Noninvasive Optical Modalities in Tissue Viability Assessment: a Review

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SMGr up Noninvasive Optical Modalities in Tissue Viability Assessment: a Review Guennadi Saiko* Department of Physics, Ryerson University, Canada

*Corresponding author: Guennadi Saiko, Department of Physics, Ryerson University, Toronto, Canada. Email: [email protected] Published Date: January 04, 2017

ABSTRACT Clinical examination alone is not always sufficient to assess wound properly. We present a review of optical modalities currently in clinical use and under development to assist wound doctors in assessing tissue viability, including laser Doppler imaging, laser speckle contrast imaging, photoplethysmography, polarization spectroscopy, hyperspectral/multispectral imaging, photoacoustic tomography, optical coherence tomography, photometric structural and chromatic assessment, endogenous fluorescence imaging, and fluorescence videoangiography.

INTRODUCTION

Chronic wounds represent a significant challenge to healthcare systems around the world. Up to 2% of the population including up to 15% of seniors suffer from chronic or compromised wounds [1]. This condition is characterized by debilitating pain and reduced quality of life for those whose health is already compromised.

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The proper diagnostic is an essential part of chronic wound treatment and management. For example, it is essential to differentiate between arterial and venous ulcers, mainly because the standard of care for venous ulcers (compression therapy) may lead to skin necrosis if applied to an ischemic leg. In addition to visual observation, which remains a gold standard for diagnostics (e.g. for burns and pressure ulcers) a number of diagnostic and adjuvant to diagnostic techniques has been developed.

These techniques rely on qualitative and quantitative assessments of the direct parameters[2] such as the length, width, depth, area, volume, healing rate, location, appearance of the ulcer, ulcer odor as well as the indirect parameters such as blood perfusion[3,4], local tissue metabolism and oxygen supply [5,6], local tissue structural atrophy [7,8,9] and pain [10]. Some of these methods have been widely adopted into the clinical practice. Among them are Transcutaneous oximetry, Doppler ultrasound, and Duplex Ultrasonography.

Transcutaneous oximetry (tcpO2) is a well-established non-invasive method used in wound healing centers to predict wound healing potential [11]. tcpO2 measurement quantifies the amount of oxygen that diffuses from the capillaries and through the skin and reflects the status of the capillaries identifying tissue that is hypoxic.

Doppler ankle-brachial pressure index (ABI) is considered the non-invasive screening test of choice to detect peripheral arterial disease (PAD) and select appropriate treatment pathway for patients with leg ulcers [12]. Duplex ultrasonography combines real-time b-mode ultrasound with pulsed-wave Doppler. It became the method of choice in patients with varicose veins and now is used in patients with leg ulcers. In particular, Duplex ultrasound imaging is useful in the assessment of venous reflux (retrograde flow of blood) in patients with chronic leg ulceration. The same technology can be used to visualize the arterial network to identify the severity of arterial disease.

With the recent advances in optics and photonics and advent of inexpensive CMOS/CCD cameras, a lot of novel optical diagnostic modalities have been developed and brought to the clinic. Most of the clinical devices aim towards monitoring or diagnostics through non-invasive or minimally invasive schemes. These novel optical techniques are the subject of this review. We will review basic principles, clinical applications, and current market offering for these techniques. It should be pointed out that the focus of this review is wide viewing area optical modalities in visible and near infrared (NIR) ranges of the spectrum. Thus, a number of microscopic modalities [13] (e.g. capillary microscopy, confocal microscopy, orthogonal polarization spectral imaging) as well as thermal imaging [14] were omitted from the current review. We also omitted some recently-developed optical modalities (e.g. lens-less imaging [15]), which are not translated to the clinic yet.

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To facilitate and highlight the importance of measuring direct and indirect parameters related to wound healing, the discussion of clinical diagnostics is categorically classified as follows:

EVALUATION OF BLOOD PERFUSION IN TISSUES

The clinical application of monitoring blood flow in tissues is of paramount importance. For maintaining a normal structural and functional state of the skin, an adequate blood perfusion is required. Evaluating blood perfusion provides opportunities to diagnose various diseased states. Many non-invasive methods have been developed to monitor blood perfusion in tissues.

Laser Doppler Perfusion Monitoring and Imaging

Biological tissues comprise of multiple stationary as well as mobile (mainly red blood cells) scattering particles. Doppler Effect occurs when a scattering particle is moving in the presence of an incident wave. Coherent light from a laser, when directed into tissues, exhibits a Doppler shift in frequency when encountering moving particles. The incident wave’s frequency is modified according to the speed and direction of motion of the particle. As a result, the backscattered signal from the tissues can be decomposed into flux, cell concentration, and cell velocity [3,16 ].

Two common geometries are employed for Laser Doppler perfusion monitoring: a) Laser Doppler Flowmetry (LDF), where a fiber optic probe (transmitting and receiving fibers) is kept in contact with skin (Figure 1) and b) Laser Doppler Imaging (LDI), where scanning (X-Y) mirrors or beamsplitters are used to transmit light to the skin and direct the received light to the photodetector to form an image [16]. Interrogation depth of 1mm can be usually achieved [17] by this method where most of the capillaries and dermal vessels are situated and flow velocities ranging from 0.01 - 0.1mm/s can be determined [7].

Figure 1: A fiber-optic based laser Doppler flowmeter placed on the skin. The red lines emanating from the fiber indicate laser illumination and the blue indicate Doppler shifted light (adapted from [18], copyrights of Perimed AB).

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Laser Doppler Flowmetry Laser Doppler Flowmetry technology is a non-invasive method in which laser light is used to detect blood perfusion in the microcirculation, identifying ischaemic tissue. The measuring depth is around 0.5 - 1 mm, reaching the superficial vessels: arterioles, venules, shunts, and capillaries. For diagnostic purposes, laser Doppler is often combined with a provocation, for example in the form of heating. The response to a provocation is a more repetitive measurement than just measuring blood perfusion at a basal level since the microcirculation itself is extremely dynamic and may vary extensively under normal conditions [18].

Laser Doppler Imaging

Laser Doppler Imaging (LDI) is a natural extension of the Laser Doppler Flowmetry to imaging. The ability of LDI to evaluate burn depth was investigated in [19]. Clinical examination correctly determined 66% of deep partial or full thickness burns between 36 and 72 h of injury compared to 90% using LDI. The LDI was also more specific; correctly diagnosing 96% of superficial partial thickness burns as opposed to 71% on clinical examination. Both LDF and LDI can be used to quantify perfusion in burn scars and monitor changes over time [20]. The moorLDI2-IR (Moor Instruments Ltd) [21] is a clinical LDI device with wide scan area (50x50cm) and high spatial resolution (up to 100µm). It uses a low-power laser beam, directed at the burn wound using a mirror. The laser beam remotely scans across the burn wound by rotating the mirror. Depending on the size of the burn wound and required resolution of the image, the scan takes from 80 seconds to about 5 minutes.

Laser Speckle Contrast Imaging

Speckle structures are produced as a result of interference of a large number of elementary waves with random phases that arise when coherent light is reflected from a rough surface or when coherent light passes through a scattering medium. If the surface or media does not change, the speckle pattern remains static. Motion gives rise to spatial or temporal fluctuations of the intensity dependent on the velocity of the motion [22]. Thus, by studying the temporal statistics of these fluctuations, velocity estimation is performed. A high-speed camera (~200fps) is used (Figure 2) to measure and analyze the temporal statistics of the speckle patterns to estimate the blood perfusion.

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Figure 2: Laser speckle imaging setup. LS: Light source, CAM: Camera (including its own focusing lenses), O: Optical element (focusing system). [Adapted from [23] with permission, copyrights of Elsevier]. This modality provides essentially the same information of skin perfusion as LDI, but its

effective penetration depth is less than a conventional laser Doppler imager. So, it collects data from the nutritive layers [24] and is less influenced by flow from deeper and larger vessels. Another difference, that this technique can provide video frame acquisition rate.

Moor Instruments (UK) offers Laser Speckle Contrast Imager moorFLPI-2 [25], which provides full-field, video frame rate blood flow imaging over the field of view from 5.6mm x 7.5mm up to 15cm x 20cm (continuously variable with zoom lens) with spatial resolution up to 10µm. Acquisition rate: from 25 images per second to 1 image every 12 hours. PeriCam PSI system (PeriMed, Sweden) [26] is another clinical tool for blood perfusion imaging that can be used for burn depth assessment.

Photoplethysmography

Photoplethysmography (PPG) uses light to detect blood volume changes occurring in the microvasculature [27]. Chromophores distributed in the skin and blood vessels are responsible for variations in light attenuation through absorption. The optical absorption coefficient of blood is higher than the surroundings and correspondingly light has a higher attenuation in regions with a higher blood volume. As the blood constantly flows through our body, the pulsatile flow of blood is registered as an AC signal superimposed on a DC signal. Together they form a PPG

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waveform (similar to an ECG waveform, PPG waveform is synchronous with the heart rate) [28]. The DC component of the signal is attributed to the attenuation from static structures and the AC component correlates to the blood flow rate. Transmission and reflectance schemes have been utilized to capture the PPG waveform and LEDs are generally used as a light source and a photodiode, CCD or CMOS is used as a sensor as depicted in Figure 3, [27].

Figure 3: Three approaches to the Photoplethysmograph setup. LED: Light emitting diode, PD: Photodetector. I) Transmission mode setup where illumination and detection are on opposite sides of the sample. II) Reflectance setup where they are on the same side, III) Area scan using a camera and illumination covering a large area. [Adapted from [23] with permission, copyrights of Elsevier]. Although Doppler ultrasound is considered a method of choice for peripheral arterial disease detection, PPG is also a suitable alternative; however, correlation and validation are yet to be established. In particular, it was found [12] that in patients with diabetes and/or chronic renal failure, Doppler ABI had lower sensitivity (69.2-71.4%) than photoplethysmography toe-finger index (TFI) (84.6-85.7%).

Polarization Spectroscopy

Specular reflection occurs when a small fraction of light is reflected at the interface of two media with a refractive index mismatch. These fractions, which are assumed to be weakly scattered do not represent much information about the perfusion states as they are superficially reflected and do not have a chance to integrate useful information. By using polarized incident light, these fractions can be isolated from the light backscattered from the epidermal or the dermal tissue matrix [16]. Strongly scattered light depolarizes while the weakly scattered light retains

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its polarization. This allows the gating of photons which integrate spectroscopic measurements over the region of interest for perfusion measurements. An example of such a setup is shown in Figure 4. Video rate polarization imaging allows monitoring blood flow velocities and vessel diameter estimations. It was applied to burn depth estimation and determined need for operative intervention with a sensitivity of 93% [29].

Figure 4: Setup depicting polarization spectroscopy. LS: Light source, P: Polarizer, OC: Optical Coupling, S: Source Fiber, D: Detector fiber, A: Analyzer (Polarizer), SPEC: Spectrometer. The analyzer is arranged to complement the polarizer in order to achieve polarization spectroscopy. [Adapted from [23] with permission, copyrights of Elsevier].

DIAGNOSTICS OF LOCAL TISSUE METABOLISM AND OXYGEN SUPPLY Hyperspectral/Multispectral Imaging Hyperspectral Imaging (HSI) is a novel imaging approach, which aims to record the spectrum for each pixel of the image. In this sense, hyperspectral imaging is the natural extension of the color (RGB) imaging. Spectrum at each pixel can be considered as a spectroscopic input, which can be decomposed and spectral signatures can be found. There are a few different techniques for acquiring the three-dimensional (x,y,λ) dataset of a hyperspectral cube (e.g. spatial scanning and spectral scanning). The choice of technique depends on the specific application.

Hyperspectral vs Multispectral: Hyperspectral imaging is related to multispectral imaging. The distinction between hyper- and multi-spectral is sometimes based on an arbitrary “number of bands”. Multispectral imaging deals with several images at discrete and somewhat narrow bands. Multispectral images do not produce the “spectrum” of an object. Hyperspectral deals with imaging narrow spectral bands over a continuous spectral range. So, a sensor with only 20 bands can also be hyperspectral when it covers the range from 500 to 700 nm with 20 bands each 10 nm Wound Healing | www.smgebooks.com

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wide. While a sensor with 20 discrete bands covering the VIS, NIR, and SWIR would be considered multispectral. In biomedical imaging hyperspectral or multispectral images can be used to extract data about components of the blood, which are chromophores in visible and NIR spectrum.

In typical biomedical applications, a monochrome CCD/CMOS sensor is used to acquire a spectral image of the area under interrogation. The multiplexing of wavelengths is achieved either by switching sources to obtain different wavelengths or switching band-pass filters to gate the photons in the case of broadband illumination. Rotary filter wheels, Liquid crystal tunable filters, and Acousto-optic tunable filters are a few examples which allow switching the band-pass wavelength range for acquiring HSI images (Figure 5). By obtaining white references, relative absorbance spectra can be calculated and by fitting the reference curves to each of the absorbance spectra, maps of oxygen saturation can be calculated [30,31].

Figure 5: HSI setup showing common approaches. HF: Hyperspectral filter (Liquid crystal, Acousto-optic etc.), O: Optical lens, MS: Mechanical scanning. I) A camera is used to take a hyperspectral image where a broadband illumination is utilized to capture an image after passing through an HF allowing spectral multiplexing (can be fast but has limited spectral resolution). II) A spectrometer taking point measurements and an image is achieved through MS (High spectral resolution, limited speed). [Adapted from [23] with permission, copyrights of Elsevier].

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Hyperspectral (or multispectral) imaging is a noninvasive technique, which does not require

injections of any dyes. Moreover, the non-contact system eliminates the potential for crosscontamination.

Oximetry imaging Tissue undergoing wound healing is characterized by varying oxygenation status, depending

on where the measurement is taken relative to the wound. This makes point measurements

poor indicators of the wound healing process. Oximetry Imaging is a spectroscopic method through which 2D maps of hemoglobin oxygen saturation (SO2) can be created. Oxygen anatomy

mapping of tissue has been used by clinicians in evaluating pathogenic conditions of localized

microcirculation, irritant-induced inflammation, ischemia-reperfusion injury, optical detection of cancer, and peripheral arterial disease.

There are a few medical device manufacturers which provide imaging systems for visualization

of oxygen distribution.

OxyVu (previously marketed by Hypermed Imaging Inc, US) is a clinical imaging system, which

provides imaging of 10x13 cm area with resolution up to 0.1mm using wavelengths between 500 and 660nm. Acquisition time: about 20 sec. The system is currently pulled from the market.

Clinical studies have shown that OxyVu may be used to help identify patients at risk for

developing critical limb ischemia missed by other modalities. The manufacturer also claimed: 92% in vivo correlation to a spectrometer, 90% PPV in predicting wound healing, 87% PPV in predicting revascularization success, and 88% PPV in amputation planning

Kent Multispectral Imaging device (Kent Imaging Inc., Canada) is another clinical imaging

system [32]. It is a NIR-based (700-980nm) multispectral imaging device, which visualizes tissue oxygenation and perfusion. Acquisition time: 1second.

Beyond tissue oximetry

In addition to oxyhemoglobin and deoxyhemoglobin, other tissue chromophores can be

extracted by hyperspectral/multispectral imaging. In particular, methemoglobin and water can be used for burn depth determination [33].

Raptor (Oxilight Inc, Canada) is a multispectral wound imaging system for visualization of total

hemoglobin, oxygenation, methemoglobin and water concentration in skin [34]. Field of view:

7x10cm, spatial resolution: 100µm. Acquisition time: 1 second. The device can be integrated with any EHR system.

Photoacoustic Tomography

Photoacoustic Tomography (PAT) uses pulsed laser light to irradiate tissues and as a result,

pressure waves are produced due to the increased temperature and volume [16]. High-frequency

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ultrasound transducer monitors these pressure waves and a 3D reconstruction is performed (Figure 6). Visible and near infrared light is used to perform PAT. The contrast in PAT images depends mainly upon the absorption properties of tissues [35]. The optical absorption is specific to tissue chromophores and changing the wavelength of light, one can tune the images to obtain enhanced contrast for specific chromophores.

Figure 6: Photoacoustic Imaging. The laser illuminates the sample and the resultant ultrasound signal is detected by an Ultrasound Transducer (US). [Adapted from [23] with permission, copyrights of Elsevier]. By projecting light at hemoglobin’s peak absorption, inflamed, hyperemic tissue appears

dark (hypoechoic) on PAT, while surrounding tissues reflecting such light waves appear bright (hyperechoic). In an experimental model of burns, PAT was able to distinguish different durations of thermal exposure within minutes of injury [36].

Other techniques such as Confocal Microscopy, Transcutaneous oximetry, Magnetic Resonance Imaging are also employed towards measuring evaluating oxygen saturation and local tissue metabolism [37].

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ESTIMATION OF LOCAL TISSUE STRUCTURES ATROPHY Optical Coherence Tomography Optical Coherence Tomography (OCT) is an interferometry method used to perform noninvasive, high-resolution, cross-sectional images of the superficial tissues. The most common approach in OCT is using an arm of a fiber-optic Michelson interferometer to irradiate the superficial tissues (Figure 7). A photodetector registers the interference generated by the combination of the reflected, backscattered light and the light from the reference arm. A translating mirror is attached to the reference arm which is moved to control the axial scan depth. Near infrared excitation (800nm - 1800nm) is generally preferred for OCT. One can perform high-resolution atrophy estimation by measuring the wound physically.

Figure 7: Standard OCT setup depicting the principle. LCS: Low-coherence light source, M: Reference Mirror, BS: Beam-splitter, O: Optical lens, PD: Photodetector. Mirror (M) is translated to achieve axial scan depth and the Sample can be translated to achieve lateral scan (this can also be achieved through the use of scanning mirrors). [Adapted from [23] with permission, copyrights of Elsevier]. In [38], Polarization-sensitive optical coherence tomography (PSOCT) showed a statistically strong mathematical correlation with absolute burn depth as determined by histology.

OTIS™ (Optical Tissue Imaging System) (Perimeter Imaging, Canada) is an intra-operative imaging tool which provides surgeons, radiologists, and pathologists with the ability to review ex vivo tissue microstructure during surgery [39]. Wound Healing | www.smgebooks.com

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PHOTOMETRIC STRUCTURAL AND CHROMATIC ASSESSMENT Manual wound size tracing has been widely practiced to assess the size/volume and physical attributes of wounds. With the advent of affordable CCD/CMOS cameras, the measurement of dimensions, volume, and chromatic characteristics have been employed to identify and grade ulcers. There have been many efforts towards using digital photography to get reliable 3D information. Photography, stereo photography, and planar wound tracing have been effectively applied towards structural characterization and measurements [2]. Typically, these systems are integrated with an EHR system or a wound database to support the overall clinical management of wounds. Colorimetric analysis has also been performed in conjunction with the structural analysis to estimate the ulcer stages [37]. The three main classes are used by clinicians: black for a necrotic eschar, yellow for slough and red for granulation tissue [37].

2D Imaging

The most traditional approach is to take a conventional RGB image using a digital camera or smartphone and track the progress in time. Wound margins can be outlined manually or automatically. Wound dimensions can be measured manually or automatically (e.g. using the reference object, e.g. a coin).

Swift App (Swift Medical, Canada) is software run on smartphones for measurement and tracking of wound healing process [40]. It uses machine vision technology, which is 10x more accurate than ruler-based methods.

3D Imaging

3D imaging is a natural extension of the traditional wound imaging, which allows measuring wound depth and calculating wound volume.

Silhouette(R) (Aranz Medical, Australia) is a 3D imaging system for wound care, which takes images, creates a 3D model of the wound based on the data acquired by the camera, derives measurements from the model, and records standardized notes [41]. A study [42] found that a Silhouette-derived measurement is likely to be within approximately 2% for the area, 1% for perimeter, 5% for average depth and 5% for volume (95% confidence interval).

FLUORESCENCE IMAGING

Fluorescence is the emission of light by a substance that has absorbed light. The human body contains numerous endogenous fluorophores (e.g. collagen, amino acids), or a fluorophore can be injected into the body (exogenous fluorophores, e.g. Indocyanine green).

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Endogenous Fluorescence Imaging All wounds contain bacteria (e.g., Staphylococcus, Streptococcus, Pseudomonas species, and Coliform bacteria including aerobic and anaerobic types), at levels ranging from contamination through critical colonization to infection. Fluorescence imaging may be used to identify particular strains in the wound, to assess (qualitatively or quantitatively) bacteria presence in the wound, or to guide sampling, debridement or antimicrobial selection.

Most of the clinically important strains (both gram- positive and negative) clearly show a distinctive double-peak of tryptophan fluorescence [43]. Unfortunately, these bands are within UVC band, which makes it problematic for the clinical use. However, some clinically relevant bacteria (S. aureus, S. epidermidis, Candida, S. marcescens, Viridans streptococci, Corynebacterium diphtheriae, S. pyogenes, Enterobacter, and Enterococcus) produces red fluorescence (from porphyrin [44]), while P. aeruginosa produced a bluish-green fluorescence (from pyoverdin [45]). MolecuLight i:X (Moleculight, Canada) is a handheld fluorescence imaging device that allows clinicians to visualize and precisely target bacterial presence and distribution in and around wounds, in real-time at the point-of-care [46].

Exogenous Fluorescence Imaging (fluorescence angiography)

Fluorescence angiography is based on the injection of fluorescent dye in the bloodstream and subsequent visualization of blood vessels. Initially, the method was developed for ophthalmology. Intravenous Fluorescein angiography (IVFA) or Fluorescent Angiography (FAG) is a technique for examining the circulation of the retina and choroid using a fluorescent dye and a specialized camera. Recently, the method has been extended to other blood vessels.

Indocyanine green (ICG) is a non-toxic, protein-bound dye that is retained within the vasculature after intravenous injection for several minutes until rapid clearance by the liver. ICG absorbs and fluoresces within the near-infrared spectrum, making deeper dermal vasculature visible using this dye. Relative to the subject’s normal skin, ICG fluorescence is markedly higher in spontaneously healing wounds, and markedly lower in those that required surgery [47]. Unlike other angiographic devices, the ICG angiography does not involve ionizing radiation. Spy Elite (Novadaq, Canada) is a fluorescence imaging system [48] for cardiovascular surgeons and wound care to visualize microvascular blood flow and perfusion in tissue intraoperatively. It is cleared by FDA for use during coronary artery bypass, cardiovascular, plastic, reconstructive, micro, organ transplant, and gastrointestinal surgery. The LUNA Fluorescence Angiography System (Novadaq, Canada) [49] produces a real-time visualization of tissue perfusion in patients being evaluated and treated for diabetic foot ulcers, arterial and venous blockages in the lower extremities, traumatic and chronic wounds. Wound Healing | www.smgebooks.com

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CONCLUSIONS The ability to assess chronic or compromised wound is a critical component of clinical treatment algorithms. Most of the current optical techniques to complement clinical exam operate on the premise that functioning blood vessels are retained in viable tissue. Various imaging modalities are available, offering different advantages and disadvantages.

References

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22. Briers JD. Laser speckle contrast imaging for measuring blood flow. Opt. Appl. 2007; 37: 139-152. 23. Schelkanova I, Pandya A, Muhaseen A, Saiko G, Douplik A. Early optical diagnosis of pressure ulcers, Ch 21 in Biophotonics for medical applications (Woodhead Publishing, 2015). 2015. 24. Forrester KR, Tulip J, Leonard C, Stewart C, Bray RC. A laser speckle imaging technique for measuring tissue perfusion. IEEE Trans. Biomed. Eng. 2004; 51: 2074-2084. 25. Moor. www.moor.co.uk/product/moorflpi-2-laser-speckle-contrast-imager/291. 26. Perimed AB. www.perimed-instruments.com/products/pericam-psi-burn-evaluation. 27. Allen J. Photoplethysmography and its application in clinical physiological measurement. Physiol. Meas. 2007; 28: R1. 28. Peláez-Coca MD, Orini M, Lázaro J, Bailón R, Gil E. Cross Time-Frequency Analysis for Combining Information of Several Sources: Application to Estimation of Spontaneous Respiratory Rate from Photoplethysmography. Comput. Math. Methods Med. 2013: 1. 29. Goertz O, Ring A, Kohlinger A, Daigeler A, Andree C, et al. Orthogonal polarization spectral imaging: a tool for assessing burn depths? Ann Plast Surg. 2010; 64: 217-221. 30. Denstedt M, Pukstad BS, Paluchowski LA, Hernandez-Palacios JE, Randeberg LL. Hyperspectral imaging as a diagnostic tool for chronic skin ulcers. Proc. SPIE 8565, Photonic Therapeutics and Diagnostics IX. 8565 (85650N–85650N–14). 2013. 31. Basiri A, Nabili M, Mathews S, Libin A, Groah S, et al. Use of a multi-spectral camera in the characterization of skin wounds. Opt. Express. 2010; 18: 3244-3257. 32. Kent Imaging. http://www.kentimaging.com/nir-spectroscopy. 33. Cross KM. Assessment of Tissue Viability in Acute Thermal Injuries Using Near Infrared Point Spectroscopy. PhD thesis, U of Toronto, Toronto, Canada. 2010. 34. Oxilight Inc. http://www.oxilight.ca. 35. Beard P. Biomedical photoacoustic imaging. Interface Focus. 2011; 1: 602-631. 36. Zhang HF, Maslov K, Stoica G, Wang LV. Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging. Nat Biotechnol. 2006; 24: 848-851. 37. Romanelli M, Dini V. “Bioengineering Techniques in Wound Assessment,” in Bioengineering Research of Chronic Wounds SE - 16, vol. 1, A. Gefen, Ed. Springer Berlin Heidelberg. 2009; 363-380. 38. Srinivas SM, de Boer JF, Park H, Keikhanzadeh K, Huang HE, et al. Determination of burn depth by polarization sensitive optical coherence tomography. J Biomed Opt. 2004; 9: 207-212. 39. Perimed. http://www.perimetermed.com/perimeters-otistrade.html. 40. Swift Medical. https://www.swiftmedical.io/. 41. Aranz Medical. http://www.aranzmedical.com/wound-assessment/. 42. Aranz Medical. http://www.aranzmedical.com/wound-measurement-accuracy-study/. 43. Dartnell LR, Roberts TA, Moore G, Ward JM, Muller JP. Fluorescence Characterization of Clinically-Important Bacteria. PLoS ONE. 2013; 8: e75270. 44. Kjeldstad B, Christensen T, Johnsson A. Porphyrin photosensitization of bacteria. Adv. Exp. Med. Biol. 1985; 193: 155-159. 45. Cody YS, Gross DC. Characterization of pyoverdin (pss), the fluorescent siderophore produced by Pseudomonas syringae pv. Syringae. Appl. Environ. Microbiol. 1987; 53: 928-934. 46. Moleculight. http://moleculight.com/product/. 47. Braue EH, Graham JS, Doxzon BF, Hanssen KA, Lumpkin HL, et al. Noninvasive methods for determine lesion depth from vesicant exposure. J Burn Care Res. 2007; 28: 275-285. 48. Novadaq. http://novadaq.com/products/spy-elite/. 49. Novadaq. http://novadaq.com/products/luna/.

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Copyright  Saiko G.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for commercial purposes, as long as the author and publisher are properly credited.