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Reduction of Light Scattering in Biological Tissue: Implications for Optical Diagnostics and Therapeutics
Abstract
The propagation of light in biological tissue provides invaluable information about structure and morphology. Recent years have seen an increase in the number of optical techniques developed to non-invasively diagnose the pathological state of tissue and to treat medical conditions. There are improvements to be made and many obstacles to be overcome. One of the biggest obstacles is how to proceed with techniques whose effectiveness is limited by the inherent properties of tissue. For instance, the scattering properties of tissue govern how deep light can penetrate into the tissue, at times greatly limiting the imaging or therapeutic potential of a given optical technique. It is this problem that the work of this dissertation attempts to address. A novel technique is described in this dissertation to reversibly alter light scattering in biological tissue in order to improve light-based diagnostic and therapeutic techniques in medicine. The method uses an organic chemical agent to reduce light scattering in skin. This significantly increases the amount of light that can be transmitted through the sample, rendering the tissue optically clear. The dominant processes were identified to be local tissue dehydration and refractive index matching. The technique of tissue optical clearing is shown to improve two clinically relevant procedures. In the first, the technique of decreased tissue scattering allows up to a 200% increase in the fluorescent signal from a deep tissue target. Additionally, glycerol was shown to significantly improve the direct visualization and optical imaging of blood vessels in skin, which is relevant to the laser treatment of cutaneous vascular lesions. The research studies contained in this dissertation are among the first in the area of optical property control in biological tissue. Results of the studies have shown the technique of reduced scattering by glycerol and other chemical agents may enhance optical applications in medicine that require light delivery deep within tissue.
... The backscattered magnitude (I) and the phase information (Q) undergo digital signal processing in a computer. The optical setup of the color Doppler optical coherence tomography system (CDOCT) is shown in Fig.3 [Vargas, 2001]. Two images are formed from the sampled data, one using only the magnitude of backscattered light to create an amplitude image and the other using the phase information to create a velocity map, called the Doppler image. ...
... The results from the speckle contrast technique are compared with the experimental results from the color Doppler OCT (CDOCT) system. These results from the Doppler system are taken from the PhD dissertation of [Vargas, 2001]. The series of figures that follow demonstrate an example of the effect of glycerol on the blood vessels of the hamster dorsal skin flap window preparation when 100% glycerol was applied in vivo. ...
... (a) (b) Fig. 9. Amplitude (a) and Doppler (b) images of native skin in the hamster dorsal skin flap window preparation taken from the subdermal side of the skin. The flow velocity in the artery (A) is 4 mm/sec and is 3.5 mm/sec in the vein (V) [Vargas, 2001]. ...
Optical changes in skin blood flow due to the presence of glycerol were
measured from a two-dimensional map of blood flow in skin blood vessels
with a dynamic imaging technique using laser speckle. In this study a
dorsal skin-flap window was implanted on the hamster skin with and
without a hyper-osmotic agent i.e. glycerol. The hyper-osmotic drug was
delivered to the skin through the open dermal end of the window model. A
two-dimensional map of blood flow in skin blood vessels were obtained
with very high spatial and temporal resolution by imaging the speckle
pattern with a CCD camera. Preliminary studies demonstrated that
hyper-osmotic agents such as glycerol not only make tissue temporarily
translucent, but also reduce blood flow. The blood perfusion was
measured every 3 minutes up to 36-60 minutes after diffusion of
anhydrous glycerol. Small capillaries blood flow reduced significantly
within 3-9 minutes. Perfusion rate in lager blood vessels i.e. all
arteries and some veins decreased (speckle contrasts increased from
0.0115 to 0.384) over time. However, the blood flow in some veins
reduced significantly in 36 minutes. After 24 hours the blood perfusion
further reduced in capillaries. However, the blood flow increased in
larger blood vessels in 24 hours compared to an hour after application
of glycerol. For further investigation the speckle contrast measurement
were verified with color Doppler optical coherence tomography.
... Specific to in vivo skin optical clearing investigation, subcutaneous injection was applied mostly [7,8]. However, it tended to cause skin lesion, such as blood coagulation, or even suppuration or scar forming [9,10]. There were also numbers of investigators attempting to breach SC barrier function by physical methods, including using sandpaper [11], lattice of islets of damage [12], microneedles [13], etc. unfortunately, these methods were harmful to in vivo skin, and it's hard to be quantified and be of poor repeatability, most of all, the clinical utility and feasibility may not be satisfying. 1 Hence, investigation of in vivo skin optical clearing faces a big challenge. ...
The inherent barrier function of the stratum corneum (SC) makes optical
clearing agents difficult to penetrate into skin. To date, several
physicochemical methods have been studied to enhance skin optical
clearing. In this study, the rat skin was initially irradiated by
various light (Carbon-Dioxide Laser, Intensed Pulse Light, Nd:YAG Laser
and its frequency-doubled laser) with different dose, and then topically
applied anhydrous glycerol. A fiber spectrometer was used to monitor the
change of skin diffuse reflectance spectrum so as to evaluate the
optical clearing effect on skin. The results showed that Nd:YAG
Laser(1,064 nm) with appropriate pulse width and energy density combined
with glycerol could improve skin optical clearing effectively, and that
Q-switched Nd:YAG Laser combining glycerol made the most significant
decrease of skin diffuse reflectance. However, after the irradiation of
Carbon-Dioxide Laser (ultra-pulsed), Intensed Pulse Light (400-700 nm)
or frequency-doubled Q-switched Nd:YAG Laser(532 nm), the following
application of glycerol didn't lead to skin optical clearing. Adversely,
higher power of the former two light could result in erythema, the later
one may harm skin apparently even lead to blood coagulation dot. This
study provids a new idea to find out a noninvasive but high-effective
approach to increase skin optical clearing, and available parameters of
laser need to be further investigated.
... Hyperosmotic agents i.e. 100% anhydrous glycerol, substantially increase the penetration depth of light in skin [1−6]. The increase in light penetration depth is accomplished by reducing light scattering in the tissue [4,6]. Thus, the primary and the secondary goals of this study were to investigate the change in morphology and optical properties of sclera due to 100% anhydrous glycerol. ...
The primary and the secondary goals of this study were to investigate the change in morphology and optical properties of sclera due to a hyperosmotic agent i.e. 100% anhydrous glycerol. We performed our experiments in vivo on the sclera of 8 rabbits and 3 miniature pigs. All the animals were under anesthetic for the entire experiment according to an approved protocol. The position of the eye was stabilized with a suture placed in the limbus. Glycerol was delivered to sclera in 2 methods (i) injection (using a hypodermic needle 27G ½), (ii) direct application after 0.3 cm incision at conjunctiva. A camera attached to a slit lamp was used to capture the morphological changes of the sclera. For the secondary goal we used a diffuse optical spectroscopy (DOS) system with a linear fiber arrangement to measure reflectance from the sclera before and after application of glycerol. The probe source-detector separation was set to 370 mum for optimal penetration depth. We fit the measured diffuse reflectance to a Lookup Table (LUT)-based inverse model specific to our probe geometry to determine the scattering and absorption properties of the sclera. This method estimated the size and density of scatterers, absorbers-blood volume fraction, melanin concentration, oxygen saturation, and blood vessel size. The results illustrated that the initial clearing of sclera started 3 minutes after injecting glycerol to sclera. The sclera became completely transparent at 8 minutes and stayed clear for 10-15 minutes. During this time the choroid layer was visible through sclera. The clear sclera became less transparent over next 11 minutes and became completely opaque once we applied 0.9% saline to hydrate the sclera. These dehydration and hydration cycles were repeated 4 times for each eye and the results were consistent for all animal models. When glycerol was applied directly to sclera after the incision at the conjunctiva, the sclera became transparent instantaneously. For the secondary goal, the changes in optical properties of sclera were monitored during the dehydration and hydration cycles. The reduced scattering coefficient decreased when glycerol was injected and it further reduced with direct application. The scattering increased after re-hydration. We also measured the blood volume fraction, melanin concentration, oxygen saturation, and blood vessels diameter to calculate absorption coefficient with the DOS system. This study provided a novel way to identify morphological changes of sclera in addition to measuring changes in optical properties due to hyper osmotic agent. The changes in optical properties were consistent with the morphological changes in sclera during the dehydration and hydration cycles.
... Addition of osmotically active agents to tissues may produce structural modifications if concentrations of osmolytes are high. For instance, Vargas demonstrated that addition of high concentration of glucose (4.2 M) to the rat skin results in 20% water loss (Vargas 2001) that may significantly decrease overall tissue scattering. However, at physiologically relevant concentrations of glucose (3-30 mM), the water loss would be only 0.005% mM −1 (assuming a linear dependence between glucose concentration and water loss). ...
Management of diabetic disease requires frequent monitoring of blood glucose concentration. Development of a noninvasive technique capable of reliable and sensitive monitoring of blood glucose concentration would considerably improve quality of life of diabetic patients and reduce mortality associated with this disease. Recently, we proposed to use Optical Coherence Tomography (OCT) technique for noninvasive glucose monitoring. In this paper, we tested in animals several aspects of specificity of noninvasive blood glucose monitoring with the OCT technique. Influence of temperature and tissue heterogeneity on the OCT signal profile is experimentally studied in this paper. We also theoretically investigated the changes in tissue scattering induced by variation of concentration of glucose and other osmolytes. Obtained results suggest that although several physical and chemical agents could potentially interfere with blood glucose concentration measurements using the OCT technique, their effect is smaller compared to that of glucose under normal physiological conditions.
... High scattering property of biological tissue for VIS-NIR light limits the penetration depth of light deep into tissue, which has become the obstacle of optical diagnosis and therapy [1]. However, the tissue scattering can be reduced and the penetration depth can be improved by injecting agents with characteristics of high permeability, high refractive index and biocompatibility into tissues, which is called tissue optical clearing technique and was proposed by Tuchin VV [2][3][4]. ...
In the past decades, laser has been widely used in clinical diagnosis and cosmetic therapy. However, there is limitation for further usage in deeper tissue for high scattering property. Skin optical clearing technique, by introducing optical clearing agents (OCAs) into tissue, will have a potential impact on optical diagnosis and therapy. In this work, anhydrous glycerol at different temperatures of 4, 25, 32 and 45°C were applied respectively to in vitro porcine skin, and reflectance and transmittance spectra were then measured dynamically using a spectrometry combined with integrating sphere system. Further, reduced scattering coefficient and penetration depth were obtained. Results showed that, glycerol at different temperatures could induce the reduced scattering coefficient of in vitro skin to decrease and the penetration depth to increase. 4 and 25°C glycerol had similar effect, decreasing the scattering by 48.2% and 49.7%, and increasing penetration depth by 37.9% and 39.5%, respectively. However, 32 and 45°C glycerol treatment could decrease scattering by 61.6% and 76.6%, and increase penetration depth by 53.3% and 84.1%, respectively. In conclusion, glycerol at higher temperature can induce greater and faster skin optical clearing efficacy.
... Hyperosmotic agents such as 100% anhydrous glycerol, substantially increase the penetration depth of light in sclera 1-10 by reducing light scattering. [11][12][13] Optical clearing provides a low attenuation path for therapeutic laser applications and prevents excessive damage to sclera. 14 However, glycerol causes a decrease in blood flow when this agent is used in skin. ...
Light scattering in the normally white sclera prevents diagnostic imaging or delivery of a focused laser beam to a target in the underlying choroid layer. In this study, we examine optical clearing of the sclera and changes in blood flow resulting from the application of glycerol to the sclera of rabbits. Recovery dynamics are monitored after the application of saline. The speed of clearing for injection delivery is compared to the direct application of glycerol through an incision in the conjunctiva. Although, the same volume of glycerol was applied, the sclera cleared much faster (5 to 10 s) with the topical application of glycerol compared to the injection method (3 min). In addition, the direct topical application of glycerol spreads over a larger area in the sclera than the latter method. A diffuse optical spectroscopy system provided spectral analysis of the remitted light every two minutes during clearing and rehydration. Comparison of measurements to those obtained from phantoms with various absorption and scattering properties provided estimates of the absorption coefficient and reduced scattering coefficient of rabbit eye tissue.
... To accelerate the permeability of OCAs, different methods, such as immersion [5], microneedles [6], ultrasound [7], and chemical penetration enhancers [8] were proposed, but most of them were performed in vitro skin. As a common method, intradermal injection of OCAs was used to improve the optical clearing of in vivo skin effectively [1,9], Nevertheless, OCAs with high concentration induced visible skin lesions [10,11], such as edema, suppuration, or even scarring. Although sandpaper abrasive is effective in facilitating transepidermal OCAs delivery [12], it is difficult to quantify and control the abrasive strength. ...
Tissue optical clearing technique based on immersion of tissues into optical clearing agents (OCAs) can reduce the scattering and enhance the penetration of light in tissue. However, the barrier function of epidermis limits the penetration of OCAs, and hence is responsible for the poor optical clearing efficacy of skin by topical action. In this study, a variety of light irradiation was applied to increase permeability of agents in skin and improve the optical clearing efficacy.
Different light sources with different dose, i.e, CO(2) laser, Nd:YAG laser (532 and 1,064 nm) with different pulse modes and Intense Pulsed Light (IPL) (400-700 and 560-950 nm) were used to irradiate rat skin in vivo, and then glycerol was applied onto the irradiated zone. VIS-NIR spectrometer was utilized to monitor the changes of reflectance. In vitro skin samples were also irradiated by Q-switched Nd:YAG laser (1,064 nm) and then treated by glycerol for 10-60 minutes. Based on the measurement of the reflectance and transmittance of the samples, the optical properties of skin and penetration depth of light were calculated.
Results show that photo-irradiation with appropriate dose combining with the following glycerol treatment is able to reduce in vivo skin reflectance. Compared with the control group, the maximal changes in reflectance are ninefold at 575 nm and eightfold at 615 nm, respectively, which were caused by Q-switched 1,064-nm Nd:YAG laser irradiation and following glycerol treatment. The results for in vitro skin demonstrate that the joint action can significantly increase the optical penetration depth in samples.
The combination of Q-switched Nd:YAG (1,064 nm) laser and glycerol could enhance optical skin clearing efficacy significantly. This study provides a non-invasive way to improve the optical clearing of skin, which will benefit the skin optical therapy.
A review of specific features and methods of optical clearing and related interaction of light with tissues is presented. Physical and molecular mechanisms of immersion, compression, and photodynamic/photothermal optical clearing of some fibrous and cellular tissues are discussed. The possibility of efficient control of the tissue optical properties, particularly, the reduction of light scattering in tissues is demonstrated, which facilitates the increased efficiency of various optical visualisation methods (optical biopsy) used in medical purposes .
The tissue optical clearing (TOC) technique could significantly improve the biomedical optical imaging depth, but most current investigations are limited to in vitro studies. For in vivo applications, the TOC method must provide a rapid treatment process, sufficient transparency, and safety for animals, which makes it more difficult. Recently developed innovative optical clearing methods for in vivo use show great potential for enhancing the contrast and resolution of laser speckle contrast imaging (LSCI) for blood flow monitoring. This paper gives an overview of recent progress in the use of TOC for vascular visualization with LSCI. First, the principle of TOC-induced improvement of LSCI and a quantitative analysis method for evaluating the improvement are described briefly. Second, the paper introduces transparent windows, including various skin windows and a cranial window, that permit LSCI to monitor dermal or cortical blood flow, respectively, with high resolution and contrast. Third, preliminary investigations of the safety of TOC demonstrate that the transparent skin window is switchable, which enables LSCI to repeatedly image blood flow. However, research on in vivo TOC is currently less advanced than that on in vitro TOC. Future work should focus on developing a highly effective, safe method and extending its applications.
Not available Biomedical Engineering
A goniometric apparatus is presented for measuring angular dependence of scattering of a HeNe laser beam by in vitro human dermis samples of various thicknesses, hydrated to an 85 percent water content. Measurements of the transmitted and reflected light as a function of angle are presented for tissue thicknesses of 20--650 microns. Extrapolation of these angular scattering patters to the limit of an incremental tissue thickness specifies the scattering phase function appropriate for use in the radiative transport equation. The scattering phase function is composed of a forward-directed scattering component (90 percent contribution) characterized as a Henyey-Greenstein function with g_{HG}=0.91, and an additional isotropic component (10 percent contribution, b=0.10). The net value for the average cosine of the scattering phase function, g=(1-b)g_{HG} is 0.82, which corresponds to an average deflection angle of 35° for a scattering event. The on-axis attenuation of the collimated HeNe laser beam indicates a total attenuation constant of 190/cm, composed of an absorption coefficient of 2.7/cm and a scattering coefficient of 187/cm.
Monte Carlo simulations of photon propagation offer a flexible yet rigorous approach toward photon transport in turbid tissues. This method simulates the “random walk” of photons in a medium that contains absorption and scattering. The method is based on a set of rules that govern the movement of a photon in tissue. The two key decisions are (1) the mean free path for a scattering or absorption event, and (2) the scattering angle. Figure 4.1 illustrates a scattering event. At boundaries, a photon is reflected or moves across the boundary. The rules of photon propagation are expressed as probability distributions for the incremental steps of photon movement between sites of photon—tissue interaction, for the angles of deflection in a photon’s trajectory when a scattering event occurs, and for the probability of transmittance or reflectance at boundaries. Monte Carlo light propagation is rigorous yet very descriptive. However, this method is basically statistical in nature and requires a computer to calculate the propagation of a large number of photons. To illustrate how photons propagate inside tissues, a few photon paths are shown in Fig. 4.2.
In this text, scientists provide a detailed description of the physical events that occur when light interacts with tissue. Their work emphasizes the optical response of tissue during treatment procedures or diagnostic applications of laser light. Supported by numerous illustrations, chapters present methods for estimating tissue optical properties from measurements of reflection and transmission in addition to methods for measuring temperature, thermal properties and rate constants. A discussion on the applications of optical and thermal tissue interactions to various medical problems is included.
This chapter describes the adding-doubling method for solving the radiative transport equation. The advantages and disadvantages of the method are presented, followed by sections describing its theory and computer implementation. A detailed example is given with intermediate numerical results. Accurate tables with values of reflection and transmission for slabs of varying thicknesses with mismatched boundaries are given.
The cornea and sclera together form the tough outer tunic of the eye, which withstands the intra-ocular pressure from within and protects the contents from mechanical injury from without. The cornea is the major component in the optical system of the eye; of the total dioptric power of the human eyeball, nearly three-quarters is contributed by the interface between the cornea and the air. To this end, it is curved and transparent, and its surfaces, particularly the external one, are smooth to good optical standards. Generally, the globe of the mammalian eye approximates to a sphere, sometimes rather flattened in the antero-posterior direction, so that the curvature of cornea and sclera is similar. Its radius ranges from 1.75 mm in the mouse to 25 mm in the horse, among the more common animals. In man, and to a lesser extent in many other species, the cornea is more curved than the eyeball as a whole.
• A flashlamp-pumped pulsed dye laser at 577 nm was evaluated in the treatment of port-wine stains. The degree of lesional lightening was compared following laser exposure with pulse durations of 20 and 360 μs. In addition, lesional therapy using the 360-μs pulse duration was evaluated for lightening and side effects following long-term patient observation and after repeated treatments of the same site. A total of 52 patients with port-wine stain were treated; their average age was 29 years, with eight patients less than 18 years, of whom 29 had comparative test site placement for the different pulse durations. Of these 29 patients, 25 demonstrated greater lightening at the 360-μs pulse duration test site. All 52 patients proceeded to receive full treatment placement with the 360-μs pulse duration, which resulted in an overall lightening of 42% after the initial treatment and 68% after re-treatment sessions. Forty-four percent of the patients had equal to or greater than 75% lesional lightening. Pretreatment anesthesia was unnecessary and only minimum posttreatment care was required. Mild adverse effects of epidermal change, depression, or pigmentary change appeared in only four cases and was limited to less than a 2% area in each of these lesions. These side effects did not recur when the lesions were re-treated at lower energy dosages. No posttreatment sclerosis or scarring appeared, even after multiple retreatment sessions to the same area, regardless of the anatomic location, color of the lesion, or age of the patient. The ability of the pulsed dye laser to increase blood vessel thermal damage selectivity has resulted in pulse duration—dependent changes that allow for favorable lesional lightening while significantly minimizing undesired side effects.
(Arch Dermatol 1988;124:889-896)