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Hyperspectral imaging is a generic imaging modality allowing high spectral and spatial resolution over a wide wavelength range from the visible to mid-infrared. Short wavelength infrared (SWIR) hyperspectral imaging is currently becoming an important supplement to spectroscopy in optical diagnostics due to the flexibility and adaptability of the technique. However, due to the complexity of hyperspectral data, the analysis requires a well planned approach. In this paper a simple but effective approach combining dimension reduction and unsupervised classification is suggested. Examples of in vivo hyperspectral data in the SWIR spectral range (950-2500 nm) from human skin bruises and porcine skin burns are presented as examples. Data are processed using the minimum noise fraction transform (MNF), and K-means clustering. K-means clustering was found to perform significantly better if applied to MNF transformed data. The classification results agree well with biopsies, spectral data and visual inspection of injuries. It is thus shown that unsupervised clustering can be a preferable technique in cases where it is challenging to use or interpret results from physics based models, or where the ground truth is lacking or not well defined. The presented results confirm that SWIR hyperspectral imaging indeed is a useful tool for optical characterization of tissue. © 2019 Society of Photo Optical Instrumentation Engineers. One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited.

The spectral and spatial resolution of hyperspectral imaging is useful for investigation of tissue autofluorescence. The low-light, noisy conditions in fluorescence imaging usually necessitates noise removal for extraction of precise spectral signatures and peak shifts. However, noise removal techniques like low-pass filtering or the Maximum Noise Fraction transform might discard information or distort spectral features. In this study, smoothing splines is proposed as an alternative technique to avoid spectral distortion in analysis of hyperspectral fluorescence images in the wavelength range 400-1000 nm. Continuous tuning parameters and use of natural cubic splines makes the method advantageous for unbiased peak extraction. The method was tested on ex vivo images of atherosclerosis lesions and simulations. The method was used to estimate autofluorescence peak shifts, and found to perform well in comparison with MNF. © 2019 Society of Photo Optical Instrumentation Engineers. One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited.

Hyperspectral imagery opens a new perspective for biomedical diagnostics and tissue characterization. High spectral resolution can give insight into optical properties of the skin tissue. However, at the same time the amount of collected data represents a challenge when it comes to decomposition into clusters and extraction of useful diagnostic information. In this study spectral-spatial classification and inverse diffusion modeling were employed to hyperspectral images obtained from a porcine burn model using a hyperspectral push-broom camera. The implemented method takes advantage of spatial and spectral information simultaneously, and provides information about the average optical properties within each cluster. The implemented algorithm allows mapping spectral and spatial heterogeneity of the burn injury as well as dynamic changes of spectral properties within the burn area. The combination of statistical and physics informed tools allowed for initial separation of different burn wounds and further detailed characterization of the injuries in short post-injury time.

We apply diffuse reflectance spectroscopy (DRS) and pulsed photothermal radiometry (PPTR) for characterization of the bruise evolution process. While DRS provides information in a wide range of visible wavelengths, the PPTR enables extraction of detailed depth distribution and concentration profiles of selected absorbers (e.g. melanin, hemoglobin). In this study, we simulate experimental DRS spectra and PPTR signals using the Monte Carlo technique and focus on characterization of a suitable fitting approach for their analysis. We find inverse Monte Carlo to be superior to the diffusion approximation approach for the inverse analysis of DRS spectra. The analysis is then augmented with information obtainable by the fitting of the PPTR signal. We show that both techniques can be coupled in a combined fitting approach. The combining of two complementary techniques improves the robustness and accuracy of the inverse analysis, enabling a comprehensive quantitative characterization of the bruise evolution dynamics.

Rheumatoid arthritis (RA) is a disease that frequently leads to joint destruction. It has a high incidence rate worldwide, and the disease significantly reduces patients’ quality of life. Detecting and treating inflammatory arthritis before structural damage to the joint has occurred is known to be essential for preventing patient disability and pain. Existing diagnostic technologies are expensive, time consuming, and require trained personnel to collect and interpret data. Optical techniques might be a fast, noninvasive alternative. Hyperspectral imaging (HSI) is a noncontact optical technique which provides both spectral and spatial information in one measurement. In this study, the feasibility of HSI in arthritis diagnostics was explored by numerical simulations and optimal imaging parameters were identified. Hyperspectral reflectance and transmission images of RA and normal human joint models were simulated using the Monte Carlo method. The spectral range was 600 to 1100 nm. Characteristic spatial patterns for RA joints and two spectral windows with transmission were identified. The study demonstrated that transmittance images of human joints could be used as one parameter for discrimination between arthritic and unaffected joints. The presented work shows that HSI is a promising imaging modality for the diagnostics and follow-up monitoring of arthritis in small joints. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Light propagation in highly scattering biological tissues is often treated in the so-called diffusion approximation (DA). Although the analytical solutions derived within the DA are known to be inaccurate near tissue boundaries and absorbing layers, their use in quantitative analysis of diffuse reflectance spectra (DRS) is quite common. We analyze the artifacts in assessed tissue properties which occur in fitting of numerically simulated DRS with the DA solutions for a three-layer skin model. In addition, we introduce an original procedure which significantly improves the accuracy of such an inverse analysis of DRS. This procedure involves a single comparison run of a Monte Carlo (MC) numerical model, yet avoids the need to implement and run an inverse MC. This approach is tested also in analysis of experimental DRS from human skin.

This is the author’s final, accepted and refereed manuscript to the article. Wound healing is a complex process not fully understood. There is a need of better methods to evaluate the different stages of healing, and optical characterization is a promising tool in this respect. In this study hyperspectral imaging was employed to characterize an in vitro wound model. The wound model was established by first cutting circular patches of human abdominal skin using an 8mm punch biopsy tool, and then creating dermal wounds in the center of the skin patches using a 5mm tool. The wounds were incubated in medium with 10% serum and antibiotics. Hyperspectral images were collected every three days using a push broom hyper spectral camera (Hyspex VNIR1600). The camera had a spectral resolution of 3.7 nm and was fitted with a close up lens giving a FOV of 2.5 cm and a spatial resolution of 29 micrometer. Samples for histology were collected throughout the measurement period, which was 21 days in total. Data were processed in ENVI and Matlab. A successful classification based on hyperspectral imaging of the implemented model is presented. It was not possible to see the healing zone in the in vitro model with the naked eye without dying. The hyperspectral results showed that newly formed epithelium could be imaged without any additional contrast agents or dyes. It was also possible to detect non-viable tissue. In vitro wound models and hyperspectral imaging can thus be employed to gain further insight in the complicated process of healing in different kinds of wounds. © (2014) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only. Copyright 2014. Society of Photo Optical Instrumentation Engineers. One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited.

Rheumatoid arthritis is a disease that frequently leads to joint destruction. It has high incidence rates worldwide, and the disease significantly reduces patient’s quality of life due to pain, swelling and stiffness of the affected joints. Early diagnosis is necessary to improve course of the disease, therefore sensitive and accurate diagnostic tools are required. Optical imaging techniques have capability for early diagnosis and monitoring of arthritis. As compared to conventional diagnostic techniques optical technique is a noninvasive, noncontact and fast way of collecting diagnostic information. However, a realistic model of light transport in human joints is needed for understanding and developing of such optical diagnostic tools. The aim of this study is to develop a 3D numerical model of light transport in a human finger. The model will guide development of a hyperspectral imaging (HSI) diagnostic modality for arthritis in human fingers. The implemented human finger geometry is based on anatomical data. Optical data of finger tissues are adjusted to represent either an arthritic or an unaffected finger. The geometry and optical data serve as input into a 3D Monte Carlo method, which calculate diffuse reflectance, transmittance and absorbed energy distributions. The parameters of the model are optimized based on HIS-measurements of human fingers. The presented model serves as an important tool for understanding and development of HSI as an arthritis diagnostic modality. Yet, it can be applied to other optical techniques and finger diseases.

Inflammatory arthritic diseases have prevalence between 2 and 3% and may lead to joint destruction and deformation resulting in a loss of function. Patient’s quality of life is often severely affected as the disease attacks hands and finger joints. Pathology involved in arthritis includes angiogenesis, hyper-vascularization, hyper-metabolism and relative hypoxia. We have employed hyperspectral imaging to study the hemodynamics of affected- and non-affected joints and tissue. Two hyperspectral, push-broom cameras were used (VNIR-1600, SWIR-320i, Norsk Elektro Optikk AS, Norway). Optical spectra (400nm – 1700nm) of high spectral resolution were collected from 15 patients with visible symptoms of arthritic rheumatic diseases in at least one joint. The control group consisted of 10 healthy individuals. Concentrations of dominant chromophores were calculated based on analytical calculations of light transport in tissue. Image processing was used to analyze hyperspectral data and retrieve information, e.g. blood concentration and tissue oxygenation maps. The obtained results indicate that hyperspectral imaging can be used to quantify changes within affected joints and surrounding tissue. Further improvement of this method will have positive impact on diagnosis of arthritic joints at an early stage. Moreover it will enable development of fast, noninvasive and noncontact diagnostic tool of arthritic joints

Measurement of diffuse reflectance spectra (DRS) is a common experimental approach for non-invasive determination of tissue optical properties, as well as objective monitoring of various tissue malformations. Propagation of light in scattering media is often treated in diffusion approximation (DA). The major advantage of this approach is that it leads to enclosed analytical solutions for tissues with layered structure, which includes human skin. Despite the fact that DA solutions were shown to be inaccurate near tissue boundaries, the practicality of this approach makes it quite popular, especially when attempting extraction of specific chromophore concentrations from measured DRS. In this study we analyze the discrepancies between DRS spectra as obtained by using the DA solutions for three-layer skin model and more accurate predictions from Monte Carlo (MC) modeling. Next, we analyze the artifacts which result from the above discrepancies when extracting the parameters of skin structure and composition by fitting the DA solutions to the MC spectra. The reliability and usefulness of such a fit is then tested also on measurements of seasonal changes in otherwise healthy human skin.

Information about the size and depth of a wound and how it is developing is an important prognostic tool in wound diagnostics. In this study a two-camera vision system has been developed to collect optical properties, shape and volume of chronic skin ulcers as tool for diagnostic assistance. This system combines the functionality of 2D imaging spectroscopy and 3D stereo-photogrammetry. A high resolution hyperspectral camera and a monochromatic video frame camera were mounted on the same scanning system. Stereo images were acquired to obtain information about the wound surface geometry. A Digital Surface Model (DSM) of the wound surface was reconstructed by applying stereophotogrammetric methods. The hyperspectral image was co-registered to the monochromatic frame image and the wound border was extracted by applying spectroscopic analysis (e.g. tissue oxygenation, pigmentation, classification). The resulting DSM of the undamaged surroundings of the wound was used to reconstruct the top surface above the wound and thus the wound volume. The analyses can, if desired, be limited to a certain depth of interest like the wound bed or wound border. Simultaneous analysis of the hyperspectral data and the surface model gives a promising, new, non-invasive tool for characterization of chronic wounds. Future work will concentrate on implementation of real time analysis and improvement of the accuracy of the system.

The healing process of chronic wounds is complex, and the complete pathogenesis is not known. Diagnosis is currently based on visual inspection, biopsies and collection of samples from the wound surface. This is often time consuming, expensive and to some extent subjective procedures. Hyperspectral imaging has been shown to be a promising modality for optical diagnostics. The main objective of this study was to identify a suitable technique for reproducible classification of hyperspectral data from a wound and the surrounding tissue. Two statistical classification methods have been tested and compared to the performance of a dermatologist. Hyperspectral images (400-1000 nm) were collected from patients with venous leg ulcers using a pushbroom-scanning camera (VNIR 1600, Norsk Elektro Optikk AS).Wounds were examined regularly over 4 - 6 weeks. The patients were evaluated by a dermatologist at every appointment. One patient has been selected for presentation in this paper (female, age 53 years). The oxygen saturation of the wound area was determined by wavelength ratio metrics. Spectral angle mapping (SAM) and k-means clustering were used for classification. Automatic extraction of endmember spectra was employed to minimize human interaction. A comparison of the methods shows that k-means clustering is the most stable method over time, and shows the best overlap with the dermatologist’s assessment of the wound border. The results are assumed to be affected by the data preprocessing and chosen endmember extraction algorithm. Results indicate that it is possible to develop an automated method for reliable classification of wounds based on hyperspectral data.

Hyperspectral imaging provides means for characterizing large biological samples with microscopic spatial resolution and a narrow spectral sampling interval. However, this approach requires having a measurable light signal in each spectral band. Overcoming the limitations imposed by working with biological samples requires the use of a highly sensitive sensor to detect weak signals. For this study we have built and compared the performance of two imaging spectrometers using optimized for low light environments: an electron-multiplying CCD (EMCCD) and a scientific CMOS (sCMOS). Both systems have been designed to lower the risk of damaging photosensitive samples, delay the bleaching of fluorophores and detect weak fluorescence signals. The cameras work within the VNIR spectral region (400 nm - 900 nm) with a spectral sampling lower than 4 nm. The produced images have scene pixel sizes smaller than 25 μm and a field of view larger than 25 mm. The systems have been tested side to side measuring the diffusion front of a fluorescent tag in samples of porcine skin in challenging light conditions. The study aimed to show the advantages and limitations of each approach. Preliminary results show good performance of the EMCCD for fluorescence applications, whereas more experimental results are needed to be able to conclude on the performance of the sCMOS sensor. However, the sCMOS appears promising for imaging scenes with high dynamics in low light settings.

Optical diagnostics of bruised skin might provide important information for characterization and age determination of such injuries. Hyperspectral imaging is one of the optical techniques that have been employed for bruise characterization. This technique combines high spatial and spectral resolution and makes it possible to study both chromophore signatures and -distributions in an injury. Imaging and spectroscopy in the visible spectral range have resulted in increased knowledge about skin bruises. So far the SWIR region has not been explored for this application. The main objective of the current study was to characterize bruises in the SWIR wavelength range. Hyperspectral images in the SWIR (950-2500nm ) and VNIR (400-850nm) spectral range were collected from 3 adult volunteers with bruises of known age. Data were collected over a period of 8 days. The data were analyzed using spectroscopic techniques and statistical image analysis. Preliminary results from the pilot study indicate that SWIR hyperspectral imaging might be an important supplement to imaging in the visible part of the spectrum. The technique emphasizes local edema and gives a possibility to visualize features that cannot easily be seen in the visible part of the spectrum.

Hyperspectral fluorescence imaging is a modality combining high spatial and spectral resolution with increased sensitivity for low photon counts. The main objective of the current study was to investigate if this technique is a suitable tool for characterization of diffusion properties in human skin. This was done by imaging fluorescence from Alexa 488 in ex vivo human skin samples using an sCMOS based hyperspectral camera. Pre-treatment with acetone, DMSO and mechanical micro-needling of the stratum corneum created variation in epidermal permeability between the measured samples. Selected samples were also stained using fluorescence labelled biopolymers. The effect of fluorescence enhancers on transdermal diffusion could be documented from the collected data. Acetone was found to have an enhancing effect on the transport, and the results indicate that the biopolymers might have a similar effect. Hyperspectral fluorescence imaging has thus been proven to be an interesting tool for characterization of fluorophore diffusion in ex vivo skin samples. Further work will include repetition of the measurements in a shorter time scale and mathematical modeling of the diffusion process to determine the diffusivity in skin for the compounds in question.

The new "scientific CMOS" (sCMOS) sensor technology has been tested for use in hyperspectral imaging. The sCMOS offers extremely low readout noise combined with high resolution and high speed, making it attractive for hyperspectral imaging applications. A commercial HySpex hyperspectral camera has been modified to be used in low light conditions integrating an sCMOS sensor array. Initial tests of fluorescence imaging in challenging light settings have been performed. The imaged objects are layered phantoms labelled with controlled location and concentration of fluorophore. The camera has been compared to a state of the art spectral imager based on CCD technology. The image quality of the sCMOS-based camera suffers from artifacts due to a high density of pixels with excessive noise, attributed to the high operating temperature of the array. Image processing results illustrate some of the benefits and challenges of the new sCMOS technology.

Psoriasis is a common autoimmune disease with inflammatory symptoms affecting skin and joints. One way of dealing with psoriasis is by controlled solar UV exposure treatment. However, this treatment should be optimized to get the best possible treatment effect and to limit negative side effects such as erythema and an increased risk of skin cancer. In this study 24 patients at Valle Marina Treatment Center in Gran Canaria were monitored throughout a treatment period of three weeks starting at the beginning of November. The total UV dose to the location was monitored by UV-meters placed on the roof of the treatment centere, and the patients wore individual film dosimeters throughout the treatment period. Skin parameters were accessed by reflection spectroscopy (400-850nm). This paper presents preliminary findings from the skin measurements in the visible part of the spectrum, such as blood oxygenation, erythema and melanin indexes. Reflection spectroscopy was found to be a good tool for such treatment monitoring.

OBJECTIVES: In this study, we evaluated histologically the effects of hexyl 5-aminolevulinateinduced photodynamic treatment in the AY-27 tumor cell induced rat bladder cancer model. MATERIAL & METHODS: The animals (fischer-344 female rats) were divided into 2 groups, half of which were orthotopically implanted with 400,000 syngeniec AY-27 urothelia1 rat bladder cancer cells and half sham implanted. 14 days post implantation 6 rats from each group were treated with hexyl 5-aminolevulinate-induced photodynamic treatment (8mM HAL and light fluence of 20 J/cm2). Additional groups of animals were only given HAL instillation, only light treatment, or no treatment. All animals were sacrificed 7 days after the PDT/only HAL/only light or no treatment. Each bladder was removed, embedded in paraffin and stained with hematoxylin, eosin, and saferin for histological evaluation at high magnification for features of tissue damage by a pathologist blinded to the sample source. RESULTS: In all animals that were AY-27 implanted and not given complete PDT treatment, viable tumors were found in the bladder mucosa and wall. In the animals treated with complete HAL-PDT only 3 of 6 animals had viable tumor. In the 3 animals with viable tumor it was significantly reduced in volume compared to the untreated animals. It was also noted that in the PDT treated animals there was a significantly increased inflammatory response (lymphocytic and mononuclear cell infiltration) in the peri-tumor area compared to implanted animals without complete HAL-PDT. CONCLUSION: Our results suggest that hexyl 5-aminolevulinate-induced photodynamic treatment in a rat bladder cancer model involves both direct effects on cell death (necrosis and apoptosis) and indirect effects to evoke the host immune-response, together contributing to tumor eradication.

Imaging modalities like hyperspectral imaging create large amounts of data. Time efficient, automated analytic techniques are therefore required to enjoy the power of such methods. In this study it was investigated if hyperspectral imaging followed by automated noise filtering and statistical image analysis is a suitable method for characterization of the macroscopic structure of atherosclerotic lesions. Ten human aorta samples (6x8 cm) were collected during autopsy. Hyperspectral white light and fluorescence images and 5 – 6 biopsies were collected from each sample. The biopsies were stained (HES, Sudan red), and grouped according to histology. All images were noise filtered and normalized. Fluorescence spectra were collected from all biopsied regions, and used to compute average spectra for each histological group. Supervised classification was performed using Spectral angle mapping (SAM) with the average spectra as endmembers. K-means- and ISO-data clustering was used for unsupervised classification. The results show that noise filtering and normalization is essential for reliable classification. Supervised classification was in general found to perform better than unsupervised classification. However, the SAM results strongly depend on the variation in the spectra used to compute the average endmember spectra. The analysis show that fatty deposits, calcifications, connective tissue and hemoglobin can be identified. The lesions were found to have a complex structure where vulnerable regions could be found next to stabile regions within the same lesion. In conclusion hyperspectral imaging, automated filtering and -analysis was found to be a suitable tool to classify advanced atherosclerotic lesions.

Hyperspectral imaging is a modality which combines spatial resolution and spectroscopy in one technique. Analysis of hyperspectral data from biological samples is a demanding task due to the large amount of data, and due to the complex optical properties of biological tissue. In this study it was investigated if depth information could be revealed from hyperspectral images using a combination of image analysis and analytic simulations of skin reflectance. It was also investigated if hyperspectral imaging could be utilized to monitor changes in the distribution of hemoglobin species after smoking. Hyperspectral data in the wavelength range 400-1000nm were collected from the forearm of 15 non-smokers and 5 smokers. The hyperspectral images were analyzed with respect to the distribution of hemoglobin species and vascular structures. Changes in the vascular system due to smoking were also evaluated. Principal component analysis (PCA), Spectral angle mapping (SAM), and Mixture tuned matched filtering (MTMF) were used to enhance vascular structures. Emphasis was put on identifying apparent and true absorption spectra for the present chromophores by combining image analysis and an analytical photon transport model. The results show that the depth resolution of hyperspectral images can be better understood using analytical simulations. Modulation of the chromophore spectra by the optical properties of overlying tissue was found to be an important mechanism causing the depth resolution in hyperspectral images. It was also found that hyperspectral imaging and image analysis can be successfully applied to quantify skin changes following smoking.

Studies of immediate skin reactions are important to understand the underlying biological mechanisms involved in traumatic or chemical damage to the skin. In this study the spatial and spectral information provided by hyperspectral images was used to identify and characterize non-penetrating skin injuries in a porcine model. A hyperspectral imaging system (Hyspex, Norsk Elektro Optikk AS) was used to monitor the temporal development of minor skin injuries in an anesthetized Norwegian domestic pig. Hyperspectral data were collected in the wavelength range 400-1000 nm (VNIR), with a spectral sampling interval of 3.7 nm. The measurements were initiated immediately after inflicting the injury, and were repeated at least five times at each site with irregular frequency. The last measurement was performed 4 hours after injury. Punch biopsies (5 mm), were collected from adjacent normal skin, and at the center and the margin of each injury. The study was approved by the national animal research authority. The hyperspectral data were analyzed with respect to oxy- and deoxyhemoglobin, and erythema index. The skin biopsies were examined to determine the extent of skin damage in the bruised zones. Preliminary results show that hyperspectral imaging allows discrimination between traumatized skin and normal skin in an early phase. The extent and location of the hemorrhages can be determined from hyperspectral images. These findings might contribute to a better understanding of immediate skin reactions to minor trauma, and thereby the development of a better diagnostic modality for non-penetrating skin injuries in forensic medicine.

Determining the cause of an injury and the force behind the impact may be of crucial importance in a court case. For non-penetrating soft tissue injuries there is a lack of information available in the literature. In this study controlled bruises were inflicted on an anesthetized pig by high speed, low-weight paintball projectiles (diameter 17.1 mm, weight 3.15 g). The speed of the object and the impact itself were monitored using a high speed camera. Punch biopsies (5 mm) were collected from the injury sites. A red and purple ring with a diameter of 1.5 cm appeared on the skin within 30 seconds after the paintball impact. The ring was visually fully established after 5-10 minutes. Numerical finite element simulations performed with ABAQUS\Explicit showed a build up of shear stresses in the skin where the ring formed. Biopsies demonstrated severe dermal vessel damage in the same area. It is concluded that considerable shear stresses during the impact will create dermal vessel damage and thereby cause a visible bruise. Larger forces are required for compressive stresses to inflict equivalent vascular damage.

Photodynamic therapy (PDT) is a treatment modality which has been shown to be effective for both malignant and non-malignant diseases. New photosensitizers such as hexyl-aminolevulinate (HAL) may increase the efficiency of PDT. HAL penetrates into the cell where the photosensitizer protoporphyrin IX (PPIX) is produced endogenously. In a previous study on HAL based PDT treatment of rat bladder cancer (AY-27 transitional cell carcinoma), a depression of the optical reflectance spectra after treatment was observed in some of the animals. This depression of the spectra was caused by metHemoglobin (metHb). MetHb is an indication of oxidative stress, and can be formed as a result of for instance UV-radiation and heating of blood. The aim of this study was to identify if metHb can be formed in vitro as a result of oxidative stress caused by singlet oxygen and ROS produced during PDT. Methemoglobin formed during PDT might thus be used as an indirect measure of the photochemical processes. This may help predict the PDT treatment outcome. Red blood cells mixed with AY-27 cells exposed to HAL, or PPIX received light treatment, and the changes in the absorption spectra were measured spectrophotometrically. The methemoglobin absorbance spectrum was also studied, and found to be strongly dependant on pH. Hemolysis of erythrocytes by PDT was found, however no metHb was formed in vitro.

Photodynamic therapy (PDT) is a treatment modality which has been shown to be effective for both malignant and non-malignant diseases. New photosensitizers such as 5-aminolevulinic acid hexylester (hALA) may increase the efficiency of PDT. Monitoring of the tissue response provides important information for optimizing factors such as drug and light dose for this treatment modality. Optical spectroscopy may be suited for this task. To test the efficacy of hALA induced PDT, a study on rats with a superficial bladder cancer model, in which a bladder cancer cell line (AY-27) is instilled, will be performed. Preliminary studies have included a PDT feasibility study on rats, fluorescence spectroscopy on AY-27 cell suspensions, and optical reflection and fluorescence spectroscopy in rat bladders in vivo. The results from the preliminary studies are promising, and the study on hALA induced PDT treatment of bladder cancer will be continued.

Bruises can be important evidence in legal medicine, for example in cases of child abuse. Optical techniques can be used to discriminate and quantify the chromophores present in bruised skin, and thereby aid dating of an injury. However, spectroscopic techniques provide only average chromophore concentrations for the sampled volume, and contain little information about the spatial chromophore distribution in the bruise. Hyperspectral imaging combines the power of imaging and spectroscopy, and can provide both spectroscopic and spatial information. In this study a hyperspectral imaging system developed by Norsk Elektro Optikk AS was used to measure the temporal development of bruised skin in a human volunteer. The bruises were inflicted by paintball bullets. The wavelength ranges used were 400 - 1000 nm (VNIR) and 900 - 1700 nm (SWIR), and the spectral sampling intervals were 3.7 and 5 nm, respectively. Preliminary results show good spatial discrimination of the bruised areas compared to normal skin. Development of a white spot can be seen in the central zone of the bruises. This central white zone was found to resemble the shape of the object hitting the skin, and is believed to develop in areas where the impact caused vessel damage. These results show that hyperspectral imaging is a promising technique to evaluate the temporal and spatial development of bruises on human skin.

An objective method for dating of soft tissue injuries is highly desired in forensic medicine. To achieve this, obtaining a fundamental understanding of extravascular blood behavior is necessary. Porcine blood mixed with fluorescein was inserted subcutaneously on an anaesthetized pig. The fluorescence build-up was measured as a function of time, using a fiber probe setup. Fluorescence measurements proved to be a suitable tool for monitoring the temporal distribution of fluid in a subcutaneous hemorrhage. Reasonable values for the fluid velocity was obtained by monitoring the fluorescein distribution around the infusion point. Further theoretical work and development of the experimental technique is necessary to obtain a good understanding of subcutaneous transport of blood.

Diffuse skin reflectance was simulated by Monte Carlo methods and diffusion theory. Diffusion theory proved to agree well with measurements, and might in some applications supersede Monte Carlo methods due to faster, more efficient algorithms.

Many methods for detecting and measuring vulnerable atherosclerotic plaques have been proposed. These include reflection spectroscopy, thermography, ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI). This paper presents an analysis and a comparison of two of these methods, near-infrared reflection spectroscopy (NIRS) and thermography. Most of the published literature evaluate methods statistically. A more analytic approach will make it easier to compare the different methods and determine if the measured signal will be strong enough in a real measurement situation. This is the approach taken in this article. Eight samples of human aorta were examined by NIRS and subsequently prepared for histology. A total of 28 measurement points were selected. A measure of the lipid content based on reflection spectra is proposed. Comparisons of this lipid measure with histology show that the lipid content in the plaques yields relatively small changes in the value of this lipid-index. Reflectance spectra from models based on the diffusion approximation for total reflectance were simulated. Temperature measurements were performed on three Watanabe heritable hyperlipidemic (WHHL) rabbits and one New Zealand white (NZW) rabbit with a thermistor-type intravascular temperature sensor. The measurements gave no significant signals which correlated with the subsequent histology. A simple analytic model was developed which indicates that a temperature increase of more than 0.01-0.04 °C at the surface of a vessel wall, due to inflammation in a plaque, is unlikely. Such a small temperature difference will probably be obscured by normal variation in the vessel wall temperature.

Determining the age of injuries is an important aspect of forensic medicine. Currently, visual inspection and colorimetric measurements are the most common techniques used to assess the age of bruises on a victim's body. Bruises are caused by trauma to the skin and vasculature, and the color will depend on the age, depth, and anatomic site of the hemorrhage. Breakdown products of hemoglobin e.g. biliverdin and bilirubin possess various colors, which can be determined spectrometrically. This study presents reflection spectra collected from bruises in otherwise healthy subjects. A total of 73 spectra of 25 bruises were measured on 13 individuals in the 400-850 nm wavelength region. All injuries were caused by sports activities such as judo and soccer. The bruises were classified according to visual appearance, bilirubin content, oxygenation, and age of the injury. Only bruises with known age and cause were included in the study. Spectral changes of each hematoma were recorded over several days. Preliminary results show large variation in the spectra, caused by differences in age and depth of the bruises. This data may provide a basis for developing an algorithm to determine the age of injuries in e.g. child abuse cases.

The applicability of reflectance spectroscopy to detect pathological changes in human liver tissue was investigated. Post mortem reflectance spectra were collected from liver tissue originating from 13 individuals. A point counting method was applied to determine relative areas of connective tissue, liver cells with or without fat vacuoles, and vascular spaces in the liver. Preliminary results show that the amount of fat and connective tissue in liver can be estimated from reflectance spectra.

Knowledge of optical and thermal properties of human tissue is essential for optimization of laser therapy and optical diagnostics. Published data are in general incomplete, and there is very limited information on the temperature dependence of optical properties. The temperature dependence of hemoglobin is important since it is the target chromophore for laser treatment of e. g. port-wine stains. The present study emphasizes the temperature dependence of the hemoglobin absorption. Erythrocyte concentrate was heated in a water bath, hemolyzed with distilled water and centrifuged before absorption spectra were collected. Measurements were performed in the range of 50-80 °C. Penetration depth measurements of the heated erythrocyte concentrate were done at 488 nm, 585 nm, 590 nm and 630 nm. For these measurements the erythrocytes were mixed with a highly scattering material (whole milk). The hemoglobin absorbance demonstrates a characteristic change during heating. This change is partly due to oxidative reactions with formation of met-hemoglobin, and also by protein denaturation. The change in the absorption coeAEcient due to met-hemoglobin has a maximum at approximately 72 °C, where the increase was measured up to 200 % at 630 nm wavelength.

A device independent method for skin color visualization has been developed. Colors reconstructed from a reflectance spectrum are presented on a computer screen by sRGB (standard Red Green Blue) color coordinates. The colors are presented as adjacent patches surrounded by a medium grey border. CIELAB color coordinates and CIE (International Commission on Illumination) color difference (Delta) E are computed. The change in skin color due to a change in average blood content or scattering properties in dermis is investigated. This is done by analytical simulations based on the diffusion approximation. It is found that an 11% change in average blood content and a 15% change in scattering properties will give a visible color change. A supposed visibility limit for (Delta) E is given. This value is based on experimental testing and the known properties of the human visual system. This limit value can be used as a tool to determine when to terminate laser treatment of port- wine stain due to low treatment response, i.e. low (Delta) E between treatments. The visualization method presented seems promising for medical applications as port-wine stain diagnostics. The method gives good possibilities for electronic transfer of data between clinics because it is device independent.