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A fast algorithm for fluorescence diffuse optical tomography is proposed. The algorithm is robust against the choice of initial guesses. We estimate the position of a fluorescent target by assuming a cuboid (rectangular parallelepiped) for the fluorophore target. The proposed numerical algorithm is verified by a numerical experiment and an experiment with a meat phantom. The target position is reconstructed with a cuboid from measurements in the time domain. Moreover, the long-time behavior of the emission light is investigated making use of the analytical solution to the diffusion equation.
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Significance: Although the depth detection limit of fluorescence objects in tissue has been studied, reports with a model including noise statistics for designing the optimum measurement configuration are missing. We demonstrate a variance analysis of the depth detection limit toward clinical applications such as noninvasively assessing the risk of aspiration. Aim: It is essential to analyze how the depth detection limit of the fluorescence object in a strong scattering medium depends on the measurement configuration to optimize the configuration. We aim to evaluate the depth detection limit from theoretical analysis and phantom experiments and discuss the source-detector distance that maximizes this limit. Approach: Experiments for detecting a fluorescent object in a biological tissue-mimicking phantom of ground beef with background emission were conducted using continuous wave fluorescence measurements with a point source-detector scheme. The results were analyzed using a model based on the photon diffusion equations. Then, variance analysis of the signal fluctuation was introduced. Results: The model explained the measured fluorescence intensities and their fluctuations well. The variance analysis showed that the depth detection limit in the presence of ambient light increased with the decrease in the source-detector distance, and the optimum distance was in the range of 10 to 15 mm. The depth detection limit was found to be â¼ 30 mm with this optimum distance for the phantom. Conclusions: The presented analysis provides a guide for the optimum design of the measurement configuration for detecting fluorescence objects in clinical applications.
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Fantasmas de Imagen , Animales , Bovinos , Límite de Detección , Espectrometría de Fluorescencia/métodos , Imagen Óptica/métodosRESUMEN
The time-dependent nature of the complexes of IR806 dye and other similar near-infrared cyanine dyes in human serum albumin (HSA) have been studied by employing absorption, emission and time-correlated single photon counting techniques. The complex formation of IR806 with HSA modifies the native structure of IR806, resulting in time-dependent changes in its optical properties. The modification process of the dye and its complex formation with HSA is very slow and takes about 90 min. The process of the formation of the new complex is irreversible and totally controlled by the initial complex of IR806 and HSA. As far as we know, this new property of cyanine dyes has not been reported before. These properties are very important for near-infrared fluorescence imaging.
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Carbocianinas/química , Colorantes/química , Albúmina Sérica/química , Absorción , Sitios de Unión , Humanos , Espectroscopía Infrarroja Corta , Factores de TiempoRESUMEN
Indocyanine green-based fluorescence imaging techniques are very powerful in clinical applications, but the imaging is restricted to the signal from the near-surface region of tissue. Here, we focus on the method to discriminate the fluorescence signal from the background using a time-domain gating technique. The contrast of the fluorescence image from a fluorescence object at more than 1 cm depth in a meat phantom could be enhanced about 4-5 times relative to the continuous wave method if the time-gate range was properly selected. Further, a Monte Carlo simulation with a simple background model indicates that a shorter source and detector distance is more effective to improve the contrast. The simple time-gating method will enable a highly sensitive fluorescence detection in thick tissue.
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We report on a simple correlation method for lifetime measurements using a random modulated excitation light source. We use an intensity correlation function of emission for lifetime analyses. In this method, no reference timing of the excitation is required. We apply the correlation method to measure phosphorescence decays and successfully demonstrate in the analysis of the phosphorescence decay from Pd(II) porphine in HeLa cells under aerobic and anaerobic conditions to understand the oxygen dynamics in individual cells. The method is applicable to faster decay time measurements down to a nanosecond range when the detection system is improved. Current fluorescence correlation setups can easily be modified for lifetime measurements, expanding the applicability in biological problems.
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Algoritmos , Consumo de Oxígeno/fisiología , Oxígeno/metabolismo , Espectrometría de Fluorescencia/métodos , Células HeLa , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Time- and space-resolved diffuse reflectance measurements were used to identify the optical parameters, the reduced scattering and absorption coefficients, of bulk living tissue in the region from 1.15 to 1.52 microm. Although in this region the detector was limited in its temporal resolution, we applied a peak-time shift analysis successfully to determine these coefficients in a human forearm, and then determined the absorption spectrum by space-resolved diffuse reflectance measurements. The absorption spectrum of a water content of 52% determined by magnetic resonance imaging experiments is in good agreement with the absorption coefficient obtained by optical measurements. Moreover, magnetic resonance imaging measurements suggest that the deviation of the absorption coefficients from the water spectrum in the strong water absorption band is caused by the heterogeneity of water distribution in tissue: the low content of water in the skin. These findings indicate that this optical method is potentially applicable to the non-invasive measurement of water in tissue, especially in a region lower than about 1.3-1.35 microm, which may be useful in monitoring oedema and tissue swelling.
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Antebrazo/diagnóstico por imagen , Óptica y Fotónica/instrumentación , Refractometría/métodos , Edema/diagnóstico , Edema/patología , Antebrazo/patología , Humanos , Imagen por Resonancia Magnética , Radiografía , Dispersión de Radiación , Traumatismos de los Tejidos Blandos/diagnóstico , Traumatismos de los Tejidos Blandos/patología , Análisis Espectral , Distribución Tisular , Agua/química , Agua/metabolismoRESUMEN
Fluorescence lifetime in heterogeneous multiple light scattering systems is analyzed by an algorithm without solving the diffusion or radiative transfer equations. The algorithm assumes that the optical properties of medium are constant in the excitation and emission wavelength regions. If the assumption is correct and the fluorophore is a single species, the fluorescence lifetime can be determined by a set of measurements of temporal point-spread function of the excitation light and fluorescence at two different concentrations of the fluorophore. This method is not dependent on the heterogeneity of the optical properties of the medium as well as the geometry of the excitationdetection on an arbitrary shape of the sample. The algorithm was validated by an indocyanine green fluorescence in phantom measurements and demonstrated by an in vivo measurement.
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Algoritmos , Espectrometría de Fluorescencia/métodos , Difusión , Fluorescencia , Fantasmas de Imagen , Dispersión de RadiaciónRESUMEN
Recent experimental studies have shown that bone marrow stromal cells (BMSC) differentiate into neural cells and reduce neurological deficits when transplanted into traumatized spinal cord. These findings have been derived primarily from histological analyses. We conducted a study directed chiefly at developing a non-invasive system for tracking BMSC transplanted into the spinal cord of living animals. In this study, we induced spinal cord injury (SCI) in rats with a pneumatic device. BMSC were harvested from transgenic mice expressing green fluorescence protein (BMSC-GFP), and were transplanted stereotactically into a control group of rats without SCI (n = 6) and a group with SCI (n = 3). At 2 and 4 weeks after transplantation, the dura mater was exposed and green fluorescence derived from the transplanted BMSC-GFP was observed. The distribution and differentiation of the transplanted cells were subsequently evaluated with immunohistochemistry. Green fluorescence could be detected around the transplantation site in three of six of the control rats. In all three rats subjected to SCI, green fluorescence was shown to spread from the site of BMSC-GFP injection toward the injury site, suggesting that the transplanted cells had migrated toward the lesion within the 4-week post-transplantation period. Histological evaluation suggested that the detected green fluorescence was emitted by cells that had distributed in the dorsal white matter, and demonstrated that some of the transplanted cells expressed neuronal or astrocytic markers. These results suggest the possibility of tracking BMSC transplanted into the spinal cord in living animals. Such noninvasive bioimaging techniques would be valuable for monitoring the fate of these transplanted cells and assessing the safety and efficacy of their transplantation.
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Células de la Médula Ósea/fisiología , Trasplante de Médula Ósea/métodos , Supervivencia de Injerto/fisiología , Traumatismos de la Médula Espinal/terapia , Células del Estroma/fisiología , Animales , Astrocitos/citología , Astrocitos/metabolismo , Biomarcadores , Células de la Médula Ósea/citología , Supervivencia Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Inmunohistoquímica , Ratones , Ratones Transgénicos , Microscopía Fluorescente/métodos , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/ultraestructura , Proteínas del Tejido Nervioso/metabolismo , Ratas , Ratas Wistar , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Coloración y Etiquetado/métodos , Células del Estroma/citología , Células del Estroma/trasplanteRESUMEN
Analysis of time-of-flight (TOF) data is sometimes limited by the instrumental response function, and optical parameters are extracted from the observed response curve by several mathematical methods, such as deconvolution. In contrast to this, we demonstrate that a method using shifts of the peak time of the response curve with different source-detector separations can yield the average path length of the light traveling in a tissue-like sample without deconvolution. In addition, combining the intensity information allows us to separate the scattering and absorption coefficients. This simple method is more robust in signal-to-noise ratio than the moment analysis, which also does not require the deconvolution procedure, because the peak position is not significantly dependent on the baseline fluctuation and the contamination of the scattering. The analysis is demonstrated by TOF measurements of an Intralipid solution at 800 nm, and is applied to the measurements at 1.29 microm, where the temporal response of photomultiplier tubes is not sufficiently good.
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Luz , Modelos Teóricos , Óptica y Fotónica , Espectroscopía Infrarroja Corta , Emulsiones Grasas Intravenosas/efectos de la radiación , Fantasmas de Imagen , Dispersión de Radiación , Soluciones/efectos de la radiación , Factores de TiempoRESUMEN
The least-squares (LS) method in fluorescence decay analyses and in time-domain analyses of the diffuse scattering light for data measured by the time-correlated single photon counting (TCSPC) technique is experimentally evaluated, and the artefact in LS analysis for data with different counting statistics is discussed. In single exponential decay analysis, the error of the decay parameter by the LS method is smaller than 10% of the expected true value when the average number of counts per bin (N/k) is more than 1, and the fitting region covers a period on the order of the decay time. In multi-exponential analysis, the decay parameters are sensitively dependent on the counting statistics. In contrast, the fitting by the maximum likelihood estimation (MLE), assuming Poissonian statistics, greatly reduces such dependence of parameters on the counting statistics. In another application, time-domain diffuse scattering measurements, the LS method is only accurate at N/k > 50 (10% error in the absorption coefficient). In particular, the absorption coefficient is largely dependent on the count. In both examples, the problem of stability in the fitting process by MLE still remains: the convergence of the fitting is critically dependent on the selection of initial guesses of the parameters in contrast to the convergence in the LS method. Thus, a hybrid method using the LS method for the determination of the initial guesses is a practical solution to this problem.
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Algoritmos , Artefactos , Mediciones Luminiscentes/métodos , Modelos Estadísticos , Fotones , Radiometría/métodos , Simulación por Computador , Semivida , Dosis de Radiación , Factores de TiempoRESUMEN
A photon timing recorder was realized in a field programmable gate array to capture all timing data of photons on multiple channels with down to a 1-ns resolution and to transfer all data to a host computer in real-time through universal serial bus with more than 10 M events/s transfer rate. The main concept is that photon time series can be regarded as a serial communication data stream. This recorder was successfully applied for simultaneous measurements of fluorescence fluctuation and lifetime of near-infrared dyes in solution. This design is not only limited to the fluorescence fluctuation measurement but also applicable to any kind of photon counting experiments in a nanosecond time range because of the simple and easily modifiable design.
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It is important to monitor mitochondrial conditions, and light scattering (LS) measurements have been applied to the detection of morphological changes in mitochondria in vivo. Little is known about the morphological and LS responses of brain mitochondria to oxygen withdrawal, a critical factor in cell death. We have therefore investigated the morphological and LS responses of isolated brain mitochondria to anoxia. Anoxia induced an increase in LS, reflecting mitochondrial matrix shrinkage. This response was reversible, but was reduced by adding digitonin, which disrupted the outer membrane selectively. This suggested that integrity of the outer membrane was necessary for the matrix response. We further examined the effects of Mg2+ and ATP on the responses because both exist in cells and modulate the changes in matrix volume. Although Mg2+ and ATP reduced the rates of increase and decrease in LS, respectively, the magnitudes of the increases in LS caused by anoxia stayed at over 80% of the control level (no Mg2+) in the presence of Mg2+ and ATP. This suggested that the increase in LS occurred in cells containing Mg2+ and ATP during anoxia. In contrast, that caused by inhibitors of the electron transport chain was reduced to below 30% of the control level in the presence of Mg2+. The present in vitro study provides a basis for interpretation of LS signals from mitochondria in brain research during oxygen withdrawal.
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Hipoxia Encefálica/metabolismo , Hipoxia Encefálica/patología , Mitocondrias/metabolismo , Mitocondrias/patología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Química Encefálica , Citoplasma/metabolismo , Citoplasma/patología , Citoplasma/ultraestructura , Digitonina/farmacología , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/fisiología , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Membranas Intracelulares/patología , Membranas Intracelulares/ultraestructura , Iluminación , Magnesio/metabolismo , Magnesio/farmacología , Masculino , Microscopía Electrónica , Mitocondrias/ultraestructura , Consumo de Oxígeno/efectos de los fármacos , Estimulación Luminosa , Ratas , Ratas Wistar , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismoRESUMEN
Recent experimental studies have indicated that bone marrow stromal cells (BMSC) improve neurological deficits when transplanted into the animal models of various neurological disorders, although precise mechanism still remains unclear. In this study, we developed a new in vivo fluorescence optical imaging protocol to sequentially track the transplanted into the brain of the living animals subjected to cerebral infarct. Mice BMSC were harvested from transgenic mice expressing green fluorescent protein (BMSC-GFP). They were stereotactically transplanted into the ipsilateral striatum of mice subjected to permanent middle cerebral artery occlusion after 7 days of ischemia (n=12). During 12 weeks after transplantation, the skull was exposed and the green fluorescence emitted from the brain surface was sequentially observed, using in vivo fluorescence optical microscopy. As the results, regional green fluorescence was detected in the ipsilateral parietal region 4-12 weeks after transplantation in all animals and became more apparent over the time. The images obtained through the skull were very similar to those acquired by thinning or removing the skull. Immunohistochemistry evaluation revealed that the transplanted cells migrated towards the ischemic boundary zone and expressed the neuronal or astrocytic marker, supporting the findings on fluorescence optical images. Sequential visualization of the BMSC transplanted into the brain of living animals would be valuable for monitoring the migration, growth and differentiation of the transplanted cells to explore the fate and safety of stem cell transplantation for various neurological disorders.
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Trasplante de Médula Ósea/métodos , Infarto Cerebral/terapia , Infarto de la Arteria Cerebral Media/terapia , Células del Estroma/trasplante , Animales , Biomarcadores , Trasplante de Médula Ósea/instrumentación , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Cuerpo Estriado/citología , Cuerpo Estriado/fisiología , Cuerpo Estriado/cirugía , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/metabolismo , Supervivencia de Injerto/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
An application of a digital storage oscilloscope for nanosecond time-resolved spectroscopic measurements is demonstrated in the range from the single-photon region to the multi-photon region. In comparison to the time-correlated single photon counting (TCSPC) method, the measurement setup can be greatly simplified by averaging the signals measured by the oscilloscope. Moreover, the multi-photon events of the fluorescence emissions can be tracked by this simple setup although there still exist some disadvantages in the dynamic range of the signal due to radio frequency noise, and the temporal response of the photo-multiplier tube. This method can simplify time-resolved optical measurements in the nanosecond range, such as fluorescence decay and time-of-flight measurements of diffusing light. Thus, this simple method will be applicable in many clinical and industrial uses.
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Conversión Analogo-Digital , Presentación de Datos , Almacenamiento y Recuperación de la Información/métodos , Nanotecnología/métodos , Rodamina 123/análisis , Procesamiento de Señales Asistido por Computador/instrumentación , Espectrometría de Fluorescencia/instrumentación , Espectrometría de Fluorescencia/métodos , Análisis de Falla de Equipo , Nanotecnología/instrumentación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
In this paper, we report on phosphorescence measurements for oxygen dynamics in cells by means of a correlation method, which is an expansion of the fluorescence correlation spectroscopy. The intensity correlation function of the emission excited by a pulsed light source was measured. With changing the pulse timing, both the fluorescence correlation function and the decay time of phosphorescence could be analyzed. This method was applied for the analysis of the oxygen dynamics in HeLa cells stained by Pd(II)-porphine. The decay function consisted of two exponential components, which might be attributed to free and protein-bound forms of Pd(II)-porphine in the cell, respectively. The relative change of the oxygen concentration under normal and uncoupled respiration conditions was also measured. The simplicity of this method is a great advantage in the biological applications. Although the current system we used was limited in the temporal resolution, the method is in principle applicable to faster decay time measurements down to the nano-second range of the fluorescence decay times.
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Oxígeno/metabolismo , Células HeLa , Humanos , Sustancias Luminiscentes , Mediciones Luminiscentes , Compuestos Organometálicos , Paladio , Porfirinas , Espectrometría de FluorescenciaRESUMEN
The dead time of the detector significantly distorts the fluorescence correlation function of fluorescent particles in solution. This distortion of the correlation function is similar to the saturation effect of the correlation function in a high-power excitation region. The correlation amplitude is significantly reduced by the dead time. The deviations in the number of molecules and the diffusion time are empirically given by the deviation of the fluorescence intensity linearity. The empirical curves of the deviations can be applied to the systematic error estimation of the parameters. The proportionality of the number of molecules to the concentration of fluorophores is no longer maintained with a large dead time, although almost all of the proportionality of the diffusion time to the inverse diffusion constant remains. This fact makes the dead-time effect different from the saturation effect, which is due to photokinetics. In practice, these distortions can be reduced by use of a smaller excitation power in which the proportionality of the fluorescence intensity is maintained.
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Algoritmos , Artefactos , Modelos Químicos , Rodamina 123/análisis , Espectrometría de Fluorescencia/métodos , Simulación por Computador , Estadística como Asunto , Factores de TiempoRESUMEN
The distortion of the fluorescence correlation function of a dye solution becomes larger with the increase in the excitation power, and eventually the parameters, such as the number of molecules and the diffusion time, obtained by the fluorescence correlation function systematically change. The most fundamental reason for this change is thought to be the distortion of the Gaussian excitation-detection field due to the saturation of the photocycle of the chromophore. The deviation from linearity of the fluorescence intensity causes the distortion of the fluorescence correlation function. Consequently, a smaller excitation power reduces the distortion and ensures an accurate measurement of the absolute value of these parameters. At the same time, the measurements at a fixed excitation power can be used to quantitatively determine the relative value of concentration and of the diffusion time. The deviation in the linearity of the fluorescence intensity and the deviation of the parameters show a high degree of correlation, and a 10% deviation of the intensity results in a prediction of a approximately 10% deviation in the number of molecules and a approximately 5% in the diffusion time.
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The distance dependence of the diffusion coefficient (DDDC) of a globular protein (cytochrome c) in aqueous hyaluronan (HA) solution, which is a model system for extracellular matrices (ECMs), was measured by a combination of three kinds of spectroscopic measurements of diffusion coefficients, the time and space samplings of which are different. The results of the three methods are plotted against the diffusion distance derived from the consideration of each experimental condition. Due to the characteristic morphology of HA with an effective mesh structure, the proteins showed two extreme diffusion modes: (1) short (<10 nm) diffusion with rare contact with polymer chains; (2) long (>100 nm) diffusion significantly disrupted by polymer chains showing an approximately 30% reduction in diffusion coefficient. The transition from the short diffusion to the long one occurs in a very narrow range (10-100 nm) of diffusion distance and this unique character of HA realizing anomalous diffusion should provide suitable environments for various bioactivities when involved in ECM.