Long-working-distance fluorescence microscope with high-numerical-aperture objectives for variable-magnification imaging in live mice from macro- to subcellular

We demonstrate the development of a long-working-distance fluorescence microscope with high-numerical-aperture objectives for variable-magnification imaging in live mice from macro- to subcellular. To observe cytoplasmic and nuclear dynamics of cancer cells in the living mouse, 143B human osteosarcoma cells are labeled with green fluorescent protein in the nucleus and red fluorescent protein in the cytoplasm. These dual-color cells are injected by a vascular route in an abdominal skin flap in nude mice. The mice are then imaged with the Olympus MVX10 macroview fluorescence microscope. With the MVX10, the nuclear and cytoplasmic behavior of cancer cells trafficking in blood vessels of live mice is observed. We also image lung metastases in live mice from the macro- to the subcellular level by opening the chest wall and imaging the exposed lung in live mice. Injected splenocytes, expressing cyan fluorescent protein, could also be imaged on the lung of live mice. We demonstrate that the MVX10 microscope offers the possibility of full-range in vivo fluorescence imaging from macro- to subcellular and should enable widespread use of powerful imaging technologies enabled by genetic reporters and other fluorophores.     

Development of morphologic heterogeneity of hepatocyte mitochondria in the mouse

The ultrastructure of mitochondria in periportal and perihepatic hepatocytes from newborn, 5-, 10-, and 20-day-old, and adult male ddY mice was analyzed by quantitative electron microscopy. In newborn and 5- and 10-day-old animals, the axial ratio (length per diameter), surface to volume ratio (area of the outer membrane per unit mitochondrial volume), and volume density were not significantly different between periportal and perihepatic cells. In 20-day-old and adult animals, the surface to volume ratio was greater in perihepatic cells than periportal cells, and the volume density was greater in periportal cells than perihepatic cells. The axial ratio became greater in perihepatic cells than periportal cells in adult animals. However, there were no differences in the surface density of the outer membrane, and of the inner membrane and cristae between the cells of both zones in all age groups examined. When the data were expressed as volume and area per cell, the patterns of subacinar distribution and age-related changes differed from the patterns seen in the volume and surface density data mainly in adult animals. This difference was generally caused by the marked increase in hepatocyte volume between 20 days of age and adulthood, especially in perihepatic cells. The results show that differences between mitochondria in periportal cells and those in perihepatic cells in the shape (the axial and surface to volume ratios), volume density, and area of the outer membrane per cell, evident in adult animals, are not present in newborn animals but arise during postnatal development.     

Intralobular heterogeneity of carbon tetrachloride-induced oxidative stress in perfused rat liver visualized by digital imaging fluorescence microscopy

Spatial and temporal alterations in carbon tetrachloride-induced oxidative stress were studied in perfused hepatic microcirculatory units of the rat by digital imaging microscopic fluorography using dichlorofluorescin diacetate, a hydroperoxide-sensitive fluorogenic probe. The surface of the liver loaded with dichlorofluorescin was microscopically visualized, and the fluorescence of dichlorofluorescin (DCF), a highly fluorescent molecule generated by hydroperoxide-mediated oxidation of dichlorofluorescin, was digitally processed. After completing the experiments, fluorescein-labeled albumin was injected into the perfusate to confirm the state of sinusoidal perfusion and the topography of hepatic microangioarchitecture in the area studied. During transportal infusion of carbon tetrachloride, DCF fluorescence was observed predominantly in the pericentral area and was attenuated by pretreatment with SKF-525A, suggesting the involvement of cytochrome P-450. After peaking at the maximum level, the pericentral DCF fluorescence gradually decreased in parallel to the loss of viability, implicating the causal role of intracellular hydroperoxide formation in hepatocellular injury. In retrogradely perfused liver, in which intralobular O2 gradient was reversed, no significant activation of DCF fluorescence was observed among hepatic lobules. It is therefore conceivable that the zonal heterogeneity of carbon tetrachloride-induced lipid peroxidation may depend on at least two factors, sinusoidal oxygenation and the intralobular distribution of cytochrome P-450. Furthermore, development of the current technique will provide a useful method to investigate the microtopography of oxidative stress in organ microcirculatory units.     

Performance of high-resolution monitors for digital chest imaging

High-resolution cathode-ray tubes (CRT's) are currently the most viable soft-copy display for digital radiography. We present here methods for measuring large-area contrast ratio and detail contrast ratio. A two-dimensional charge coupled device (ccd) array signal-averaged with a video frame buffer permits linear microradiometric measure of individual beam lines. Results from three different 1000-line monitors demonstrate the shift variance of resolution. The detail contrast ratio (or modulation depth) was found to vary from 100% to less than 10% across the face of one CRT. Dynamic focus in both the horizontal and vertical deflection circuitry proved effective in reducing this shift variance. Comparisons of three phosphors demonstrate the utility of long persistence phosphors (P164) for static display in producing brighter images with less flicker. Recommendations for CRT design and selection for high-resolution digital radiography are included.     

Molecular diagnosis reveals genetic heterogeneity for the overlapping MKKS and BBS phenotypes

Hydrometrocolpos and polydactyly diagnosed in the prenatal period or early childhood may raise diagnostic dilemmas especially in distinguishing McKusick-Kaufman syndrome (MKKS) and the Bardet-Biedl syndrome (BBS). These two conditions can initially overlap. With time, the additional features of BBS appearing in childhood, such as retinitis pigmentosa, obesity, learning disabilities and progressive renal dysfunction allow clear differentiation between BBS and MKKS. Genotype overlap also exists, as mutations in the MKKS-BBS6 gene are found in both syndromes. We report 7 patients diagnosed in the neonatal period with hydrometrocolpos and polydactyly who carry mutations in various BBS genes (BBS6, BBS2, BBS10, BBS8 and BBS12), stressing the importance of wide BBS genotyping in patients with this clinical association for diagnosis, prognosis and genetic counselling.     

Sequence-based HLA high-resolution retyping of a bone marrow donor/recipient pair reveals the novel HLA allele DQB1*0322

In this paper, we characterize the novel human leukocyte antigen (HLA)-DQB1*0322. We found this novel allele in a hematopoietic stem cell donor. The donor and the recipient were high-resolution HLA retyped using sequence-based typing. Both, the female patient and her donor were previously typed HLA identical, which was confirmed with the exception of the novel DQB1 allele. The novel allele is characterized by a nucleotide exchange 'G' to 'A' at position 485 in exon 3. This affected codon 130-arginine (CGG), which is replaced by glutamine (CAG) in the new allele DQB1*0322. The transplant was performed because of an acute myeloid leukemia at first remission.     

Clinical exome sequencing reveals locus heterogeneity and phenotypic variability of cohesinopathies

PurposeDefects in the cohesin pathway are associated with cohesinopathies, notably Cornelia de Lange syndrome (CdLS). We aimed to delineate pathogenic variants in known and candidate cohesinopathy genes from a clinical exome perspective.MethodsWe retrospectively studied patients referred for clinical exome sequencing (CES, N = 10,698). Patients with causative variants in novel or recently described cohesinopathy genes were enrolled for phenotypic characterization.ResultsPathogenic or likely pathogenic single-nucleotide and insertion/deletion variants (SNVs/indels) were identified in established disease genes including NIPBL (N = 5), SMC1A (N = 14), SMC3 (N = 4), RAD21 (N = 2), and HDAC8 (N = 8). The phenotypes in this genetically defined cohort skew towards the mild end of CdLS spectrum as compared with phenotype-driven cohorts. Candidate or recently reported cohesinopathy genes were supported by de novo SNVs/indels in STAG1 (N = 3), STAG2 (N = 5), PDS5A (N = 1), and WAPL (N = 1), and one inherited SNV in PDS5A. We also identified copy-number deletions affecting STAG1 (two de novo, one of unknown inheritance) and STAG2 (one of unknown inheritance). Patients with STAG1 and STAG2 variants presented with overlapping features yet without characteristic facial features of CdLS.ConclusionCES effectively identified disease-causing alleles at the mild end of the cohensinopathy spectrum and enabled characterization of candidate disease genes.     

Supernormal vision, high-resolution retinal imaging, multiphoton imaging and nanosurgery of the cornea--a review.

Wavelength-corrected, adaptive optics and their relevance for diagnostic procedures of the human retina are considered. First, it is shown that the compensation of errors of the dioptric apparatus of the eye allows sharp and high-contrast images of retinal elements, such as the photoreceptors, to be generated. This technology is expected to enable on the one hand an improved laser therapy by the application of laser spots of the size of single receptors as well as on the other a further understanding of the mechanisms of vision, in particular of colour vision by using colour stimuli not larger than the cones. Second, femtosecond laser pulses, emitted from lasers working in the near infrared, based on multiphoton effects allow both imaging and laser effects to be generated which are in the submicron range and which do not cause collateral damage (nanoimaging and nanosurgery). These procedures, related to experimental ophthalmology may be considered a milestone for the research of cell physiology, in particular in the subcellular range.     

Toward the clinical application of time-domain fluorescence lifetime imaging

High-speed (video-rate) fluorescence lifetime imaging (FLIM) through a flexible endoscope is reported based on gated optical image intensifier technology. The optimization and potential application of FLIM to tissue autofluorescence for clinical applications are discussed.     

星形细胞肿瘤磁敏感成像与动态对比剂MR灌注加权成像的相关性研究

目的：探讨星形细胞肿瘤磁敏感加权成像（ SWI)半量化与动态对比剂增强MR灌注加权成像（ PI)的相关性。方法回顾性分析98例经手术病理证实星形细胞肿瘤患者的术前SWI及PI检查资料。测量SWI中肿瘤内磁敏感低信号区（ ITSHIA)半量化数据,以及PI中肿瘤内实性部分最大相对脑血流量值（ rrCBV瘤内max )和瘤周区最大相对CBV值（ rrCBV瘤周max )。应用Kruskal-Wallis H检验比较不同病理分级星形细胞肿瘤间rrCBV瘤内max与rrCBV瘤周max的差异,比较不同级别肿瘤间灌注热点区与ITSHIA形态的对应情况;应用Spearman相关性检验比较不同级别肿瘤间SWI中各半量化指标与PI中rrCBV瘤内max与rrCBV瘤周max的相关性。结果星形细胞肿瘤rrCBV瘤内max值（ rs =0．662,P<0．01)及rrCBV瘤周max值（rs =0．794,P<0．01)与其分级显著相关。毛细胞型星形细胞瘤rrCBV瘤内max高于Ⅱ级星形细胞瘤,与Ⅲ级肿瘤类似;而 rrCBV瘤周max与Ⅱ级星形细胞瘤差异无统计学意义（ P >0．05),却低于高级别肿瘤。星形细胞肿瘤的ITSHIA半量化指标与rrCBV瘤内max与rrCBV瘤周max值呈显著线性正相关。星形细胞肿瘤内灌注热点区与ITSHIA不完全对应。结论星形细胞肿瘤SWI指标与PI指标密切相关,二者对于术前评估星形细胞肿瘤的病理分级同样具有较高价值。灌注热点区与ITSHIA并不完全相同,可能与二者显示肿瘤内血管生成的机制不同有关。     

Effect of PAF on erythrocyte membrane heterogeneity: A fluorescence study

The effect of platelet activating factor, PAF, on the erythrocyte membrane heterogeneity was investigated by measuring the steady-state fluorescence anisotropy and fluorescence decay of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated in hemoglobin-free erythrocyte membranes. PAF induced a significant increase of the lipid order in the exterior part of the membrane and a decrease in membrane heterogeneity. These results indicate that PAF may cause changes in the physico-chemical structure of the erythrocyte membrane.     

Intravital imaging of thymopoiesis reveals dynamic lympho-epithelial interactions

T cell development occurs in the thymus. The thymic microenvironment attracts hematopoietic progenitors, specifies them toward the T cell lineage, and orchestrates their differentiation and egress into the periphery. The anatomical location of the thymus and the intrauterine development of mouse embryos have so far precluded a direct visualization of the initial steps of thymopoiesis. Here, we describe transgenic zebrafish lines enabling the in vivo observation of thymopoiesis. The cell-autonomous proliferation of thymic epithelial cells, their morphological transformation into a reticular meshwork upon contact with hematopoietic cells, and the multiple migration routes of thymus-settling cells could be directly visualized. The unexpectedly dynamic thymus homing process is chemokine driven and independent of blood circulation. Thymocyte development appears to be completed in less than 4 days. Our work establishes a versatile model for the in vivo observation and manipulation of thymopoiesis.     

Analysis of sampling volume and tissue heterogeneity on the in vivo detection of fluorescence

The effect of sampling region size and tissue heterogeneity is examined using fluorescence histogram assessment in a rat prostate tumor model with benzoporphyrin derivative fluorophore. Spatial heterogeneity in the fluorescence signal occurs on both macroscopic and microscopic scales. The periphery of the tumor is more fluorescent than the center. Fluorescence is also highest nearest the blood vessels immediately after injection, but over time this fluorescence becomes uniform through the tumor tissue. Using microscopy analysis, the fluorescence intensity histogram distributions follow a normal distribution, yet as the sampling area is increased from the micron scale to the millimeter scale, the variance of the distribution decreases. The mean fluorescence intensity is accurately measured with a millimeter size scale, but this cannot provide accurate measurements of the microscopic variance of drug in tissue. Fiber probe measurements taken in vivo are used to confirm that the variance observed is smaller than would be expected with microscopic sampling, but that the average fluorescence can be measured with fibers. Sampling tissue with fibers smaller than the intercapillary spacing could provide a way to estimate the spatial variance more accurately. In summary, sampling fiber size affects the fluorescence intensities detected and use of multiple region microscopic sampling could provide better information about the distribution of values that occur.     

A study of equivalent source techniques for high-resolution EEG imaging

High-resolution EEG imaging has been an important topic in recent EEG research, and much work has been done on the two equivalent source imaging techniques: the equivalent distributed dipole-layer source imaging technique (EST) and the equivalent multipole source imaging technique (SAT). In this paper we first develop a forward density formula for a spherical equivalent distributed dipole layer of an arbitrary dipole in a three-concentric-sphere head model. It is clarified using the derived forward formula that the equivalent dipole-layer source and equivalent multipole source are interrelated in theory. Finally, simulation comparisons are conducted, the results of which suggest that EST has a higher spatial resolution than SAT when both of them are implemented by a truncated singular value decomposition algorithm. This is due to the different singularities of the inversion equations involved in the two techniques. An empirical VEP data study also shows that EST is better than SAT in providing higher spatial resolution EEG imaging.     

Widefield and high-resolution reflectance imaging of gold and silver nanospheres

Metallic nanoparticles have unique optical properties that can be exploited for molecular imaging in tissue. Image contrast depends on the nature of the particles, properties of the target tissue, and the imaging system. Maximizing image contrast for a particular application requires an understanding of the interplay of these factors. We demonstrate an approach that integrates the use of reflectance spectroscopy and imaging of particles in water and various tissue phantoms to evaluate the expected image contrast. We illustrate the application of this methodology for gold and silver nanospheres targeted against a biomarker expressed in epithelial tissue; predictions of contrast properties using diffuse reflectance spectroscopy were compared with widefield and high-resolution images of labeled tissue phantoms. The results show that the predicted image contrast based on spectroscopy agrees well with widefield and high-resolution imaging, and illustrate that gold and silver nanospheres at subnanomolar concentration are sufficient to produce contrast in both imaging modes. However, the effective contrast achieved with a particular type of nanoparticle can differ dramatically depending on the imaging modality. The ability to predict and optimize image contrast properties is a crucial step in the effective use of these nanomaterials for biomedical imaging applications.     

Comparison of high-resolution echo-planar spectroscopic imaging with conventional MR imaging of prostate tumors in mice

High spectral and spatial resolution (HiSS) MRI of rodent tumors has previously been performed using conventional spectroscopic imaging to obtain images with improved contrast and anatomic detail. The work described here evaluates the use of much faster echo-planar spectroscopic imaging (EPSI) to acquire HiSS data from rodent tumor models of prostate cancer. A high-resolution EPSI pulse sequence was implemented on a 4.7 T Bruker scanner. Three-dimensional EPSI data were Fourier-transformed along the k-space and temporal (free-induction decay) axes to produce detailed water and fat spectra associated with each small image voxel. The data were used to generate images of spectral parameters, e.g. peak-height images for each small voxel. Two variants of EPSI were performed; gradient-echo or spin-echo excitation with EPSI readout. These imaging methods were tested in commonly used rodent prostate cancers, including seven mice implanted with non-metastatic AT2.1 (n=3) and metastatic AT3.1 (n=4) prostate tumors on the hind leg, and 10 mice implanted with LNCaP prostate cancers in situ. The peak-height images derived from EPSI datasets provide more detailed tumor anatomy, improved signal-to-noise and contrast-to-noise ratios compared with the gradient-echo or spin-echo images at all echo times. The results suggest that HiSS MRI data from small animal models of prostate cancer can be acquired using EPSI, and that this approach improves imaging of heterogeneous tissue and vascular environments inside the tumors compared with conventional MR techniques.     

Ageing and subcellular distribution of mitochondria: role of mitochondrial DNA deletions and energy production

The rapid growing population of elderly illustrates the importance of understanding the mechanisms responsible for ageing and the detrimental effects on health associated with increasing age. One of the primary mechanisms may be because of the accumulation of mtDNA damage and oxidative damage with age. Previous studies have examined this correlation in post-mitotic tissues such as skeletal muscle, heart and brain with decreased mitochondrial function, such as enzymatic activities of the electron transport chain and ATP production. However, regional differences in the subcellular location of mitochondria exist and most studies have failed to differentiate the effects of these two autonomous fractions, the subsarcolemmal and intermyofibrillar populations. Hence, while future research attempts to explain the mechanisms responsible for ageing in the mitochondrion, it should also take into account the independent pathways of these two distinctly different populations.     

Real-time quantitative PCR analysis of regions involved in gene amplification reveals gene overdose in low-grade astrocytic gliomas.

We have studied gene amplification of genes located in 1q32 (GAC1, ELF3, MDM4, and ren1), 4q11 (PDGFR-alpha), and in 12q13-14 (MDM2 and CDK4) using quantitative real-time PCR in a group of 86 tumors consisting of 44 WHO grade IV glioblastomas (GBM) (34 primary and 10 secondary tumors), 21 WHO grade III anaplastic astrocytomas (AA), and 21 WHO grade II astrocytomas (AII). Gene amplification was present in 56 of the 86 samples (65%) in at least 1 gene in our series. GAC1 (51%) and MDM4 (27%) were the most frequently amplified genes within the 1q32 amplicon, and their higher amplification frequency was statistically significant (P<0.05, chi) in the low-grade astrocytomas. Concordant co-amplification was determined for ELF3 and ren1 or ren1 and MDM4 in the grade III-IV tumors. MDM2 amplification was significantly more frequent in primary GBM (16%) than was in secondary GBM (0%). The present study shows that gene amplification in the studied regions is already present in low-grade astrocytic tumors and that amplification of some genes may represent another molecular marker to differentiate primary from secondary GBM.