A working hypothesis visualization method for fNIRS measurements using Monte Carlo simulation.

In neuroscience, clarifying the functional localization of the cerebrum using functional near-infrared spectroscopy (fNIRS) is one of the important works. To better understand and trust fNIRS data, neuroscientists formulate hypothesis about the underlying neural processes. However, visualizing and validating these hypotheses is not easy due to the complex nature of brain activity and the limitations of fNIRS measurements. In this paper, we suggest the novel Monte Carlo tool designed to assist fNIRS study for neuroscientists and to deal with these problems. The tool provides a user-friendly interface for generating realistic virtual brain activity patterns based on a specified hypothesis. By setting up a region of interest in the standard brain based on the hypothesis, the simulation models the propagation of light through the brain accurately and mimics the hemodynamic response observed in fNIRS measurements. By visually displaying simulation data and identifying the major activation point, neuroscientists can validate and refine hypothesis and obtain a better understanding of the neural mechanisms underlying the fNIRS signals.•A Monte Carlo simulation method reflecting the functional localization of the cerebrum for fNIRS measurements.•Method for setting ROI corresponding to the functional localization of the cerebrum in the standard brain.•Visualization of Monte Carlo simulation results and anatomical evaluation method of activation points.

Monte Carlo Modeling of Shortwave-Infrared Fluorescence Photon Migration in Voxelized Media for the Detection of Breast Cancer.

Recent progress regarding shortwave-infrared (SWIR) molecular imaging technology has inspired another modality of noninvasive diagnosis for early breast cancer detection in which previous mammography or sonography would be compensated. Although a SWIR fluorescence image of a small breast cancer of several millimeters was obtained from experiments with small animals, detailed numerical analyses before clinical application were required, since various parameters such as size as well as body hair differed between humans and small experimental animals. In this study, the feasibility of SWIR was compared against visible (VIS) and near-infrared (NIR) region, using the Monte Carlo simulation in voxelized media. In this model, due to the implementation of the excitation gradient, fluorescence is based on rational mechanisms, whereas fluorescence within breast cancer is spatially proportional to excitation intensity. The fluence map of SWIR simulation with excitation gradient indicated signals near the upper surface of the cancer, and stronger than those of the NIR. Furthermore, there was a dependency on the fluence signal distribution on the contour of the breast tissue, as well as the internal structure, due to the implementation of digital anatomical data for the Visible Human Project. The fluorescence signal was observed to become weaker in all regions including the VIS, the NIR, and the SWIR region, when fluorescence-labeled cancer either became smaller or was embedded in a deeper area. However, fluorescence in SWIR alone from a cancer of 4 mm diameter was judged to be detectable at a depth of 1.4 cm.

Optimal focus evaluated using Monte Carlo simulation in non-invasive neuroimaging in the second near-infrared window.

Adjusting the focal plane through the intact scalp of mice is crucial in novel angiography of cerebral vasculature using quantum dots emitting second near-infrared light at a wavelength of 1100 nm. Reagents were administered through the caudal vein. When we focused 0.4 mm below the scalp surface, based on the anatomical properties of mice reported previously, the intensity of clear fluorescence images observed transiently under a microscope became very weak within several seconds. The remaining time was extremely short to repeat adjustment of the focal plane. To investigate focus, photons exciting quantum dots at depths of 0.4, 0.8, 1.4, and 2.0 mm and emission photons were tracked in a four-layered Monte Carlo model including the scalp, skull, cerebrospinal fluid, and cortex. Based on the most near-ballistic photons emitted from quantum dots at 0.4 mm depth and specification of the microscope used, including numerical aperture and depth of field, the optimal focus plane was set. •Novel angiography for cerebrovascular structures was proposed using quantum dots with second near-infrared fluorescence.•Anatomical properties reported previously allowed focusing 0.4 mm below the surface of intact scalp before observation under fluorescence.•Clear images of cerebrovascular structures were attributed to many near-ballistic photons emitted from quantum dots at 0.4 mm depth.

Near infrared imaging of intrinsic signals in cortical spreading depression observed through the intact scalp in hairless mice.

Brain cooling was inevitable in both thinned and intact skull windows in neuroimaging in vivo of mice. Thus we proposed the novel imaging method leaving intact scalp on the skull using the light at 670, 785, and 975 nm. In this study, we used hairless mice (Hos:HR-1) since the deterioration of image quality was resulted from the hair. Cortical spreading depression was induced by KCl application through small incision and burr hole on the frontal bone. Intrinsic optical signals through the intact scalp in the observation area were detected. Time course of the signal showed a triphasic feature which was consistent with the intrinsic optical signals through the intact skull. In three pairs of signal amplitudes at the different wavelengths, no significant differences were observed. Although the intact scalp weakened the amplitudes significantly, e.g., 4.0 from 6.9 at 975 nm, the signals during cortical spreading depression were sufficient to be detected.

Image analysis of colocalization of nuclear DNA and GFP labelled HIF-1α in stable transformants.

HIF-1α is regarded as a target for drug development in several diseases such as cancer. For high throughput screening of HIF-1α-targeted drug, we need to examine the activity quantitatively. In the present study, we proposed a method where stable expression system of HIF-1α was combined with image correlation analysis. When the stable transformants were labeled with DRAQ5, we could detect Co2+-induced nuclear translocation by the use of cross-correlation analysis of the dual labeling images. In the case of high throughput screening for HIF-1α-targeted drug, we should use Pearson's correlation coefficient to judge nuclear translocation.

Direct quantification of mitochondria and mitochondrial DNA dynamics.

Mitochondria are known to be one of major organelles within a cell and to play a crucial role in many cellular functions. These organelles show the dynamic behaviors such as fusion, fission and the movement along cytoskeletal tracks. Besides mitochondria, mitochondrial DNA is also highly motile. Molecular analysis revealed that several proteins are involved in mitochondria and mitochondrial DNA dynamics. In addition to the degeneration of specific nerves with high energy requirement, mutation of genes coding these proteins results in metabolic diseases. During the last few years, a significant amount of relevant data has been obtained on molecular basis of these diseases but mitochondrial dynamics in cells derived from the patients is poorly understood. So far time-lapse fluorescence microscopy, fluorescence recovery after photo bleaching and image correlation methods have been used to study organellar motion. Especially, image correlation method has possibility to evaluate diffusion coefficient of mitochondria and mitochondrial DNA simultaneously and directly. When we search candidates for compounds that modulate mitochondrial dynamics by high throughput screening, image correlation method may be useful although the careful interpretation is required for crowded and heterogeneous environment within a cell.

Comparison of mRNA expression of transcriptional factors and intercalated disk constituent proteins between in vivo and cultured cardiomyocytes.

The weak contractile force exerted by engineered cardiac muscle is a big problem in cardiac muscle tissue engineering, even though the field has made great progress over the past decade. We believe that one major reason for the weak contractile force is that the expression of genes regulating cardiomyocyte differentiation and cardiac tissue syncytium may be different for in vivo and cultured cells. In the present study, we investigated the difference of mRNA expression under in vivo and culture conditions in order to seek a target for further gene transfer treatment in the process of cardiac tissue construction. To this end, mRNA expression of four major transcriptional factors (SRF, p300, Nkx2.5, and myocardin) and two intercalated disk constituent proteins (N-cadherin and connexin43) in rat cardiomyocytes was measured by means of ratiometric reverse-transcription polymerase chain reaction. Cardiomyocytes were harvested from the hearts of 18-day (about 3 days before birth) Wistar-rat embryos (embryonic cells), 12-day neonatal rat hearts (neonatal cells), or 14-day successive dish culture of the embryonic cells harvested from 18-day embryos (cultured cells). The results indicated that, except for SRF, the mRNAs had a lower expression tendency in cultured cells than in embryonic and in neonatal cells; in particular, the mRNA expression of myocardin, N-cadherin, and connexin43 of cultured cardiomyocytes was significantly lower than that of neonatal cells. Therefore, myocardin is a candidate for forced gene up-expression during the construction of engineered cardiac tissue; in addition, a plausible reason for the weak contractile force of engineered cardiac tissue is the weak constitution of intercalated disk, because it was elucidated that mRNA expression of proteins related to intercalated disk were lower in culture.

Direct quantification of gene expression using fluorescence correlation spectroscopy.

Among the methods for single molecule detection in the field of medicinal chemistry, the importance of fluorescence correlation spectroscopy (FCS) is growing. FCS has the advantage of permitting us to determine the number of fluorescent molecules and the diffusion constant dependent on the molecular weight without any physical separation process such as gel electrophoresis. Thus this method is appropriate for studies on the hybridization of fluorescence-labeled oligonucleotides with RNA or DNA as well as gene expression through translation of a target protein linked with green fluorescent protein. Indeed, several groups have employed FCS for evaluation of gene expression in different ways. Many investigators are particularly interested in using FCS to quantitatively analyze mRNA just after transcription in the living cell. Technical advances in FCS have broadened the research spectrum in medicinal chemistry since it can also be used to study SNPs and molecular interactions between transcription factors and promoter sequences, as well as gene expression in living cells.

Construction of fibroblast-collagen gels with orientated fibrils induced by static or dynamic stress: toward the fabrication of small tendon grafts.

As a step toward the fabrication of small tendon grafts, fibroblast-collagen gels were constructed with orientated fibrils induced by static or dynamic loading. Three groups of gel samples, each consisting of 1.0 x 10(6) fibroblasts and 2 mg type I collagen, were fabricated: freely contracted gels formed the control group; contraction-directed gels made up the static group (the gel contraction was directed perpendicular to an axis made by two anchors buried in the gels so that the constraint stress exerted by the two anchors was imposed on the gel); and for the dynamic group, a specific loading pattern (free contraction followed by cyclic stretching using a tensile bioreactor) was employed. Mechanical properties were evaluated by means of the uniaxial tension test. The gels of the static group had an ultimate stress of 350 +/- 43.6 kPa and a material modulus of 548.8 +/- 61.6 kPa, which were almost 5.2 times and 15.6 times, respectively, greater than those of the controls. The dynamic gels had an ultimate stress of 256.8 +/- 80.7 kPa and a material modulus of 118.6 +/- 23.5 kPa. These results show that the ultimate stress and material modulus of the static samples are much greater than those of the dynamic samples, which is the opposite of our expectations. Therefore, studies under other dynamic loading patterns and long-term culture are needed to clarify whether dynamic loading is superior to static loading.

Constraint stress, microstructural characteristics, and enhanced mechanical properties of a special fibroblast-embedded collagen construct.

Cell-contracted collagen gels could provide rejection-free biomaterials for tissue engineering, but their application is limited by relatively low mechanical strength. We developed a special type I collagen construct (based on embedded fibroblasts) that was formed into a gel thread by using two anchors to constrain gel contraction in one direction. Each gel thread contained 2 mg of type I collagen and 1.0 x 10(6) fibroblasts, and had an initial volume of 3 mL. After 9 days in culture, this preparation was transformed into a thread-like construct measuring 26 x 2.3 x 0.21 mm. Investigation of the microstructure showed that the collagen fibrils longitudinally between two cells had most aligned with the direction of the constraint stress and had assumed higher density than those in the freely contracted controls. During culturing, the constraint stress first increased then decreased, with implications for the nature of the interaction between the embedded cells and collagen matrix. Under uniaxial tensile testing, the ultimate stress and material modulus increased by factors of 6 and 16, respectively, compared with controls, while the maximal strain decreased by 590%. Compared with the similar constructs in the literature, the thread gel was fabricated by means of a novel mold configuration so that it contracted to thread shape much faster, and more importantly, the constraint force was firstly reported in this article. The improved mechanical properties show that the gel thread could be an effective biomaterial for such tissue engineering applications as the fabrication of blood vessels, ligaments, and tendon grafts.

In vivo oxygen imaging using green fluorescent protein.

In vivo oxygen measurement is the key to understanding how biological systems dynamically adapt to reductions in oxygen supply. High spatial resolution oxygen imaging is of particular importance because recent studies address the significance of within-tissue and within-cell heterogeneities in oxygen concentration in health and disease. Here, we report a new technique for in vivo molecular imaging of oxygen in organs using green fluorescent protein (GFP). GFP-expressing COS-7 cells were briefly photoactivated with a strong blue light while lowering the oxygen concentration from 10% to <0.001%. Red fluorescence (excitation 520-550 nm, emission >580 nm) appeared after photoactivation at <2% oxygen (the red shift of GFP fluorescence). The red shift disappeared after reoxygenation of the cell, indicating that the red shift is stable as long as the cell is hypoxic. The red shift of GFP fluorescence was also demonstrated in single cardiomyocytes isolated from the GFP knock-in mouse (green mouse) heart. Then, we tried in vivo molecular imaging of hypoxia in organs. The red shift could be imaged in the ischemic liver and kidney in the green mouse using macroscopic optics provided that oxygen diffusion from the atmospheric air was prevented. In crystalloid-perfused beating heart isolated from the green mouse, significant spatial heterogeneities in the red shift were demonstrated in the epicardium distal to the coronary artery ligation. We conclude that the present technique using GFP as an oxygen indicator may allow in vivo molecular imaging of oxygen in organs.

Detection of oxidative stress-induced mitochondrial DNA damage using fluorescence correlation spectroscopy.

Using fluorescence correlation spectroscopy (FCS), we tested the feasibility of rapid detection of oxidative damage of mitochondrial DNA (mtDNA) in a small volume. The complete mtDNA genome was amplified by long polymerase chain reaction (LPCR), and the product was fluorescently labeled with an intercalating dye, YOYO-1. The fluorescence autocorrelation function was analyzed using a simple two-component model with the diffusion time of 0.21 ms for the LPCR primer and 18 ms for the mtDNA LPCR product. When human embryonic kidney 293 (HEK-293) cells were exposed to 0.4 mM H2O2, the fraction of the mtDNA LPCR product decreased significantly. In contrast, the fraction of the nuclear-encoded beta-globin LPCR product remained unchanged. The analysis time of FCS measurement was very short (5 min) compared with that of gel electrophoresis (3 h). Thus, FCS allowed the rapid detection of the vulnerability of mtDNA to oxidative stress within a small volume element at the subfemtoliter level in solution. These results suggest that the LPCR-FCS method can be used for epidemiological studies of diseases caused by mtDNA damage.

Quantification of size distribution of restriction fragments in mitochondrial genome using fluorescence correlation spectroscopy.

A crucial investigation is to quantify restriction fragment length polymorphisms without gel electrophoresis, as the distribution of fragment size is mainly evaluated on the gel, which cannot be easily quantified. We developed a method to determine the fragmentation of the mitochondrial genome caused by restriction enzymes using fluorescence correlation spectroscopy (FCS). Distribution of fragment size was evaluated by the decrease in amplitude of the fluorescence correlation function while the mitochondrial genome PCR product was digested with Hga I or Hae III. Using a multicomponent model, which was considered as a fragment length-weighted correlation function, we calculated the correlation amplitude theoretically expected and compared it to that measured by FCS. These amplitudes for Hga I were coincident, whereas the measured amplitude for Hae III was more than the theoretical one. Because of tetra-nucleotide recognition by Hae III, there were many more fragments than with Hga I. Therefore, the amplitude measured by FCS would be a very useful index for primary screening for alterations in the entire mitochondrial genome with restriction enzymes that have several polymorphic restriction sites in the genome.

Effect of cobalt on the liver glycogen content in the streptozotocin-induced diabetic rats.

Cobalt decreases blood glucose in diabetic rats but the mechanisms involved are unclear. To determine the contribution of glycogen metabolism to glycemia-lowering effect, glycogen contents of liver and muscle in the streptozotocin-induced diabetic rats were determined. The liver glycogen was depleted in diabetic rats. But when cobalt was administered to the rats, the glycogen returned to the level of healthy rats, concomitantly with the decrease in blood glucose. The cobalt treatment had no effect on the muscle glycogen in the diabetic rats. The tissue-specific responses of glycogen metabolism suggest the involvement of suppressed glucagon signaling due to cobalt treatment.

Genetic oxygen sensor: GFP as an indicator of intracellular oxygenation.

We report in this article a new method for in vivo oxygen measurement using green fluorescence protein (GFP). COS7 cells were transiently transfected with an expression vector, pCMX-GFP, using a polyethylenimine reagent and cultured for 48 hrs. After exposure of the cell to anoxic gas (O2 < .001%), a 1 min illumination of the cell to strong 470-490 nm light evoked a significant red fluorescence (excitation 520-550 nm, emission > 580 nm) that had been negligible before the photoactivation. This red shift of (green) GFP fluorescence was never observed in normoxia. We then examined the validity of this method in transgenic mice in which GFP is stably expressed (green mice). All the ventricular myocytes isolated from the green mice showed significant green fluorescence, although the intensity was approximately 1/200 of the transiently GFP-expressing COS7 cells. The photoactivation in anoxia increased the red fluorescence in these cells, but the magnitude was much smaller than expected. In summary, GFP can be used as an in situ probe for hypoxia. In GFP-expressing transgenic animals, in vivo imaging of anoxic loci with a submicron spatial resolution may be possible.

Relationship between the gene expression of c-fos and degree of hypoxia in rat brain, as revealed by near-infrared spectroscopy.

Hypoxic induction of c-fos was studied in rat brains as a function of the cerebral oxygenation state using near-infrared spectroscopy by which the hemoglobin oxygenation state and redox state of mitochondrial cytochrome oxidase can be monitored noninvasively. Following reoxygenation after hypoxia, the expression of c-fos and MAP2 mRNAs was determined by reverse transcription-coupled PCR. The expression of MAP2 remained unchanged throughout all conditions from 21 to 8% FiO2. Under the mildly hypoxic conditions, c-fos mRNA was not induced. Hemoglobin was partially deoxygenated but cytochrome oxidase remained fully oxidized. Severe hypoxia, where cytochrome oxidase was reduced, caused a significant induction of c-fos mRNA. At this stage, the oxygen concentration in cerebral tissue fell to lower than 10(-7) M. These data suggest that the decline in oxidative phosphorylation might be a trigger for the induction of c-fos mRNA.