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A critical need exists for early, accurate diagnosis of burn wound severity to help identify the course of treatment and outcome of the wound. Laser speckle imaging (LSI) is a promising blood perfusion imaging approach, but it does not account for changes in tissue optical properties that can occur with burn wounds, which are highly dynamic environments. Here, we studied optical property dynamics following burn injury and debridement and the associated impact on interpretation of LSI measurements of skin perfusion. We used spatial frequency domain imaging (SFDI) measurements of tissue optical properties to study the impact of burn-induced changes in these properties on LSI measurements. An established preclinical porcine model of burn injury was used (n = 8). SFDI and LSI data were collected from burn wounds of varying severity. SFDI measurements demonstrate that optical properties change in response to burn injury in a porcine model. We then apply theoretical modeling to demonstrate that the measured range of optical property changes can affect the interpretation of LSI measurements of blood flow, but this effect is minimal for most of the measured data. Collectively, our results indicate that, even with a dynamic burn wound environment, blood-flow measurements with LSI can serve as an appropriate strategy for accurate assessment of burn severity.

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.

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.

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.

Spatial Frequency Domain Imaging (SFDI) is a non-invasive diffuse optical imaging technology that quantifies tissue optical properties by projecting spatially modulated light onto a region of interest. By detecting and fitting the diffuse reflectance to a light transport model, tissue absorption (μa) and scattering (μs') coefficients are calculated. However, when measuring skin, bulk μa and μs' from a homogeneous light transport model may not correspond with the actual properties of individual skin layers, especially for highly pigmented skin. To obtain physiologically accurate skin optical property values, we propose an iterative method based on a two- layered Monte Carlo model. We initially assume that μs' in darker skin is the same as that in lighter skin, and set the epidermal a as the free parameter when fitting the diffuse reflectance. To test this algorithm, we analyzed data from the forearms of 6 subjects having various levels of pigmentation, at 8 visible-to-near-infrared wavelengths. We compared the measured reflectance to both the homogeneous model and our layered model to quantify fit accuracy. At 471 nm and 526 nm for patients of Fitzpatrick skin types IV-VI (relatively dark skin), the two-layered model provided a 10-20% improvement in fit to the AC reflectance as a function of spatial frequency, compared to the homogeneous model. These improved fits yielded epidermal absorption coefficients that were notably higher than the bulk μa from the homogeneous model. Fitting the extracted epidermal μa to a melanin extinction spectrum1 enabled estimation of the melanin concentration in the epidermis.

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

We present an approach to quantify drug–target engagement using in vivo fluorescence endomicroscopy, validated with in vitro measurements. Doxorubicin binding to chromatin changes the fluorescence lifetime of histone-GFP fusions that we measure in vivo at single-cell resolution using a confocal laparo/endomicroscope. We measure both intra- and inter-tumor heterogeneity in doxorubicin chromatin engagement in a model of peritoneal metastasis of ovarian cancer, revealing striking variation in the efficacy of doxorubicin–chromatin binding depending on intra-peritoneal or intravenous delivery. Further, we observe significant variations in doxorubicin–chromatin binding between different metastases in the same mouse and between different regions of the same metastasis. The quantitative nature of fluorescence lifetime imaging enables direct comparison of drug–target engagement for different drug delivery routes and between in vitro and in vivo experiments. This uncovers different rates of cell killing for the same level of doxorubicin binding in vitro and in vivo., The engagement of DNA-binding drugs to their target is difficult to study, particularly in vivo. Here the authors develop an in vivo fluorescence lifetime imaging confocal laparo/endomicroscope to show intra- and inter-tumor heterogeneity in doxorubicin binding to peritoneal metastases, which depends on the route of administration.

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.

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.

We introduce a method for quantitative hyperspectral optical imaging in the spatial frequency domain (hs-SFDI) to image tissue absorption (μa) and reduced scattering (μs') parameters over a broad spectral range. The hs-SFDI utilizes principles of spatial scanning of the spectrally dispersed output of a supercontinuum laser that is sinusoidally projected onto the tissue using a digital micromirror device. A scientific complementary metal-oxide-semiconductor camera is used for capturing images that are demodulated and analyzed using SFDI computational models. The hs-SFDI performance is validated using tissue-simulating phantoms over a range of μa and μs' values. Quantitative hs-SFDI images are obtained from an ex-vivo beef sample to spatially resolve concentrations of oxy-, deoxy-, and met-hemoglobin, as well as water and fat fractions. Our results demonstrate that the hs-SFDI can quantitatively image tissue optical properties with 1000 spectral bins in the 580- to 950-nm range over a wide, scalable field of view. With an average accuracy of 6.7% and 12.3% in μa and μs', respectively, compared to conventional methods, hs-SFDI offers a promising approach for quantitative hyperspectral tissue optical imaging. Funding agencies: National Institute of Health (NIH) NIBIB Biomedical Technology Research Center LAMMP [P41EB015890]; NIH [R21EB020953]; Military Medical Photonics Program: AFOSR [FA9550-17-1-0193]; Arnold and Mabel Beckman Foundation

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.

Spatial frequency domain imaging (SFDI) utilizes a digital light processing (DLP) projector for illuminating turbid media with sinusoidal patterns. The tissue absorption (μ a ) and reduced scattering coefficient (μ , s ) are calculated by analyzing the modulation transfer function for at least two spatial frequencies. We evaluated different illumination strategies with a red, green and blue light emitting diodes (LED) in the DLP, while imaging with a filter mosaic camera, XiSpec, with 16 different multi-wavelength sensitive pixels in the 470-630 nm wavelength range. Data were compared to SFDI by a multispectral camera setup (MSI) consisting of four cameras with bandpass filters centered at 475, 560, 580 and 650 nm. A pointwise system for comprehensive microcirculation analysis was used (EPOS) for comparison. A 5-min arterial occlusion and release protocol on the forearm of a Caucasian male with fair skin was analyzed by fitting the absorption spectra of the chromophores HbO 2 , Hb and melanin to the estimatedμ a . The tissue fractions of red blood cells ( f RBC), melanin (/mel) and the Hb oxygenation ( S 0 2 ) were calculated at baseline, end of occlusion, early after release and late after release. EPOS results showed a decrease in S 0 2 during the occlusion and hyperemia during release ( S 0 2 = 40%, 5%, 80% and 51%). The f RBC showed an increase during occlusion and release phases. The best MSI resemblance to the EPOS was for green LED illumination ( S 0 2 = 53%, 9%, 82%, 65%). Several illumination and analysis strategies using the XiSpec gave un-physiological results (e.g. negative S 0 2 ). XiSpec with green LED illumination gave the expected change in /RBC , while the dynamics in S 0 2 were less than those for EPOS. These results may be explained by the calculation of modulation using an illumination and detector setup with a broad spectral transmission bandwidth, with considerable variation in μ a of included chromophores. Approaches for either reducing the effective bandwidth of the XiSpec filters or by including their characteristic in a light transport model for SFDI modulation, are proposed.

Hyperspectral Imaging (HSI) is a growing field in tissue optics due to its ability to collect continuous spectral features of a sample without a contact probe. Spatial Frequency Domain Imaging (SFDI) is a non-contact wide-field spectral imaging technique that is used to quantitatively characterize tissue structure and chromophore concentration. In this study, we designed a Hyperspectral SFDI (H-SFDI) instrument which integrated a supercontinuum laser source to a wavelength tuning optical configuration and a sCMOS camera to extract spatial (Field of View: 2cm×2cm) and broadband spectral features (580nm-950nm). A preliminary experiment was also performed to integrate the hyperspectral projection unit to a compressed single pixel camera and Light Labeling (LiLa) technique.

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.

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 ...

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.

Polydimethylsiloxane (PDMS) has been a popular medium to fabricate tissue simulating optical phantoms. Recently, its use has significantly expanded in instrument calibration and performance testing, validation of advanced models of light transport of complex tissue geometries and evaluation of novel measurement modalities. To meet these demands, fabrication methods of these optical phantoms have become more refined and its structure and constituent components (i.e. dyes and scattering agents) have evolved to better mimic optical properties of tissue spanning both visible and near infrared regimes. We present efforts at the Beckman Laser Institute that address these challenges through PDMS phantoms.

Fluorescence lifetime imaging (FLIM) is a novel wide-field imaging technique that measures the rate of decay (or ‘lifetime’) of fluorescence at each point in the image field following pulsed laser excitation. The decay kinetics of fluorescence are dependent not only on the specific fluorophore but also on the fluorophore’s surrounding micro-environment. As a result of the sensitivity of FLIM to the composition and the function of tissue it can be used to generate tissue contrast in unstained tissue both at the microscopic and the macroscopic level. We present our results using wide-field FLIM of freshly resected surgical specimens to demonstrate lifetime contrast between normal and malignant tissue

We describe a fluorescence lifetime imaging endomicroscope employing a fibre bundle probe and time correlated single photon counting. Preliminary images of stained pollen grains, eGFP-labelled cells exhibiting Förster resonant energy transfer and tissue autofluorescence are presented.

We model the capabilities of several commercially available microelectromechanical deformable mirrors to compensate for the typical optical wavefront aberrations that occur in the human eye. The simulations include the nonlinear effects of actuator clipping, ocular pupil diameter, beam diameter on the mirror and the number of mirror modes.

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.

Uracil DNA glycosylase plays a key role in DNA maintenance via base excision repair. Its role is to bind to DNA, locate unwanted uracil, and remove it using a base flipping mechanism. To date, kinetic analysis of this complex process has been achieved using stopped-flow analysis but, due to limitations in instrumental dead-times, discrimination of the “binding” and “base flipping” steps is compromised. Herein we present a novel approach for analyzing base flipping using a microfluidic mixer and two-color two-photon (2c2p) fluorescence lifetime imaging microscopy (FLIM). We demonstrate that 2c2p FLIM can simultaneously monitor binding and base flipping kinetics within the continuous flow microfluidic mixer, with results showing good agreement with computational fluid dynamics simulations.

The spin relaxation times of electrons in InGaAs(P)/InGaAsP multiple quantum wells have been directly measured as a function of well width using an ultrashort pulse colour centre laser. The times measured range from 20 to 5 ps, considerably faster than previously determined in GaAs quantum wells. In addition, a fast, all-optical polarization switch based on a spin-sensitive optical nonlinearity is demonstrated at a wavelength of . This operating wavelength and the observed switch recovery time of 5.5 ps make it a potentially useful element for future all-optical soliton communication systems.

We report the use of impurity-free vacancy disordering techniques to control the nonlinear optical properties of GaAs-AlGaAs multiple quantum wells. These processes result in a shift in the position of the absorption edge to higher energy and have been used to modify the second- and third-order nonlinear coefficients. Working at photon energies just below the half bandgap we have observed a reduction of in the value of nonlinear refractive coefficient for a bandgap shift of around 40 nm. This change arises due to the combined effects of increasing the bandgap and increasing the detuning. The process can also result in a modulation in the magnitude of the second-order susceptibility coefficient and provides a potential mechanism for realizing quasi-phase-matched structures.

In this paper, we present experimental results which demonstrate that quantum-well intermixing techniques can be used to modulate the magnitude of the second-order nonlinear coefficient /spl chi//sup (2)/. Impurity-free vacancy disordering with SiO/sub 2/ and Ga/sub 2/O/sub 3/ caps was used to modulate the position of the band edge and hence, the magnitude of /spl chi//sub eff//sup (2)/. Using a coupled quantum-well structure we were able to demonstrate modulation of the d/sub 33/ tensor components associated with the asymmetric structure and of the d/sub 14/ component associated with the bulk crystal structure.

We describe the operation and characterization of Ti:sapphire laser-pumped femtosecond and picosecond optical parametric oscillators based the new quasi-phase-matched nonlinear materials of periodically poled LiNbO/sub 3/ and RbTiOAsO/sub 4/ with broad tunability in the near- to mid-infrared. We discuss the merits of the two materials for use in ultrafast optical parametric oscillators (OPOs) and compare and contrast their properties to the birefringent materials. We demonstrate an extended spectral coverage from 5 /spl mu/m, pump power thresholds as low as 45 mW, average mid-infrared output powers in excess of 100 mW, and pulse durations of 100-200 fs and 1-2 ps at /spl sim/80 MHz repetition rate. We also report the efficient operation of Ti:sapphire-pumped femtosecond OPOs in all-solid-state configurations by utilizing diode-laser-based input pump sources.

We report the development and application of instrumentation to measure and image tissue autofluorescence lifetime for the study and diagnosis of disease including cancer and osteoarthritis.

Cartilage is a vital organ to maintain joint function. Upon arthritis, proteolytic enzymes initiate degradation of cartilage extracellular matrix (ECM) resulting in eventual loss of joint function. However, there are only limited ways of non-invasively monitoring early chemical changes in cartilage matrix. Here we report that the autofluorescence decay profiles of cartilage tissue are significantly affected by proteolytic degradation of cartilage ECM and can be characterised by measurements of the autofluorescence lifetime (AFL). A compact multidimensional fluorometer coupled to a fibre-optic probe was developed for single point measurements of AFL and applied to cartilage that was treated with different proteinases. Upon treating cartilage with bacterial collagenase, trypsin or matrix metalloproteinase 1, a significant dose and time dependent decrease of AFL was observed. Our data suggest that AFL of cartilage tissue is a potential non-invasive readout to monitor cartilage matrix integrity that may contribute to future diagnosis of cartilage defects as well as monitoring the efficacy of anti-joint therapeutic agents.

We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that mTFP-based probes are more suitable for FLIM experiments than CFP-based probes.

Very few nonlinear optical materials are actually useful for high throughput all-optical devices. However, AlGaAs does satisfy all of the nonlinear optical figures of merit when used with photons of energy less than one half the semiconductor bandgap. Here we review our measurements of the pertinent nonlinear coefficients in waveguides and various device applications to all-optical switching in the communications band around 1550 nm.

A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated.