Laser tweezers and multiphoton microscopes in life sciences

Near infrared (NIR) laser microscopy enables optical micromanipulation, piconewton force determination, and sensitive fluorescence studies by laser tweezers. Otherwise, fluorescence images with high spatial and temporal resolution of living cells and tissues can be obtained via non-resonant fluorophore excitation with multiphoton NIR laser scanning microscopes. Furthermore, NIR femtosecond laser pulses at TW/cm2 intensities can be used to realize non-invasive contact-free surgery of nanometer-sized structures within living cells and tissues. Applications of these novel versatile NIR laser-based tools for the determination of motility forces, coenzyme and chlorophyll imaging, three-dimensional multigene detection, non-invasive optical sectioning of tissues ("optical biopsy"), functional protein imaging, and nanosurgery of chromosomes are described.     

Development of biological tissue-equivalent phantoms for optical imaging

Optical characteristics of freshly isolated tissues depend on their color and composition. The surface backscattered profile, which account for the tissue compositional variation in fresh excised sheep's heart, lungs, bone and muscle, were measured by multi-probe reflectometer. Optical phantoms were prepared from paraffin wax by mixing a specific combination of wax color materials till the surface backscattered profile of these matched with that of the biological tissues. The optical parameters absorption coefficient (micro(a)), reduced scattering coefficient (micro(s)) and anisotropy factor (g) of these phantoms, are the same as that of biological tissues and are obtained by matching their surface backscattered profiles with that as simulated by Monte Carlo procedure. The maximum and minimum values of absorption coefficient are for the phantoms of lungs (1.0 cm(-1)) and muscle (0.02 cm(-1)), whereas, for scattering coefficient these values are for muscle (21.2 cm(-1)) and bone (13.08 cm(-1)).     

Non destructive analysis of the wax layer of apple (Malus domestica Borkh.) by means of confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) was used to non-destructively analyse the changes in the structure and thickness of the cuticle during storage of apples (Malus domestica Borkh.). Interpretation of the confocal images was performed by comparison with scanning electron microscopy and environmental scanning electron microscopy images. The natural reflectance of the wax and the auto-fluorescence of the underlying cells made it possible with CLSM to distinguish the wax from the underlying layers without any pretreatment of the fruit. The thickness of the consecutive layers (wax, cutin, cells) could be estimated from measurements of the reflection and fluorescence intensities as a function of the number of pixels. The mean wax-layer thickness measured in this way amounted to 2.58 microm, 3.41 microm or 4.14 microm for the cultivars Jonagold, Jonagored and Elstar, respectively. Changes in the wax structure and cells of the same important Belgian apple cultivars as mentioned above were monitored during nine months of storage in ultra low oxygen and after exposure to ambient conditions. The changes in the wax ultrastructure and cell morphology are likely related to water losses and specific protection of the apple cultivars against water losses during storage and shelf life.     

Epicuticular Wax Accumulation and Fatty Acid Elongation Activities Are Induced during Leaf Development of Leeks

Epicuticular wax production was evaluated along the length of expanding leek (Allium porrum L.) leaves to gain insight into the regulation of wax production. Leaf segments from the bottom to the top were analyzed for (a) wax composition and load; (b) microsomal fatty acid elongase, plastidial fatty acid synthase, and acyl-acyl carrier protein (ACP) thioesterase activities; and (c) tissue and cellular morphological changes. The level of total wax, which was low at the bottom, increased 23-fold along the length of the leaf, whereas accumulation of the hentriacontan-16-one increased more than 1000-fold. The onset of wax accumulation was not linked to cell elongation but, rather, occurred several centimeters above the leaf base. Peak microsomal fatty acid elongation activity preceded the onset of wax accumulation, and the maximum fatty acid synthase activity was coincident with the onset. The C16:0- and C18:0-ACP-hydrolyzing activities changed relatively little along the leaf, whereas C18:1-ACP-hydrolyzing activity increased slightly prior to the peak elongase activity. Electron micrographic analyses revealed that wax crystal formation was asynchronous among cells in the initial stages of wax deposition, and morphological changes in the cuticle and cell wall preceded the appearance of wax crystals. These studies demonstrated that wax production and microsomal fatty acid elongation activities were induced within a defined and identifiable region of the expanding leek leaf and provide the foundation for future molecular studies.     

Image reconstruction of optical attenuation coefficient variation in biological tissues

A procedure for non-invasive imaging of the optical attenuation coefficient variation of in vivo thick organs/tissues is developed. The laser back-scattered surface profiles at various locations of human forearm, by multi-probe reflectometer, are measured. These profiles are matched by iterative procedure, with that as obtained by Monte Carlo simulation and the corresponding values of attenuation coefficient (equal to the sum of absorption and reduced scattering coefficients) are determined. By interpolation of this data a 100 x 100 grid is constructed and after median filtering of this data a color-coded image of the variability of the optical attenuation coefficient of the forearm is obtained. These images in different subjects show variation due to change in overall tissue composition and blood pooling. This non-invasive imaging procedure may help in identifying the diseased affected regions in healthy tissues and in application of photodynamic therapy.     

In situ NADH laser fluorimetry during muscle contraction in humans

The aim of the present study was to use nicotinamide adenine dinucleotide phosphate, reduced (NADH) fluorimetry, to investigate in situ NADH changes during muscle contraction in humans on an isokinetic dynamometer. Thirteen healthy male subjects each performed one maximal voluntary contraction (MVC) with the knee extensor muscle. The NADH muscle fluorescence was monitored by a double beam laser fluorimeter which uses an optical fibre, percutaneously inserted through a needle into the vastus lateral muscle, to guide the light. The NADH fluorescence was continuously measured at a wavelength of 337 nm. To estimate the haemodynamic artefact, blood backscattering was simultaneously determined at a wavelength of 586 nm. The fluorescence signal was recorded before, during and after contractions at 50% of MVC. The fibre was kept out of contact with the muscle during contractions at 100% of MVC and was only put into contact with it at the end of the contraction. At the onset of contractions at 50% of MVC, NADH fluorescence increased rapidly for 3 s and remained stable thereafter until exhaustion. After a muscle measurement had been made, the optical fibre was put successively into solutions of increasing NADH concentration to ascertain the relationship between the muscle fluorescence signal and the muscle NADH level. This procedure yielded estimated mean values for muscle NADH of 0.172 mmol.kg-1, SEM 0.028 and of 0.184 mmol.kg-1, SEM 0.027 after contractions at 50% and 100% of MVC, respectively, from a resting value of 0.087 mmol.kg-1, SEM 0.015. These results indicated that in situ laser fluorimetry could be used to evaluate NADH changes in humans during muscle contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

激光共聚焦显微镜与光学显微镜之比较

激光扫描共聚焦显微镜在活细胞的动态检测、光学切片和三维结构重建等方面较光学显微镜有质的飞跃。本文对激光扫描共聚焦显微镜和光学显微镜进行了比较和讨论,并简单介绍多光子激光扫描显微镜。     

Mechanistic Evaluation of the Effect of Sintering on Compritol® 888 ATO Matrices

The present research studied the effect of sintering technique in the development of a controlled release formulation for ketorolac tromethamine. The method consisted of mixing drug and wax powder (Compritol® 888 ATO) along with lactose as diluent and talc as lubricant followed by direct compression at room temperature. The compressed fluffy matrices were kept at 80°C for 1, 2, and 3 h for sintering. The sintered tablets were characterized by their physical parameters and in vitro dissolution profile. The sintering time markedly affected the drug release properties of Compritol® 888 ATO matrices. It is notable that the release rate of ketorolac tromethamine from matrices was inversely related to the time of sintering. This may be due to the increase in the extent and firmness of sintering which further compacts the mass so that drug release is affected. Contact angle measurement and scanning electron microscopy analysis indicated that heat treatment caused the wax to melt and redistribute. This redistributed wax formed a network-like structure in which the drug along with lactose is entrapped. This particular formed matrix is responsible for retarding the drug release. Fourier transform infrared spectroscopy results did not show any drug–wax interaction due to sintering. Differential scanning calorimetric and powder X-ray diffraction studies ruled out the occurrence of solid solution and polymorphic changes of the drug. Drug release from the wax tablets with or without sintering was best described by the Higuchi equation.     

Biochemical analysis of the bone of SHC rats with spontaneous hypercholesterolemia and renal dysfunction.

Spontaneously hypercholesterolemic (SHC) rats exhibit severe abnormalities in renal function and bone metabolism at old ages, in addition to hypercholesterolemia. SHC rats were also found to show endocrine abnormalities such as hyperthyroidism from young ages. In this study, biochemical and microdensitometric analyses were carried out using femurs to characterize further the abnormality in bone metabolism: whether biochemical markers of the bone may be affected by these abnormalities. At 6 weeks of age, the ashed weight and calcium content of the dried femurs were slightly lower in SHC rats than in age-matched Sprague-Dawley (SD) rats. None of the markers of microdensitometric analysis was changed. At 24 weeks of age, the ashed weight of dried femurs and the density of the marrow region of femurs were lower in the SHC rats. The results indicate that SHC rats exhibit severe abnormality in bone metabolism leading to biochemical changes in the bone at old ages whereas changes in bone markers were minimal at young ages before the onset of severe renal dysfunction.     

Nonideal mixing and phase separation in phosphatidylcholine-phosphatidic acid mixtures as a function of acyl chain length and pH.

The miscibilities of phosphatidic acids (PAs) and phosphatidylcholines (PCs) with different chain lengths (n = 14, 16) at pH 4, pH 7, and pH 12 were examined by differential scanning calorimetry. Simulation of heat capacity curves was performed using a new approach that incorporates changes of cooperativity of the transition in addition to nonideal mixing in the gel and the liquid-crystalline phase as a function of composition. From the simulations of the heat capacity curves, first estimates for the nonideality parameters for nonideal mixing as a function of composition were obtained, and phase diagrams were constructed using temperatures for onset and end of melting, which were corrected for the broadening effect caused by a decrease in cooperativity. In all cases the composition dependence of the nonideality parameters indicated nonsymmetrical mixing behavior. The phase diagrams were therefore further refined by simulations of the coexistence curves using a four-parameter approximation to account for nonideal and nonsymmetrical mixing in the gel and the liquid-crystalline phase. The mixing behavior was studied at three different pH values to investigate how changes in headgroup charge of the PA influences the miscibility. The experiments showed that at pH 7, where the PA component is negatively charged, the nonideality parameters are in most cases negative, indicating that electrostatic effects favor a mixing of the two components. Partial protonation of the PA component at pH 4 leads to strong changes in miscibility; the nonideality parameters for the liquid-crystalline phase are now in most cases positive, indicating clustering of like molecules. The phase diagram for 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid:1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine mixtures at pH 4 indicates that a fluid-fluid immiscibility is likely. The results show that a decrease in ionization of PAs can induce large changes in mixing behavior. This occurs because of a reduction in electrostatic repulsion between PA headgroups and a concomitant increase in attractive hydrogen bonding interactions.     

In vivo imaging of subcutaneous structures using functional photoacoustic microscopy

Functional photoacoustic microscopy (fPAM) is a hybrid technology that permits noninvasive imaging of the optical absorption contrast in subcutaneous biological tissues. fPAM uses a focused ultrasonic transducer to detect high-frequency photoacoustic (PA) signals. Volumetric images of biological tissues can be formed by two-dimensional raster scanning, and functional parameters can be further extracted from spectral measurements. fPAM is safe and applicable to animals as well as humans. This protocol provides guidelines for parameter selection, system alignment, imaging operation, laser safety and data processing for in vivo fPAM. It currently takes approximately 100 min to carry out this protocol, including approximately 50 min for data acquisition using a 10-Hz pulse-repetition-rate laser system. The data acquisition time, however, can be significantly reduced by using a laser system with a higher pulse repetition rate.     

Reconstruction of Laser-scanned 3D Torso Topography and Stereoradiographical Spine and Rib-cage Geometry in Scoliosis

Assessments of scoliosis are routinely done by means of clinical examination and full spinal x-rays. Multiple exposure to ionization radiation, however, can be hazardous to the child and is costly. Here, we explain the use of a noninvasive imaging technique, based on laser optical scanning, for quantifying the three-dimensional (3D) trunk surface topography that can be used to estimate parameters of 3D deformity of the spine. The laser optical scanning system consisted of four BIRIS laser cameras mounted on a ring moving along a vertical axis, producing a topographical mapping of the entire torso. In conjunction with the laser scans, an accurate 3D reconstruction of the spine and rib cage were developed from the digitized x-ray images. Results from 14 scoliotic patients are reported. The digitized surfaces provided the foundation data to start studying concordance of trunk surface asymmetry and spinal shape in idiopathic scoliosis.     

Reconstruction of laser-scanned 3D torso topography and stereoradiographical spine and rib-cage geometry in scoliosis

Assessments of scoliosis are routinely done by means of clinical examination and full spinal x-rays. Multiple exposure to ionization radiation, however, can be hazardous to the child and is costly. Here, we explain the use of a noninvasive imaging technique, based on laser optical scanning, for quantifying the three-dimensional (3D) trunk surface topography that can be used to estimate parameters of 3D deformity of the spine. The laser optical scanning system consisted of four BIRIS laser cameras mounted on a ring moving along a vertical axis, producing a topographical mapping of the entire torso. In conjunction with the laser scans, an accurate 3D reconstruction of the spine and rib cage were developed from the digitized x-ray images. Results from 14 scoliotic patients are reported. The digitized surfaces provided the foundation data to start studying concordance of trunk surface asymmetry and spinal shape in idiopathic scoliosis.     

Label-free 3D visualization of cellular and tissue structures in intact muscle with second and third harmonic generation microscopy

Second and Third Harmonic Generation (SHG and THG) microscopy is based on optical effects which are induced by specific inherent physical properties of a specimen. As a multi-photon laser scanning approach which is not based on fluorescence it combines the advantages of a label-free technique with restriction of signal generation to the focal plane, thus allowing high resolution 3D reconstruction of image volumes without out-of-focus background several hundred micrometers deep into the tissue. While in mammalian soft tissues SHG is mostly restricted to collagen fibers and striated muscle myosin, THG is induced at a large variety of structures, since it is generated at interfaces such as refraction index changes within the focal volume of the excitation laser. Besides, colorants such as hemoglobin can cause resonance enhancement, leading to intense THG signals. We applied SHG and THG microscopy to murine (Mus musculus) muscles, an established model system for physiological research, to investigate their potential for label-free tissue imaging. In addition to collagen fibers and muscle fiber substructure, THG allowed us to visualize blood vessel walls and erythrocytes as well as white blood cells adhering to vessel walls, residing in or moving through the extravascular tissue. Moreover peripheral nerve fibers could be clearly identified. Structure down to the nuclear chromatin distribution was visualized in 3D and with more detail than obtainable by bright field microscopy. To our knowledge, most of these objects have not been visualized previously by THG or any label-free 3D approach. THG allows label-free microscopy with inherent optical sectioning and therefore may offer similar improvements compared to bright field microscopy as does confocal laser scanning microscopy compared to conventional fluorescence microscopy.     

Diel rhythmicity of lipid-body formation in a coral-Symbiodinium endosymbiosis

The biogenesis of intracellular lipid bodies (LBs) is dependent upon the symbiotic status between host corals and their intracellular dinoflagellates (genus Symbiodinium), though aside from this observation, little is known about LB behavior and function in this globally important endosymbiosis. The present research aimed to understand how LB formation and density are regulated in the gastrodermal tissue layer of the reef-building coral Euphyllia glabrescens. After tissue fixation and labeling with osmium tetroxide, LB distribution and density were quantified by imaging analysis of serial cryo-sections, and a diel rhythmicity was observed; the onset of solar irradiation at sunrise initiated an increase in LB density and size, which peaked at sunset. Both LB density and size then decreased to basal levels at night. On a seasonal timescale, LB density was found to be significantly positively correlated with seasonal irradiation, with highest densities found in the summer and lowest in the fall. In terms of LB lipid composition, only the concentration of wax esters, and not triglycerides or sterols, exhibited diel variability. This suggests that the metabolism and accumulation of lipids in LBs is at least partially light dependent. Ultrastructural examinations revealed that the LB wax ester concentration correlated with the number of electron-transparent inclusion bodies. Finally, there was a directional redistribution of the LB population across the gastroderm over the diel cycle. Collectively, these data reveal that coral gastrodermal LBs vary in composition and intracellular location over diel cycles, features which may shed light on their function within this coral–dinoflagellate mutualism.     

Crystallization and Gelation Behavior of Low- and High Melting Waxes in Rice Bran Oil: a Case-Study on Berry Wax and Sunflower Wax

Low-melting berry wax (BEW) has proven to be a good oil gelator with a positive contribution to the consistency and flexibility of the structured oil. Nevertheless, the properties of BEW and the corresponding oleogel have not yet been investigated in-depth. In this research, the difference in crystallization and gelling behavior between sunflower wax (SW), a high melting wax, and BEW, a low-melting wax, in rice bran oil (RBO) was investigated. The difference in melting and crystallization temperatures can be explained by the different chemical composition (long-chain wax esters in SW and short-chain fatty acids in BEW). The heterogeneity in crystal habits (unidirectional platelets versus microcrystalline particles) and polymorphism (orthorhombic versus hexagonal) are responsible for the varying gel strength and hardness of the respective SW- and BEW-oleogels. The microcrystalline BEW particles aligned and reorganized during 1-month storage at 5 °C, which leaded to an increase in the gel strength and hardness of BEW-oleogel. The gelling property of SW-oleogel however did not significantly differ after 4 weeks at 5 °C, despite of the appearance of spherulitic crystalline clusters. The changes in the physical properties of wax-based oleogels during storage time were further explored using differential scanning calorimetry, polarized light microscope, powder X-ray diffraction and rheology.     

N+离子注入活性污泥辅助处理焦化废水的初步研究

利用离子束对活性污泥进行不同剂量的辐照处理。分析测定了辐照前后活性污泥的废水处理效果、性能参数与污染负荷的变化。结果表明离子束照射剂量在40×2.6×1013N /cm2~200×2.6×1013N /cm2时,污泥浓度、30 min沉降后的污泥沉降比、污泥指数和活性污泥增长率均略低于对照。40×2.6×1013N /cm2以上的辐照剂量即会导致污泥增长率下降,特别当剂量达到200×2.6×1013N /cm2时,污泥量出现负增长(-1.2%和-2.00%)。N 离子注入的单位活性污泥氨氮、化学需氧量去除率均有提高,在辐照剂量40×2.6×1013N /cm2~160×2.6×1013N /cm2时,处理效果随辐照剂量增加有增强趋势。酚的单位活性污泥处理能力明显优于对照,在辐照剂量为160×2.6×1013N /cm2时系统处理率达到99.76%。     

Antenatal ultrasonography to detect fetal renal abnormalities: a prospective screening programme.

OBJECTIVE--To evaluate screening for abnormalities of the fetal renal tract by ultrasonography and to determine the incidence of such abnormalities in a population. DESIGN--A 12 month prospective population study. Follow up of infants to between 9 and 18 months. SETTING--A district general hospital. PARTICIPANTS--6292 Pregnant women reaching 28 weeks'' gestation within the study period. INTERVENTIONS--Antenatal ultrasound scanning was offered to all of the women. Babies in whom an abnormality of the renal tract had been detected antenatally underwent ultrasound scanning at the end of the first week. If the abnormality was confirmed contrast radiography was performed. END POINT--Confirmation of suspected renal abnormality by postnatal investigations. Detection of abnormality in children thought to be normal antenatally. MEASUREMENTS AND MAIN RESULTS--Of the 92 babies who had abnormal antenatal scans, 42 had abnormalities confirmed postnatally. Four of them died and 21 had had or were awaiting an operation at 18 months'' follow up. Seven children had renal abnormalities that were missed antenatally. The incidence of abnormalities detected by screening antenatally was 0.65%, and the overall incidence at 18 months'' follow up was 0.76%. CONCLUSIONS--The incidence of structural renal abnormalities in babies is higher than reported previously. Antenatal ultrasonography is an effective way of detecting such abnormalities.     

Chemical composition of the epicuticular and intracuticular wax layers on adaxial sides of Rosa canina leaves

BACKGROUND AND AIMS: The waxy cuticle is the first point of contact for many herbivorous and pathogenic organisms on rose plants. Previous studies have reported the average composition of the combined wax extract from both sides of rose leaves. Recently, the compositions of the waxes on the adaxial and abaxial surfaces of Rosa canina leaves were determined separately. In this paper, a first report is made on the compositions of the epicuticular and intracuticular wax layers of Rosa canina leaves. The methods described enable the determination of which compounds are truly available at the surface for plant-organism interactions. METHODS: An adhesive was used to mechanically strip the epicuticular wax from the adaxial leaf surface and the removal was visually confirmed using scanning electron microscopy. After the epicuticular wax had been removed, the intracuticular wax was then isolated using standard chemical extraction. Gas chromatography, flame ionization detection and mass spectrometry were used to identify and quantify compounds in the separated wax mixtures. KEY RESULTS: The epicuticular wax contained higher concentrations of alkanes and alkyl esters but lower concentrations of primary alcohols and alkenols when compared to the intracuticular wax. In addition, the average chain lengths of these compound classes were higher in the epicuticular wax. Secondary alcohols were found only in the epicuticular layer while triterpenoids were restricted mainly to the intracuticular wax. CONCLUSIONS: A gradient exists between the composition of the epi- and intracuticular wax layers of Rosa canina leaves. This gradient may result from polarity differences, in part caused by differences in chain lengths. The outer wax layer accessible to the phyllosphere showed a unique composition of wax compounds. The ecological consequences from such a gradient may now be probed.     

Image quality for radiotherapy CT simulators with different scanner bore size

PurposeWe compare image quality parameters derived from phantom images taken on three commercially available radiotherapy CT simulators. To make an unbiased evaluation, we assured images were obtained with the same surface dose measured using XR-QA2 model GafChromic™ film placed at the imaging phantom surface for all three CT-simulators.MethodsRadiotherapy CT simulators GE LS 16, Philips Brilliance Big Bore, and Toshiba Aquilion LB were compared in terms of spatial resolution, low contrast detectability, image uniformity, and contrast to noise ratio using CATPHAN-504 phantom, scanned with Head and Pelvis protocols. Dose was measured at phantom surface, with CT scans repeated until doses on all scanners were within 2%.ResultsIn terms of spatial resolution, the GE simulator appears slightly better, while Philips CT images are superior in terms of SNR for both scanning protocols. The CNR results show that Philips CT images appear to be better, except for high Z material, while Toshiba appears to fit in between the two simulators.ConclusionsWhile the image quality parameters for three RT CT simulators show comparable results, the scanner bore size is of vital importance in various radiotherapy applications. Since the image quality is a function of a large number of confounding parameters, any loss in image quality due to scanner bore size could be compensated by the appropriate choice of scanning parameters, including the exposure and by balancing between the additional imaging dose to the patient and high image quality required in highly conformal RT techniques.