Goro Nishimura

A simple and novel algorithm for time-resolved multiwavelength oximetry

Using time-resolved spectroscopy, we have developed an experimental approach to obtain the absolute changes in concentration in the scattering medium of living tissues. The time-resolved Beer-Lambert equation can be applied to living tissue due to the fact that the optical attenuation by absorption can be separated from that by scattering, and the intensity of the light along the non-linear scattered optical path is exponentially attenuated by the absorption. Based on the above, the absolute concentration of haemoglobin as well as oxygen saturation in the rat head can be determined in situ under various respiratory conditions where multiwavelength measurements were performed. The optically assessed values agree with those determined directly by the gas analysis of our previous report. The present method is very simple and therefore opens up wide applications for time-resolved spectrophotometry in clinical medicine as a technique for quantitative near-infrared oxygen monitoring.

Synthesis and optical properties of emission-tunable PbS/CdS core–shell quantum dots for in vivo fluorescence imaging in the second near-infrared window

Near-infrared (NIR) fluorescence imaging at wavelengths from 1000 to 1500 nm (2nd-NIR window) is a promising modality for in vivo fluorescence imaging because of the deeper tissue penetration with lower tissue scattering of the 2nd-NIR light. For such in vivo fluorescence imaging, highly fluorescent probes in the 2nd-NIR wavelength region are needed. Although single-walled carbon nanotubes and Ag2S quantum dots (QDs) have recently appeared as 2nd-NIR fluorescent probes, their fluorescence brightness is relatively low (quantum yields <6%). In this study, we developed a synthetic method for preparing highly fluorescent PbS/CdS core–shell QDs (quantum yields, 17% in water) with narrow band widths (<200 nm) that emit in the 2nd-NIR region. By overcoating of a CdS shell onto a PbS QD core, we could easily control the emission wavelengths of the PbS/CdS QDs at 1000 to 1500 nm. To use the QDs for in vivo imaging, we investigated the optical properties of QDs (penetration depth and blurring of fluorescence images in slices of skin, brain, and heart in mice) in the 2nd-NIR region. We found that the 2nd-NIR fluorescence imaging at ca.1300 nm using the PbS/CdS QDs results in the highest signal to background ratio with a low blurring for in vivo imaging. To confirm the capabilities of the PbS/CdS QDs for in vivo imaging, we conducted fluorescence angiography imaging of a mouse head.

Artefacts in the analysis of temporal response functions measured by photon counting

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.

Experimental evidence of distance-dependent diffusion coefficients of a globular protein observed in polymer aqueous solution forming a network structure on nanometer scale

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.

Anoxia induces matrix shrinkage accompanied by an increase in light scattering in isolated brain mitochondria

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.

Optical tomography by the temporally extrapolated absorbance method.

The concept of the temporally extrapolated absorbance method (TEAM) for optical tomography of turbid media has been verified by fundamental experiments and image reconstruction. The TEAM uses the time-resolved spectroscopic data of the reference and object to provide projection data that are processed by conventional backprojection. Optical tomography images of a phantom consisting of axisymmetric double cylinders were experimentally obtained with the TEAM and time-gating and continuous-wave (CW) methods. The reconstructed TEAM images are compared with those obtained with the time-gating and CW methods and are found to have better spatial resolution.

Quantitation of absolute concentration change in scattering media by the time-resolved microscopic Beer-Lambert law.

In a scattering media like living tissue, there have been two lines of critical argument concerning the effect of optical absorption on the distribution of’ the optical pathlength. One is that the optical pathlength is constant when the absorption changes, though it differs markedly from the physical pathlength, such as the thickness of the tissue because of considerable scattering. The other is that the distribution of the path-length depends on the absorption (1,2). Previously, we have shown the validity of the Beer-Lambert law in rat heads (3) and thigh muscles (4). The requirement of’ the Beer-Lambert law is the independence of the attenuation of incident light by absorption from that by scattering. This was confirmed by measuring the time of flight of picosecond length light pulses in several rat tissues (5) as well as model systems (6). Equation(1), which was derived from our time-resolved study on the Beer-Lambert law, shows that light intensity along the non-linear path taken by photons through scattering media is exponentially attenuated by absorption. Monte Carlo simulation also confirmed this (7). The present paper expands equation(1) into time-resolved multiwavelength photometry for determination of the absolute concentration of absorber coexisting in scattering media, by which the optical pathlength was directly estimated.

Characterization of optical parameters with a human forearm at the region from 1.15 to 1.52 µm using diffuse reflectance measurements

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.

Diffusing-wave absorption spectroscopy in homogeneous turbid media

Abstract The intensity autocorrelation function of light through a dense intralipid suspension in the presence of an absorber was investigated by the photon correlation method. Then, we demonstrated that the parameter of the scattering can be obtained through the absorption effect with the diffusing-wave spectroscopy: diffusing-wave absorption spectroscopy. Firstly, the relation of the Laplace transform between the autocorrelation and path distribution functions was confirmed by using the time-of-flight method. Then, the linear shifts along the time axis of the autocorrelation function due to the absorption were also validated and the transport mean free path was obtained. Furthermore, this was also estimated from the intensity change by absorption.

Absorbance measurements in turbid media by the photon correlation method

Optical absorption in highly turbid media was quantified by the time shift of the electric field autocorrelation function of diffused photons. The intensity autocorrelation function was analyzed by the third-order cumulant expansion and a linear relationship between the time shift and the absorber concentration was observed. The slope of the fitted line gave the molecular extinction coefficient of the absorber. The absorption spectra were also obtained from the time shift. Applicability to dual-wavelength absorption measurement is also discussed. We demonstrate for the first time, as far as we know, the feasibility of absorbance quantification in turbid media by the photon correlation method.