently, multispectral and hyperspectral imaging optical features have been combined with machine learning to classify burn severity. Here, we investigated the use of SFDI reflectance data at multiple wavelengths and spatial frequencies, with a support vector machine (SVM), to predict severity in a porcine model of graded burns. Burn severity predictions using SVM were compared to burn grade determined using histology techniques. Results suggest that the combination of spatial frequency data with machine learning models has the potential for accurately predicting burn severity at the 24 hr postburn time point.

Spatial frequency domain imaging (SFDI) is a wide-field imaging technique that enables quantification of in vivo tissue optical properties. SFDI was employed to evaluate burn wounds in pigs where incomplete debridement resulted in skin graft failure. We observed increase in the magnitude of the reduced-scattering coefficient in the wound beds after debridement, prior to grafting in wounds that failed compared to the reduced scattering measured in the wound beds that did not fail. We anticipate that SFDI may be a useful means for determining appropriate debridement prior to grafting.

Burn wounds and wound healing invoke several biological processes that may complicate the interpretation of spectral imaging data. Through analysis of spatial frequency domain spectroscopy data (450 to 1000nm) obtained from longitudinal investigations using a graded porcine burn wound healing model, we have identified features in the absorption spectrum that appear to suggest the presence of hemoglobin breakdown products, e.g., methemoglobin. Our results show that the calculated concentrations of methemoglobin directly correlate with burn severity, 24h after the injury. In addition, tissue parameters such as oxygenation (StO2) and water fraction may be underestimated by 20% and 78%, respectively, if methemoglobin is not included in the spectral analysis.

The ability to predict success or failure of wound healing strategies in burns has the potential to enable customized treatments tailored for the individual patient that would ultimately shorten recovery times, reduce the need for repeated grafting, and aid in achieving full rehabilitation. To this end, we are investigating the potential for spatial frequency domain imaging (SFDI) to non-invasively assess wound severity and healing status. SFDI is a wide-field diffuse optical imaging technique that enables non-invasive quantitative determination of in vivo tissue optical properties associated with physiologic structure and function. We have employed SFDI in a longitudinal study of wound healing using a controlled porcine burn model. Regions of wound repair using split thickness skin grafts were imaged using SFDI at multiple time points over a period of 60 days. The reduced scattering and absorption coefficients were determined at nine wavelengths spanning 470–970 nm, enabling determination of oxy- and deoxy-hemoglobin concentration, total hemoglobin concentration, oxygen saturation, and water fraction. These values obtained were compared to unburned control regions, undebrided burns, and debrided regions without treatment by grafting. Changes in the reduced scattering associated with structural changes in tissue correlate with histology as the wound heals. Compared to unburned tissue, the reduced scattering coefficient associated with repaired wounds is depressed and is spatially heterogeneous immediately following grafting. Over the course of healing, the scattering values increase and converge toward values of normal tissue and become more spatially homogeneous. Variations in chromophore concentrations are also characterized.

Author(s): Ponticorvo, Adrien; Taydas, Eren; Mazhar, Amaan; Ellstrom, Christopher L; Rimler, Jonathan; Scholz, Thomas; Tong, June; Evans, Gregory R D; Cuccia, David J; Durkin, Anthony J | Abstract: Detecting failing tissue flaps before they are clinically apparent has the potential to improve postoperative flap management and salvage rates. This study demonstrates a model to quantitatively compare clinical appearance, as recorded via digital camera, with spatial frequency domain imaging (SFDI), a noninvasive imaging technique using patterned illumination to generate images of total hemoglobin and tissue oxygen saturation (stO2).Using a swine pedicle model in which blood flow was carefully controlled with occlusion cuffs and monitored with ultrasound probes, throughput was reduced by 25%, 50%, 75%, and 100% of baseline values in either the artery or the vein of each of the flaps. The color changes recorded by a digital camera were quantified to predict which occlusion levels were visible to the human eye. SFDI was also used to quantify the changes in physiological parameters including total hemoglobin and oxygen saturation associated with each occlusion.There were no statistically significant changes in color above the noticeable perception levels associated with human vision during any of the occlusion levels. However, there were statistically significant changes in total hemoglobin and stO2 levels detected at the 50%, 75%, and 100% occlusion levels for arterial and venous occlusions.As demonstrated by the color imaging data, visual flap changes are difficult to detect until significant occlusion has occurred. SFDI is capable of detecting changes in total hemoglobin and stO2 as a result of partial occlusions before they are perceivable, thereby potentially improving response times and salvage rates.

Tissue simulating phantoms provide a valuable platform for quantitative evaluation of the performance of diffuse optical devices. In this paper we report the development of a poly(dimethylsiloxane) (PDMS) tissue phantom that mimics the spectral characteristics of tissue water. We have developed these phantoms to mimic different water fractions in tissue for testing new devices within the context of clinical applications such as burn wound triage. Compared to liquid phantoms, PDMS phantoms are easier to transport and use, and have a longer usable life than gelatin based phantoms. The pthalocyanine dye 9606 was used to provide an absorption feature of in the vicinity of 970 nm. Scattering properties were independently determined by adding titanium dioxide powder to obtain reduced scattering coefficients similar to that of tissue in the near infrared. Phantom properties were characterized using the techniques of inverse adding doubling and spatial frequency domain imaging. Results presented here demonstrate that we can fabricate solid phantoms that can be used to simulate different water fractions.

Burn wounds are often characterized by injury depth, which then dictates wound management strategy. While most superficial burns and full thickness burns can be diagnosed through visual inspection, clinicians experience difficulty with accurate diagnosis of burns that fall between these extremes. Accurately diagnosing burn severity in a timely manner is critical for starting the appropriate treatment plan at the earliest time points to improve patient outcomes. To address this challenge, research groups have studied the use of commercial laser Doppler imaging (LDI) systems to provide objective characterization of burn-wound severity. Despite initial promising findings, LDI systems are not commonplace in part due to long acquisition times that can suffer from artifacts in moving patients. Commercial LDI systems are being phased out in favor of laser speckle imaging (LSI) systems that can provide similar information with faster acquisition speeds. To better understand the accuracy and usefulness of commercial LSI systems in burn-oriented research, we studied the performance of a commercial LSI system in three different sample systems and compared its results to a research-grade LSI system in the same environments. The first sample system involved laboratory measurements of intralipid (1%) flowing through a tissue simulating phantom, the second preclinical measurements in a controlled burn study in which wounds of graded severity were created on a Yorkshire pig, and the third clinical measurements involving a small sample of clinical patients. In addition to the commercial LSI system, a research grade LSI system that was designed and fabricated in our labs was used to quantitatively compare the performance of both systems and also to better understand the “Perfusion Unit” output of commercial systems.

The current standard for diagnosis of burn severity and subsequent wound healing is through clinical examination, which is highly subjective. Several new technologies are shifting focus to burn care in an attempt to help quantify not only burn depth but also the progress of healing. While accurate early assessment of partial thickness burns is critical for dictating the course of treatment, the ability to quantitatively monitor wound status over time is critical for understanding treatment efficacy. SFDI and LSI are both non-invasive imaging modalities that have been shown to have great diagnostic value for burn severity, but have yet to be tested over the course of wound healing.In this study, a hairless rat model (n = 6, 300-450 g) was used with a four pronged comb to create four identical partial thickness burns (superficial n = 3 and deep n = 3) that were used to monitor wound healing over a 28 days period. Weekly biopsies were taken for histological analysis to verify wound progression. Both SFDI and LSI were performed weekly to track the evolution of hemodynamic (blood flow and oxygen saturation) and structural (reduced scattering coefficient) properties for the burns.LSI showed significant changes in blood flow from baseline to 220% in superficial and 165% in deep burns by day 7. In superficial burns, blood flow returned to baseline levels by day 28, but not for deep burns where blood flow remained elevated. Smaller increases in blood flow were also observed in the surrounding tissue over the same time period. Oxygen saturation values measured with SFDI showed a progressive increase from baseline values of 66-74% in superficial burns and 72% in deep burns by day 28. Additionally, SFDI showed significant decreases in the reduced scattering coefficient shortly after the burns were created. The scattering coefficient progressively decreased in the wound area, but returned towards baseline conditions at the end of the 28 days period. Scattering changes in the surrounding tissue remained constant despite the presence of hemodynamic changes.Here, we show that LSI and SFDI are capable of monitoring changes in hemodynamic and scattering properties in burn wounds over a 28 days period. These results highlight the potential insights that can be gained by using non-invasive imaging technologies to study wound healing. Further development of these technologies could be revolutionary for wound monitoring and studying the efficacy of different treatments. Lasers Surg. Med. 49:293-304, 2017. © 2017 Wiley Periodicals, Inc.

IntroductionThe current standard for diagnosis of burn severity and subsequent wound healing is through clinical examination, which is highly subjective. Several new technologies focus on burn care ...

Author(s): Rowland, Rebecca; Ponticorvo, Adrien; Jarrin Lopez, Alberto; Li, Shiri; Li, Xiaodong; Ichii, Hirohito; Durkin, Anthony | Abstract: Transplantation of kidneys results in delayed graft function in as many as 40% of cases. During the organ transplantation process, donor kidneys undergo a period of cold ischemic time (CIT), where the organ is preserved with a cold storage solution to maintain tissue viability. Some complications observed after grafting may be due to damage sustained to the kidney during CIT. However, the effects due to this damage are not apparent until well after transplant surgery has concluded. To this end, we have used spatial frequency domain imaging (SFDI) to measure spatially resolved optical properties of porcine kidneys over the course of 80-h CIT. During this time, we observed an increase in both reduced scattering (μsa') and absorption (μa) coefficients. The measured scattering b parameter increased until 24nh of CIT, then returned toward baseline during the remaining duration of the imaging sequence. These results show that the optical properties of kidney tissue change with increasing CIT and suggest that continued investigation into the application of SFDI to kidneys under CIT may lead to the development of a noninvasive method for assessing graft viability.

The ability to accurately assess burn wound severity in a timely manner is a critical component of wound management as it dictates the course of treatment. While full thickness and superficial burns can be easily diagnosed through visual inspection, burns that fall in between these categories are difficult to classify. Additionally, the ability to better quantify different stages of wound healing from a burn of any severity would be important for evaluating the efficacy of different treatment options. Here we present a longitudinal (28 day) study that employs spatial frequency domain imaging (SFDI) and laser speckle imaging (LSI) as non-invasive technologies to characterize in-vivo burn wounds and healing in a murine model. Burn wounds were created using an established technique of a brass comb heated to a given temperature and applied for a set amount of time. They were imaged immediately after the initial injury and then at 2, 4, 7, 14, 21, and 28 days following the injury. Biopsies were taken on the day of the injury in order to verify the extent of the burn damage as well as at different time points after the injury in order to visualize different stages of inflammation and healing. The results of this study suggest that the reduced scattering coefficient measured using SFDI and blood flow as measured using LSI have the potential to provide useful metrics for quantifying the severity of burn injuries as well as track the different stages associated with wound healing progression.

Tissue-simulating phantoms provide the opportunity to evaluate the performance of optical and spectroscopic instruments under controlled experimental conditions. Recent efforts have advanced phantom fabrication methods to provide more tissue realistic phantoms, both in terms of a) incorporating absorbing agents that more faithfully mimic in vivo tissue chromophores spanning visible and near infrared regimes and b) accounting for multi-layer tissue structures with distinct optical properties. The spectral scattering properties in these phantoms, however, are typically based only on a single scattering agent, thereby locking the spectral scattering properties to a single particle size distribution. However, in both healthy tissue as well as pathologic tissue, regions of distinct and differentiated scattering may be present. With differing mean size and distribution of scattering objects in these tissue regions, the relative wavelength-dependent scattering spectra may vary. For example, partial thickness burns exhibit significant cellular damage and collagen denaturation that will significantly alter the wavelength-dependent scattering properties resembling large Mie-like scatterer distributions in both visible and near infrared regimes. We present a low-cost method to fabricate silicone tissue-simulating phantoms with tunable scattering spectra properties that span visible and near infrared wavelengths. We use optical polishing agents (white aluminum oxides powders) at various grit sizes to approximate Mie scattering across multiple mean particle sizes. Mean particle sizes used in this study range from 17-3 micron. The optical properties of these phantoms are verified using an integrating sphere in combination with inverse adding-doubling methods. The tolerances of this fabrication method will be discussed.

Laser speckle imaging (LSI) is a wide-field optical technique that enables superficial blood flow quantification. LSI is normally performed in a mounted configuration to decrease the likelihood of motion artifact. However, mounted LSI systems are cumbersome and difficult to transport quickly in a clinical setting for which portability is essential in providing bedside patient care. To address this issue, we created a handheld LSI device using scientific grade components. To account for motion artifact of the LSI device used in a handheld setup, we incorporated a fiducial marker (FM) into our imaging protocol and determined the difference between highest and lowest speckle contrast values for the FM within each data set ([Formula: see text] and [Formula: see text]). The difference between [Formula: see text] and [Formula: see text] in mounted and handheld setups was 8% and 52%, respectively, thereby reinforcing the need for motion artifact quantification. When using a threshold FM speckle contrast value ([Formula: see text]) to identify a subset of images with an acceptable level of motion artifact, mounted and handheld LSI measurements of speckle contrast of a flow region ([Formula: see text]) in in vitro flow phantom experiments differed by 8%. Without the use of the FM, mounted and handheld [Formula: see text] values differed by 20%. To further validate our handheld LSI device, we compared mounted and handheld data from an in vivo porcine burn model of superficial and full thickness burns. The speckle contrast within the burn region ([Formula: see text]) of the mounted and handheld LSI data differed by [Formula: see text] when accounting for motion artifact using the FM, which is less than the speckle contrast difference between superficial and full thickness burns. Collectively, our results suggest the potential of handheld LSI with an FM as a suitable alternative to mounted LSI, especially in challenging clinical settings with space limitations such as the intensive care unit.

Though laser speckle contrast imaging enables the measurement of scattering particle dynamics with high temporal resolution, the subsequent processing has previously been much slower. In prior studies, generating a laser speckle contrast image required about 1 s to process a raw image potentially collected in 10 ms or less. In this paper, novel algorithms are described which are demonstrated to convert 291 raw images per second to laser speckle contrast images and as many as 410 laser speckle contrast images per second to relative correlation time images. As long as image processing occurs during image acquisition, these algorithms render processing time irrelevant in most circumstances and enable real-time imaging of blood flow dynamics.

Baseline optical properties are typically assumed in calculating the differential pathlength factor (DPF) of mouse brains, a value used in the modified Beer-Lambert law to characterize an evoked stimulus response. We used spatial frequency domain imaging to measure in vivo baseline optical properties in 20-month-old control ([Formula: see text]) and triple transgenic APP/PS1/tau (3xTg-AD) ([Formula: see text]) mouse brains. Average [Formula: see text] for control and 3xTg-AD mice was [Formula: see text] and [Formula: see text], respectively, at 460nm; and [Formula: see text] and [Formula: see text], respectively, at 530nm. Average [Formula: see text] for control and 3xTg-AD mice was [Formula: see text] and [Formula: see text], respectively, at 460nm; and [Formula: see text] and [Formula: see text], respectively, at 530nm. The calculated DPF for control and 3xTg-AD mice was [Formula: see text] and [Formula: see text] OD mm, respectively, at 460nm; and [Formula: see text] and [Formula: see text] OD mm, respectively, at 530nm. In hindpaw stimulation experiments, the hemodynamic increase in brain tissue concentration of oxyhemoglobin was threefold larger and two times longer in the control mice compared to 3xTg-AD mice. Furthermore, the washout of deoxyhemoglobin from increased brain perfusion was seven times larger in controls compared to 3xTg-AD mice ([Formula: see text]).

© 2015 Elsevier Ltd and ISBI. All rights reserved. Surgical intervention of second degree burns is often delayed because of the difficulty in visual diagnosis, which increases the risk of scarring and infection. Non-invasive metrics have shown promise in accurately assessing burn depth. Here, we examine the use of spatial frequency domain imaging (SFDI) and laser speckle imaging (LSI) for predicting burn depth. Contact burn wounds of increasing severity were created on the dorsum of a Yorkshire pig, and wounds were imaged with SFDI/LSI starting immediately after-burn and then daily for the next 4 days. In addition, on each day the burn wounds were biopsied for histological analysis of burn depth, defined by collagen coagulation, apoptosis, and adnexal/vascular necrosis. Histological results show that collagen coagulation progressed from day 0 to day 1, and then stabilized. Results of burn wound imaging using non-invasive techniques were able to produce metrics that correlate to different predictors of burn depth. Collagen coagulation and apoptosis correlated with SFDI scattering coefficient parameter (μs) and adnexal/vascular necrosis on the day of burn correlated with blood flow determined by LSI. Therefore, incorporation of SFDI scattering coefficient and blood flow determined by LSI may provide an algorithm for accurate assessment of the severity of burn wounds in real time.

Tissue simulating phantoms can provide a valuable platform for quantitative evaluation of the performance of diffuse optical devices. While solid phantoms have been developed for applications related to characterizing exogenous fluorescence and intrinsic chromophores such as hemoglobin and melanin, we report the development of a poly(dimethylsiloxane) (PDMS) tissue phantom that mimics the spectral characteristics of tissue water. We have developed these phantoms to mimic different water fractions in tissue, with the purpose of testing new devices within the context of clinical applications such as burn wound triage. Compared to liquid phantoms, cured PDMS phantoms are easier to transport and use and have a longer usable life than gelatin-based phantoms. As silicone is hydrophobic, 9606 dye was used to mimic the optical absorption feature of water in the vicinity of 970 nm. Scattering properties are determined by adding titanium dioxide, which yields a wavelength-dependent scattering coefficient similar to that observed in tissue in the near-infrared. Phantom properties were characterized and validated using the techniques of inverse adding-doubling and spatial frequency domain imaging. Results presented here demonstrate that we can fabricate solid phantoms that can be used to simulate different water fractions.

Author(s): Rimler, J; Ponticorvo, A; Taydas, E; Mazhar, A; Scholz, T; Ellstrom, CL; Tong, J; Evans, GR; Cuccia, DJ; Durkin, AJ

© 2014 Optical Society of America. Accurate and timely assessment of burn wound severity is a critical component of wound management and has implications related to course of treatment. While most superficial burns and full thickness burns are easily diagnosed through visual inspection, burns that fall between these extremes are challenging to classify based on clinical appearance. Because of this, appropriate burn management may be delayed, increasing the risk of scarring and infection. Here we present an investigation that employs spatial frequency domain imaging (SFDI) and laser speckle imaging (LSI) as non-invasive technologies to characterize in-vivoburn severity. We used SFDI and LSI to investigate controlled burn wounds of graded severity in a Yorkshire pig model. Burn wounds were imaged starting at one hour after the initial injury and daily at approximately 24, 48 and 72 hours post burn. Biopsies were taken on each day in order to correlate the imaging data to the extent of burn damage as indicated via histological analysis. Changes in reduced scattering coefficient and blood flow could be used to categorize burn severity as soon as one hour after the burn injury. The results of this study suggest that SFDI and LSI information have the potential to provide useful metrics for quantifying the extent and severity of burn injuries.

Background and Objective: Spatial Frequency Domain Imaging (SFDI) is a non-contact wide-field optical imaging technology currently being developed to investigate the feasibility of quantitative non-invasive evaluation of burn wound severity in a rat model. Our objective is to determine the potential of SFDI for mapping quantitative changes in spatially resolved tissue oxygen saturation and water concentration may be indicative of burn wound severity, healing, and further complications. In this portion of the investigation, we focus on the development of a rat burn model and the acute response of tissue to burn wounds. Study Design/Materials and Methods: A controlled burn protocol involving a heated brass comb was applied to 6 rats. Imaging was acquired at 17 evenly spaced wavelengths in the near-infrared from 650 to 970 nm. Over the course of the 3 hour post-burn period, we were able to map quantitative changes in spatially resolved chromophores. Burn severities were verified post-experiment using standard H and E histology and optical microscopy. Results/Conclusion: In total, we were able to induce 12 superficial-partial thickness burns, 8 deep-partial thickness burns, and 4 full thickness burns in our rat models. While several tissue chromophores were tracked, we found that changes in oxygen saturation and water concentration to be sensitive indicators of burn severity. Future work will include additional longitudinal studies over a period of days in order to investigate which parameters are correlated to tissue healing. © 2013 SPIE.

The use of tissue transfer flaps has become a common and effective technique for reconstructing or replacing damaged tissue. While the overall failure rate associated with these procedures is relatively low (5-10%), the failure rate of tissue flaps that require additional surgery is significantly higher (40-60%). The reason for this is largely due to the absence of a technique for objectively assessing tissue health after surgery. Here we have investigated spatial frequency domain imaging (SFDI) as a potential tool to do this. By projecting wide-field patterned illumination at multiple wavelengths onto a tissue surface, SFDI is able to quantify absolute concentrations of oxygenated and deoxygenated hemoglobin over a large field of view. We have assessed the sensitivity of SFDI in a swine pedicle flap model by using a controlled vascular occlusion system that reduced blood flow by 25%, 50%, 75%, or 100% of the baseline values in either the vein or artery. SFDI was able to detect significant changes for oxygenated hemoglobin, deoxygenated hemoglobin, or tissue oxygen saturation in partial arterial occlusions of at least 50% and partial venous occlusions of at least 25%. This shows SFDI is sensitive enough to quantify changes in the tissue hemoglobin state during partial occlusions and thus has the potential to be a powerful tool for the early prediction of tissue flap failure.

Little numerical analysis has been done on in vivo vascular fluorescence imaging. Here, we use a 3D fluorescence Monte Carlo model to evaluate a microvasculature geometry obtained via two-photon microscopy. We found that a bulk-vascularization assumption does not pro- vide an accurate picture of penetration depth of the collected fluorescence signal. Instead the degree of absorption difference between extravascular and intravascular space, and the degree of stokes shift must be taken into account to determine the depth distribution. Additionally, we found that using targeted illumination can provide for superior surface vessel sensi- tivity over wide-field illumination, with small area detection offering an even greater amount of sensitivity to surface vasculature. Depth sensitivity can be enhanced by either increasing the detector area or increasing the illumination area. Finally, we see that excitation wavelength and vessel size can affect intra-vessel sampling distribution, as well as the amount of signal that originates from inside the vessel under targeted illumination conditions.

In vivo surface imaging of fluorescently labeled vasculature has become a widely used tool for functional brain imaging studies. Techniques such as phosphorescence quenching for oxygen tension measurements and indocyanine green fluorescence for vessel perfusion monitoring rely on surface measurements of vascular fluorescence. However, the depth dependence of the measured fluorescence signals has not been modeled in great detail. In this paper, we investigate the depth dependence of the measured signals using a three-dimensional Monte Carlo model combined with high resolution vascular anatomy. We found that a bulk-vascularization assumption to modeling the depth dependence of the signal does not provide an accurate picture of penetration depth of the collected fluorescence signal in most cases. Instead the physical distribution of microvasculature, the degree of absorption difference between extravascular and intravascular space, and the overall difference in absorption at the excitation and emission wavelengths must be taken into account to determine the depth penetration of the fluorescence signal. Additionally, we found that using targeted illumination can provide for superior surface vessel sensitivity over wide-field illumination, with small area detection offering an even greater amount of sensitivity to surface vasculature. Depth sensitivity can be enhanced by either increasing the detector area or increasing the illumination area. Finally, we see that excitation wavelength and vessel size can affect intra-vessel sampling distribution, as well as the amount of signal that originates from inside the vessel under targeted illumination conditions.

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow. The technique analyzes fluctuations in a dynamic speckle pattern to detect the movement of particles similar to how laser Doppler analyzes frequency shifts to determine particle speed. Because it can be used to monitor the movement of red blood cells, LSCI has become a popular tool for measuring blood flow in tissues such as the retina, skin, and brain. It has become especially useful in neuroscience where blood flow changes during physiological events like functional activation, stroke, and spreading depolarization can be quantified. LSCI is also attractive because it provides excellent spatial and temporal resolution while using inexpensive instrumentation that can easily be combined with other imaging modalities. Here we show how to build a LSCI setup and demonstrate its ability to monitor blood flow changes in the brain during an animal experiment.

Mapping of absolute oxygen levels in the brain is critical during stroke and other disorders. One of the standard methods for measuring oxygen tension is through oxygen dependent quenching of phosphorescence. Typically these measurements are limited to a single spatial location due to the need to measure phosphorescence lifetimes. We have developed an instrument to obtain spatial maps oxygen tension in the brain by combining a DMD with phosphorescence lifetime measurements. Blood flow is measured simultaneously with laser speckle contrast imaging. In this paper we describe this instrument and demonstrate its use in studying stroke progression in animal models.

We present a new Multi-Exposure Speckle Imaging instrument and a new speckle model to predict correlation times of flow in vivo that are unaffected by the presence of static scatterers such as an intact/thinned skull.