The hippocampus (HC) is a subcortical brain region that plays essential roles in learning and memory. It is strongly associated with Alzheimer’s disease (AD), an incurable and deadly neurodegenerative disease which is progressive and requires longitudinal observation. Two-photon microscopy (2PM) is applied here to investigate hippocampal alterations in living mouse models and better understand pathological changes during neurodegeneration. The common procedure is to surgically expose the rodent cortex and have it sealed with a coverslip to allow optical access. However, in some studies, repeated tissue injections are needed to deliver exogenous contrast agents or pharmacological agents, and current injection strategies are not compatible with subcortical imaging, which limits the ability to study subcortical lesions longitudinally. To tackle this issue, we developed a technique where both imaging and injection can be conducted. Our previous development enabled 2PM imaging in the HC using a gradient-index (GRIN) lens. We engineered a customized cannula using polyimide tubing and transparent acrylic, and implanted it into mouse brain. It allows removable insertion of a GRIN lens and enables longitudinal investigation. In this study, we improved our cannula design to enable imaging and injections. The acrylic window is replaced with an optically-transparent, biocompatible, oxygen impermeable plastic, which maintains seal after needle penetration. Here we report injection and imaging results in phantoms and animals. Our design opens opportunities for comprehensive longitudinal imaging of subcortical lesions.
The Neurovascular Unit (NVU) dynamically regulates oxygen supply to satisfy neural metabolic demand. Amyloid-β (Aβ) accumulation and hyperphosphorylated Tau in Alzheimer’s disease (AD) disrupt the NVU. Empirical evidence strongly indicates that physical activity (PA) reduces the rate of cognitive impairment, but the physiological mechanism(s) PA’s neuroprotective benefits remain unclear. We propose PA improves the brain parenchymal oxygenation and reduces metabolic deficits. Using the novel oxygen sensitizer, Oxyphor 2P, and 2-photon phosphorescence lifetime imaging (2P-PLIM), our results indicate that the PA shows the potential to curtail AD progression by increasing microvascular oxygenation and preserving NVU function.
SignificanceThe accurate large-scale mapping of cerebral microvascular blood flow velocity is crucial for a better understanding of cerebral blood flow (CBF) regulation. Although optical imaging techniques enable both high-resolution microvascular angiography and fast absolute CBF velocity measurements in the mouse cortex, they usually require different imaging techniques with independent system configurations to maximize their performances. Consequently, it is still a challenge to accurately combine functional and morphological measurements to co-register CBF speed distribution from hundreds of microvessels with high-resolution microvascular angiograms.AimWe propose a data acquisition and processing framework to co-register a large set of microvascular blood flow velocity measurements from dynamic light scattering optical coherence tomography (DLS-OCT) with the corresponding microvascular angiogram obtained using two-photon microscopy (2PM).ApproachWe used DLS-OCT to first rapidly acquire a large set of microvascular velocities through a sealed cranial window in mice and then to acquire high-resolution microvascular angiograms using 2PM. The acquired data were processed in three steps: (i) 2PM angiogram coregistration with the DLS-OCT angiogram, (ii) 2PM angiogram segmentation and graphing, and (iii) mapping of the CBF velocities to the graph representation of the 2PM angiogram.ResultsWe implemented the developed framework on the three datasets acquired from the mice cortices to facilitate the coregistration of the large sets of DLS-OCT flow velocity measurements with 2PM angiograms. We retrieved the distributions of red blood cell velocities in arterioles, venules, and capillaries as a function of the branching order from precapillary arterioles and postcapillary venules from more than 1000 microvascular segments.ConclusionsThe proposed framework may serve as a useful tool for quantitative analysis of large microvascular datasets obtained by OCT and 2PM in studies involving normal brain functioning, progression of various diseases, and numerical modeling of the oxygen advection and diffusion in the realistic microvascular networks.
Chronic inflammation is one of the most prominent features of Alzheimer's disease (AD). Little is known about how brain hemodynamics and oxygenation are affected by AD-related inflammation. Here, we use two-photon phosphorescence lifetime imaging with an oxygen-sensitive dye “Oxyphor 2P” to measure the partial pressure of oxygen (pO2) before and during endotoxin-induced neuroinflammation in cortical vessels of a mouse model of AD. Capillary red blood cell flux (RBC flux) was measured through two-photon phosphorescence intensity microscopy. To induce chronic inflammation, we injected lipopolysaccharide (LPS) intraperitoneally, daily for two weeks, in female APPswe: PS1dE9 mice and age-matched wild-type (WT) controls. Intravascular pO2 and RBC flux were measured in the somatosensory cortex before the LPS injection, on week 1 (day 7), and week 2 (day 15) during the LPS injection. Our results demonstrate that LPS-induced systemic inflammation leads to significant decreases in cortical intravascular pO2 while showing a negligible effect on capillary RBC flux. Moreover, AD mice are more susceptible to inflammation with more pronounced cortical pO2 reduction in comparison to WT mice. Our findings suggest that inflammation plays a key role in AD-related disruptions of cerebral tissue metabolism of oxygen.
Dementia afflicts more than 55 million patients worldwide, and Alzheimer’s disease (AD) accounts for around 60% to 80% of total cases1. During the onset of AD, the hippocampus (HC) is among the first-affected brain regions to experience pathological changes. Therefore, identifying changes to HC in AD subjects will be extremely helpful in providing early-stage diagnosis and interventions. We are developing in vivo techniques to investigate microscopic alterations to hippocampal structure and function in animal models of AD. Imaging will be performed using removable GRIN (gradient-index) lenses to chronically access subcortical brain structures with two-photon microscopy. Here, we report the development and characterization of a customized cannula (1 mm diameter, 6 – 7 mm length) to repeatedly insert a GRIN lens for two-photon imaging. The cannula allows for easy removal of the lens after imaging sessions and enables detailed investigations of hippocampal changes during AD progression in mouse models. The cannula is made of polyimide tubing and tipped with transparent acrylic coverslip. We compare acrylic coverslips and existing glass coverslips in terms of physical and optical properties. Acrylic coverslips display comparable imaging quality and therefore serves as a reliable alternative to glass coverslips which is more economical, reproducible, and mechanically stable. We also present preliminary hippocampal images collected in vivo with our custom cannula. These results will guide more extensive efforts to measure hippocampal metabolic and hemodynamic alterations in awake animal models of AD.
Fluorescence lifetime imaging microscopy (FLIM) and phosphorescence lifetime imaging microscopy (PLIM) often require complex and computationally intensive processes for analysis. For time-domain based measurements, computation of fluorescence and phosphorescence lifetimes conventionally involves nonlinear curve fitting techniques to model the time-resolved profiles as mono- or multi-exponential decays. The phasor, or “polar plot”, analysis method has recently gained attention as a simple method to characterize variations in fluorescence lifetime. The technique involves calculations of the intensity-normalized Fourier transform of the fluorescence profiles. The phasor can be visualized by plotting the real and imaginary components on a 2-dimensional plot. We have adapted the phasor analysis method for absolute quantitation of phosphorescence lifetimes of oxygen-sensitive phosphors. We utilize the phasor-derived lifetime values to quantify oxygen partial pressure in cortical micro vessels of awake mice. Here, we describe the modifications to adapt the technique for longer-duration phosphorescence decays. Our results demonstrate that oxygen measurements obtained from phasor analysis are in strong agreement with traditional curve fitting calculations. Using simulated phosphorescence decays, we also compare the effectiveness of the phasor method to nonlinear curve fitting techniques. To our knowledge, these findings constitute the first application of the phasor analysis method for characterizing phosphorescence measurements on the microsecond time scale. The method shows promise for monitoring cerebral metabolism and pathological changes in preclinical rodent models.
Quantifying intestinal microvascular oxygenation may shed light on origin and progression of intestinal diseases as well as the role of the gut microbiota. In this work, we developed a method for depth-resolved microscopic measurements of partial pressure of oxygen (pO2) and Red Blood Cell (RBC) flux in the capillaries of intestinal wall in mouse cecum. Capillary pO2 and RBC flux were measured in healthy mice as a function of depth during normoxia and hyperoxia. Our results show low mean capillary RBC flux and relatively uniform capillary pO2 across the cecum wall depth.
Disruptions and alterations to cerebral energy metabolism play a vital role in the onset and progression of many neurodegenerative disorders and cerebral pathologies. In order to precisely understand the complex alterations underlying Alzheimer’s disease (AD) progression, in vivo imaging at the microscopic level is required in preclinical animal models. Utilizing two-photon fluorescence lifetime imaging microscopy and the phasor analysis method, we have observed AD-related variations of endogenous fluorescence of reduced nicotinamide adenine dinucleotide (NADH) in vivo. We collected NADH FLIM images from the cerebral cortices of both APPswe:PS1dE9 mice to model amyloid β plaque accumulation and corresponding age-matched wildtype controls. Distinct variations in NADH fluorescence lifetime between wildtype and AD mice, as well as variations related to proximity from amyloid plaques, are obvervable via the phasor method. The combination of NADH FLIM and phasor analysis allows for a minimally invasive, high-resolution technique to characterize the adverse effects of amyloid β accumulation on mitochondrial energy metabolism and could guide our understanding of preclinical AD pathology.
Impaired oxygen delivery and/or consumption in the retinal tissue underlies the pathophysiology of many retinal diseases. However, the essential tools for measuring oxygen concentration in retinal capillaries and studying oxygen transport to retinal tissue are still lacking. We show that two-photon phosphorescence lifetime microscopy can be used to map absolute partial pressures of oxygen (pO2) in the retinal capillary plexus. Measurements were performed at various retinal depths in anesthetized mice under systemic normoxic and hyperoxic conditions. We used a newly developed two-photon phosphorescent oxygen probe, based on a two-photon absorbing platinum tetraphthalimidoporphyrin, and commercially available optics without correction for optical aberrations of the eye. The transverse and axial distances within the tissue volume were calibrated using a model of the eye’s optical system. We believe this is the first demonstration of in vivo depth-resolved imaging of pO2 in retinal capillaries. Application of this method has the potential to advance our understanding of oxygen delivery on the microvascular scale and help elucidate mechanisms underlying various retinal diseases.
Cortical capillary blood flow and oxygenation are highly heterogeneous. Mapping absolute capillary blood flow and oxygenation along capillary path is a key step towards understanding how oxygen is transported and delivered in a complex microvascular network to enable adequate tissue oxygenation. In this work, we applied two-photon microscopic imaging of intravascular oxygen partial pressure (PO2) to measure both oxygen concentration and red blood cell (RBC) flux in cortical arterioles, capillaries, and venules. Imaging was performed in awake, head-restrained C57BL/6 mice (n=15), through a chronic sealed cranial window centered over the E1 whisker barrel. We obtained a detailed mapping of the resting state cortical microvascular PO2 in all arterioles and venules, and both PO2 and RBC flux in most capillaries down to 600 μm depth from the cortical surface (n=6,544 capillaries across all mice). Capillary RBC speed and density were also extracted and all measurements were co-registered with the microvascular angiograms. We characterized the distributions of capillary PO2 and flow as a function of branching order and cortical depth. The results show strong positive correlation between oxygenation and flow in the capillary segments, with an increased correlation in downstream capillaries. We have also observed homogenization of both oxygenation and flow in deeper cortical layers, which may imply a mechanism to improve oxygen delivery without increasing global blood flow in the area with increased metabolism.
We characterized cortical microvascular PO2 and blood flow changes in response to whisker stimulation in awake mice. The measurements were performed by combining two-photon microscopy imaging of the cortical oxygenation and optical coherence tomography imaging of the cerebral blood flow. In order to perform fast spatio-temporally resolved measurements of PO2, we used a newly-developed oxygen-sensitive probe PtG-2P, which has significantly higher brightness than the established two-photon-enhanced oxygen sensor PtP-C343. We characterized the performance of the new probe in vivo and mapped the amplitudes and shapes (e.g. initial dip, overshoot, and post stimulus undershoot) of the PO2 changes as a function of the vessel type (e.g., arterioles, capillaries, and venules) and a distance from the activation center. The measurements in the awake mice are not affected by the confounding factors of anesthesia on the animal physiology, including the level of cerebral metabolism and the amplitude and speed of neuronal and vascular responses. Our results will help to understand changes in oxygenation and blood flow on the cortical microvascular scale, will lead to improved understanding of the cerebral physiology, pathophysiology and will improve quantitative interpretation of fMRI signals.
The cerebral metabolic rate of oxygen (CMRO2) is an essential parameter for evaluating brain function and pathophysiology. Measurements of CMRO2 with high spatio-temporal resolution are critically important for understanding how the brain copes with metabolic and blood perfusion changes associated with various clinical conditions, such as stroke, periinfarct depolarizations, and various microvasculopathies (e.g., Alzheimer’s disease, chronic hypertension). CMRO2 measurements are also important for understanding the physiological underpinnings of functional Magnetic Resonance Imaging signals. However, the currently available approaches for quantifying CMRO2 rely on complex multimodal imaging and mathematical modeling. Here, we introduce a novel method that allows estimation of CMRO2 based on a single measurement modality - two-photon phosphorescence lifetime microscopy (2PLM) imaging of the partial pressure of oxygen (PO2) in cortical tissue. CMRO2 is estimated by fitting the changes of tissue PO2 around cortical penetrating arterioles with the Krogh cylinder model of oxygen diffusion. We measured the baseline CMRO2 in anesthetized rats, and modulated tissue PO2 levels by manipulating the depth of anesthesia. This method has a spatial resolution of approximately 200 μm and it may provide CMRO2 measurements in individual cortical layers or within confined cortical regions such as in ischemic penumbra and the foci of functional activation.
KEYWORDS: Mode conditioning cables, Fluorescence lifetime imaging, Luminescence, Microscopy, Data modeling, Brain, In vivo imaging, Single photon, Multimodal imaging, Imaging systems
Monitoring cerebral energy metabolism at a cellular level is essential to improve our understanding of healthy brain function and its pathological alterations. In this study, we resolve specific alterations in cerebral metabolism utilizing minimally-invasive 2-Photon fluorescence lifetime imaging (2P-FLIM) measurements of reduced nicotinamide adenine dinucleotide (NADH) fluorescence, collected in vivo from anesthetized rats and mice. Time-resolved lifetime measurements enables distinction of different components contributing to NADH autofluorescence. These components reportedly represent different enzyme-bound formulations of NADH. Our observations from this study confirm the hypothesis that NADH FLIM can identify specific alterations in cerebral metabolism. Using time-correlated single photon counting (TCSPC) equipment and a custom-built multimodal imaging system, 2-photon fluorescence lifetime imaging (FLIM) was performed in cerebral tissue with high spatial and temporal resolution. Multi-exponential fits for NADH fluorescence lifetimes indicate 4 distinct components, or 'species.' We observed distinct variations in the relative proportions of these components before and after pharmacological-induced impairments to several reactions involved in anaerobic glycolysis and aerobic oxidative metabolism. Classification models developed with experimental data correctly predict the metabolic impairments associated with bicuculline-induced focal seizures in separate experiments. Compared to traditional intensity-based NADH measurements, lifetime imaging of NADH is less susceptible to the adverse effects of overlying blood vessels. Evaluating NADH measurements will ultimately lead to a deeper understanding of cerebral energetics and its pathology-related alterations. Such knowledge will likely aid development of therapeutic strategies for neurodegenerative diseases such as Alzheimer's Disease, Parkinson's disease, and stroke.
The cerebral metabolic rate of oxygen (CMRO2) is an essential parameter for evaluating brain function and pathophysiology. However, the currently available approaches for quantifying CMRO2 rely on complex multimodal imaging and mathematical modeling. Here, we introduce a method that allows estimation of CMRO2 based on a single measurement modality—two-photon imaging of the partial pressure of oxygen (PO2) in cortical tissue. We employed two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen-sensitive nanoprobe PtP-C343 to map the tissue PO2 distribution around cortical penetrating arterioles. CMRO2 is subsequently estimated by fitting the changes of tissue PO2 around arterioles with the Krogh cylinder model of oxygen diffusion. We measured the baseline CMRO2 in anesthetized rats and modulated tissue PO2 levels by manipulating the depth of anesthesia. This method provides CMRO2 measurements localized within ∼200 μm and it may provide oxygen consumption measurements in individual cortical layers or within confined cortical regions, such as in ischemic penumbra and the foci of functional activation.
Neuroanatomical pathways form the basis for functional activity of brain circuits. In the past, we developed a polarization-sensitive optical coherence tomography with serial scanning to achieve large-scale brain imaging. The system was able to visualize 3D fiber tracts of ~20 um in diameter. To investigate the neuroanatomical pathways at finer scales, we have now built a polarization-maintaining fiber based ultra-high resolution polarization-sensitive optical coherence microscope (PS-OCM) at 1300 nm. The PS-OCM has an axial resolution of 3.5 um in tissue. The detection setup consists of two spectrometers, acquiring spectral interference on orthogonal polarization channels. With a single measurement, the setup generates four contrasts: reflectivity, cross-polarization, retardance and optic axis orientation. To investigate the capability of PS-OCM at different resolutions, we used three microscope objectives that yield lateral resolutions of 6.0 um, 3.4 um and 1.3 um. Blocks of formalin fixed mouse brain and human brain were scanned. The cross-polarization and retardance images clearly depict the neuronal fiber structures, which are comparable with that generated by the maximum projection of volumetric reflectivity data. The optic axis orientation quantifies the in-plane fiber orientation. With the lateral resolution of 1.3 um, the retardance contrast is weak in white matter due to the shallow depth of focus. Overall, the ultra-high resolution PS-OCM provides a new tool to reveal neuroanatomical maps in the brain at cellular resolution.
Due to a lack of imaging tools for high-resolution imaging of cortical tissue oxygenation, the detailed maps of the oxygen partial pressure (PO2) around arterioles, venules, and capillaries remain largely unknown. Therefore, we have limited knowledge about the mechanisms that secure sufficient oxygen delivery in microvascular domains during brain activation, and provide some metabolic reserve capacity in diseases that affect either microvascular networks or the regulation of cerebral blood flow (CBF). To address this challenge, we applied a Two-Photon PO2 Microscopy to map PO2 at different depths in mice cortices. Measurements were performed through the cranial window in the anesthetized healthy mice as well as in the mouse models of microvascular dysfunctions. In addition, microvascular morphology was recorded by the two-photon microscopy at the end of each experiment and subsequently segmented. Co-registration of the PO2 measurements and exact microvascular morphology enabled quantification of the tissue PO2 dependence on distance from the arterioles, capillaries, and venules at various depths. Our measurements reveal significant spatial heterogeneity of the cortical tissue PO2 distribution that is dominated by the high oxygenation in periarteriolar spaces. In cases of impaired oxygen delivery due to microvascular dysfunction, significant reduction in tissue oxygenation away from the arterioles was observed. These tissue domains may be the initial sites of cortical injury that can further exacerbate the progression of the disease.
Computing microvascular cerebral blood flow (μCBF) in real cortical angiograms is challenging. Here, we investigated whether the use of Doppler optical coherence tomography (DOCT) flow measurements in individual vessel segments can help in reconstructing μCBF across the entire vasculature of a truncated cortical angiogram. A μCBF computational framework integrating DOCT measurements is presented. Simulations performed on a synthetic angiogram showed that the addition of DOCT measurements, especially close to large inflowing or outflowing vessels, reduces the impact of pressure boundary conditions and estimated vessel resistances resulting in a more accurate reconstruction of μCBF. Our technique was then applied to reconstruct microvascular flow distributions in the mouse cortex down to 660 μm by combining two-photon laser scanning microscopy angiography with DOCT.
Measuring oxygen delivery in brain tissue is important for identifying the pathophysiological changes associated with
brain injury and various diseases such as cancer, stroke, and Alzheimer's disease. We have developed a multi-modal
imaging system for minimally invasive measurement of cerebral oxygenation and blood flow in small animals with high
spatial resolution. The system allows for simultaneous measurement of blood flow using Fourier-domain optical
coherence tomography, and oxygen partial pressure (pO2) using either confocal or multiphoton phosphorescence lifetime
imaging with exogenous porphyrin-based dyes sensitive to dissolved oxygen. Here we present the changes in pO2 and
blood flow in superficial cortical vessels of Sprague Dawley rats in response to conditions such as hypoxia, hyperoxia,
and functional stimulation. pO2 measurements display considerable heterogeneity over distances that cannot be resolved
with more widely used oxygen-monitoring techniques such as BOLD-fMRI. Large increases in blood flow are observed
in response to functional stimulation and hypoxia. Our system allows for quantification of cerebral metabolic rate of
oxygen (CMRO2) with high spatial resolution, providing a better understanding of metabolic dynamics during functional
stimulation and under various neuropathologies. Ultimately, better insight into the underlying mechanisms of
neuropathologies will facilitate the development of improved therapeutic strategies to minimize damage to brain tissue.
Optoacoustic (OA) tomography has demonstrated utility in identifying blood-rich malignancies in breast tissue. We describe the development and characterization of a laser OA imaging system for the prostate (LOIS-P). The system consists of a fiber-coupled Q-switched laser operating at 757 nm, a commercial 128-channel ultrasonic probe, a digital signal processor, and software that uses the filtered radial back-projection algorithm for image reconstruction. The system is used to reconstruct OA images of a blood-rich lesion induced in vivo in a canine prostate. OA images obtained in vivo are compared to images acquired using ultrasound, the current gold standard for guiding biopsy of the prostate. Although key structural features such as the urethra could be identified with both imaging techniques, a bloody lesion representing a highly vascularized tumor could only be clearly identified in OA images. The advantages and limitations of both forward and backward illumination modes are also evaluated by collecting OA images of phantoms simulating blood vessels within tissue. System resolution is estimated to be 0.2 mm in the radial direction of the acoustic array. The minimum detectable pressure signal is 1.83 Pa. Our results encourage further development toward a dual-modality OA/ultrasonic system for prostate imaging and image-guided biopsy.
Ultrasound imaging is the current gold standard for guiding biopsy of prostate. Optoacoustic imaging yields higher
contrast in detection of malignant tissues. The two techniques provide complementary information. We are currently
developing a hybrid laser optoacoustic and ultrasound imaging system for prostate tumor detection (LOUIS-P). The
optoacoustic part consists of a fiber-coupled Q-switched laser operating at either 757 nm or 1064 nm attached to a
commercially-available 128-channel ultrasonic probe modified for optimal detection of optoacoustic signals, a digital
signal processor with 128 independent channels, and software that uses the radial (filtered) backprojection algorithm to
reconstruct tomographic images. We evaluated system-imaging performance using test objects submerged in milky
water, and poly(vinyl-chloride) plastisol tissue phantoms simulating malignant lesions. LOUIS-P demonstrates potential
as a clinical technique for minimally invasive imaging and diagnosis of prostate cancer.
The biocompatibility and high absorption in the near IR range of indocyanine green (ICG) have made it a suitable candidate chromophore for optical imaging and laser-mediated therapy of superficial tumors. However, its clinical efficacy remains limited by factors such as rapid circulation kinetics, lack of target specificity, and molecular instability. Such drawbacks motivated us to encapsulate ICG into carrier particles to improve target specificity and retention time. We use absorbance spectroscopy to investigate the effects of encapsulating ICG within dextran-coated capsules. The mesocapsules (MCs) containing ICG are synthesized using a previously reported charge-assembly technique. Both freely dissolved ICG and ICG-MCs are prepared with ICG concentrations of either 50 or 10 µg/ml. Samples are exposed either to a broadband light source or incubated at 3, 23, or 40°C. Absorbance spectra are recorded at various time points up to 96 h. At the lower concentration of 10 µg/ml, ICG within MCs experiences less light-induced degradation. The MC system also protects ICG from thermal degradation at all tested temperatures. The polymer-salt aggregate core of the MCs hinders the mobility of ICG molecules. The MC system shields ICG from vibrational and translational agitation as well as molecular changes such as fragmentation.
Indocyanine Green (ICG) is a photosensitive dye frequently used in clinical settings for retinal imaging and measuring
cardiac output function. Its low toxicity, high absorption in the near infrared region, and vascular retention have
generated much interest in its utility for several therapeutic and diagnostic applications. Its potential, however, is limited
by factors such as rapid circulation kinetics and lack of target specificity. Additionally, the optical properties of ICG vary
significantly with a wide range of influences such as concentration, solvent, pH, and temperature. In an effort to
overcome these limitations and improve ICG's effectiveness as a chromophore for photothermal treatment, we have
developed a novel nanometer-sized capsule system as a delivery vehicle for ICG. In our capsules, the ICG molecules are
encapsulated within a polymer-salt aggregate core coated with dextran. In this study, we report how the optical
properties of ICG are affected by its entrapment within the nano-assembled complexes (NACs). The absorption profiles
of both freely dissolved ICG and encapsulated ICG were recorded under various conditions to determine the effects of
encapsulation on ICG's light sensitivity and stability at different temperatures. Dilute preparations of freely dissolved
ICG demonstrated more rapid degradation kinetics in response to intense broadband light (360 - 1100 nm, 56 mW/ cm2)
than NACs containing ICG. Encapsulated ICG also demonstrated greater stability at room and elevated temperatures
than freely dissolved ICG. The results suggest that the entrapment of ICG within the capsules influences its ability to
undergo physicochemical transformations, including oligomerization and degradation to leucoforms.
KEYWORDS: Skin, Cryogenics, Injuries, Blood vessels, Temperature metrology, Ear, Laser irradiation, Laser therapeutics, In vivo imaging, In vitro testing
Cutaneous laser treatment in dark skin patients is challenging due to significant light absorption by the melanin at the basal layer of epidermis, which can result in irreversible nonspecific thermal injury to the epidermis. Cryogen spray cooling (CSC) with R-134a (boiling point –26.2°C at 1 atm), which is currently used during cutaneous laser treatment, has shown poor efficacy in protecting dark human skin. We investigated the potential of CSC with R-404a (boiling point –46.5°C at 1 atm), which has a lower boiling point than R-134a, for improved therapeutic outcome in dark human skin at three levels: in vitro (epoxy resin skin phantom), ex vivo (normal dark human skin sample), and in vivo (skin of the rabbit external ear). The skin phantom was used to acquire the surface and internal temperature profiles in response to CSC with R-134a or R-404a at various spurt durations, based upon which CSC-induced heat removal from the skin phantom was estimated using an algorithm that solved a one-dimensional inverse heat conduction problem. CSC with R-404a increased the temperature reductions within the phantom and subsequently the amount of heat removal from the phantom in comparison to that with R-134a. Normal ex vivo Fitzpatrick types V-VI human skin samples were used to investigate the thermal response of dark human skin epidermis to CSC (R-134a or R-404a) at various spurt durations in conjunction with 595-nm pulsed dye laser irradiation at various radiant exposures. Cryogen R-404a increased the threshold radiant exposures for irreversible thermal injury to the epidermis in dark pigmentation skin. No obvious CSC-induced morphological changes to human skin was observed when sprayed with R404-a spurts using durations up to 300 ms. In vivo rabbit ear vasculature was used as a model of cutaneous anomalies to assess the influences of CSC (with R-134a or R-404a) on the photothermolysis of dermal blood vessels. CSC (R-134a or R-404a) with the spurt durations of 100 to 300 ms increased the most superficial depth of ...
While initially confined to the epidermis, squamous cell carcinoma can eventually penetrate into the underlying tissue if not diagnosed early and treated. The noninvasive early detection of the carcinoma is important to achieve a complete treatment of the disease. Of the various non-invasive optical techniques, the synchronous fluorescence (SF) technique is considered to provide a simplified spectral profile with more sharp spectral signatures of the endogenous fluorophores in complex systems. The potential use of the SF technique in the characterization of the sequential tissue transformation in 7,12-dimethylbenz(a)anthracene–12-O-tetradecanoylphorbol-13-acetate (DMBA-TPA)-induced mouse skin tumor model in conjunction with simple statistical analysis is explored. The SF spectra show distinct differences during the earlier weeks of the tumor-induction period. Intensity ratio variables are calculated and used in three discriminant analyses. All the discriminant analyses show better classification results with accuracy greater than 80%. From the observed differences in the spectral characteristics and the ratio variables that resulted in better classification between groups, it is concluded that tryptophan, collagen, and NADH are the key fluorophores that undergo changes during tissue transformation process and hence they can be targeted as tumor markers to diagnose normal from abnormal tissues using the SF technique.
The use of autofluorescence technique in the characterization of the sequential tissue transformation process in 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate (DMBA & TPA) induced two-stage mouse skin carcinogenesis model in conjunction with a suitable statistical method is being explored. The fluorescence excitation emission matrix (EEM) from experimental group (n=40; DMBA/TPA application), control group (n=6; acetone application) and the blank group (n=6; no application of DMBA/TPA or acetone) were measured every week using Fluoromax3 spectrofluorometer coupled with a waveguide fiber optic bundle (JY Horiba, NJ). The EEM was recorded for 19 excitation wavelengths from 280 to 460 nm at 10 nm intervals and the fluorescence emission was scanned from 300 to 750 nm. During the tissue transformation the epithelial tissues underwent biochemical and structural changes that are manifested in the tissue fluorescence. To correlate the tissue morphology with the observed fluorescence differences in the fluorescence emission, animals were sacrificed and the tissue biopsies were subjected to histopathological evaluation. The fluorescence emission corresponding to different fluorophores was extracted from the EEM, and the spectral data were used in multivariate statistical algorithm for the earliest diagnosis of the onset of tissue transformation. The intrinsic fluorescence from tryptophan, NADH and prophyrins showed distinct differences in the spectral signature during the tissue transformation, due to the altered metabolic activities of the cells. The statistical analysis of the spectral data corresponding to each excitation wavelength showed better classification accuracy at 280, 320, 350 and 405 nm excitations, corresponding to tryptophan, collagen, NADH and porphyrins with the classification accuracy of 74.3, 68.1, 64.6 and 74.7 % respectively. The variations in the spectral signature and the results of the statistical analysis suggest that porphyrins, tryptophan and NADH can be targeted as potential tumor markers in the early detection of the tissue transformation process.
Indocyanine Green (ICG) is clinically used as a fluorescent dye for imaging purposes. Its rapid circulation kinetics and minimal toxicity has prompted investigation into ICG's utility as a photosentitizer for therapeutic applications. Traditionally, optically mediated tumor therapy has focused on photodynamic therapy, which employs a photochemical mechanism resulting from the absorption of low intensity CW laser light by localized photosensitizers such as Photofrin II, Benzoporphyrin Derivative (BPD), ICG. Treatment of cutaneous vascular malformations such as port-wine stains, on the other hand, is based on a photothermal mechanism resulting from the absorption of high intensity pulsed laser light by hemoglobin. In this study, we compared the effectiveness of combining photochemical and photothermal mechanisms during application of ICG in conjunction with laser irradiation with the intention that the combined approach may lead to a reduction in the threshold dose of pulsed laser light required to treat hypervascular malformations. The blood vessels in rabbit ears were used as an in vivo model for targeted vasculature. Irradiation of the ears with IR light (λ=785 nm, Δτ = 3 min, Io = 120 mW) was used to elicit photochemical damage, while photothermal damage was brought about using pulses from a ruby laser (λ=694 nm, τ = 3 ms) with different fluences. For the combined modality, photochemical damage was induced first and followed by photothermal irradiation. This modality was compared with photothermal irradiation alone. The effectiveness of each irradiation scheme was assessed using histopathological analysis. We present preliminary data that suggests that pretreatment with photodynamic therapy before photothermal coagulation results in more severe vascular damage with lower photothermal fluence levels. The results of this study provide the foundation work for further exploration of the therapeutic potentials of photochemical and photothermal effects during application of ICG in conjunction with laser irradiation.
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