Detection and Sizing of Submicron Particles in Biologics With Interferometric Scattering Microscopy.

We demonstrate the application of interferometric scattering microscopy (IFS) for characterizing submicron particles in stir-stressed monoclonal antibody. IFS uses a layered silicon sensor and modified optical microscope to rapidly visualize and determine the particle size distribution (PSD) of submicron particles based on their scattering intensity, which is directly proportional to particle mass. Limits for particle size and optimal solution concentration were established for IFS characterization of submicron particles. We critically compare IFS data with dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) and find IFS is superior to NTA and DLS for determining the realistic shape of the number-based PSD, whereas NTA and DLS provide superior information about absolute particle size. Together, IFS, NTA, and DLS provide complementary information on submicron particles and enable quantitative characterization of the PSD of submicron aggregates. Finally, we explore quantifying particle size with IFS by developing a calibration curve for particle scattering intensity based on correlative scanning electron microscopy imaging. We found that only a subset of isotropic-shaped particles followed the expected proportionality between IFS intensity and particle mass. Overall, this study demonstrates IFS is a simple approach for detecting and quantifying submicron aggregate PSD in protein-based therapeutics.

Assessment of non-contacting optical methods to measure wear and surface roughness in ceramic total disc replacements.

This study presents a method for measuring the low volumetric wear expected in ceramic total disc replacements, which can be used to replace intervertebral discs in the spine, using non-contacting optical methods. Alumina-on-alumina ball-on-disc tests were conducted with test conditions approximating those of cervical (neck region of the spine) total disc replacement wear tests. The samples were then scanned using a three-dimensional non-contacting optical profilometer and the data used to measure surface roughness and develop a method for measuring the wear volume. The results showed that the magnification of the optical lens affected the accuracy of both the surface roughness and wear volume measurements. The method was able to successfully measure wear volumes of 0.0001 mm(3), which corresponds to a mass of 0.0001 mg, which would have been undetectable using the gravimetric method. A further advantage of this method is that with one scan the user can measure changes in surface topography, volumetric wear and the location of the wear on the implant surface. This method could also be applied to more severe wear, other types of orthopaedic implants and different materials.

Auxin Biosynthesis, Accumulation, Action and Transport are Involved in Stress-Induced Microspore Embryogenesis Initiation and Progression in Brassica napus.

Isolated microspores are reprogrammed in vitro by stress, becoming totipotent cells and producing embryos and plants via a process known as microspore embryogenesis. Despite the abundance of data on auxin involvement in plant development and embryogenesis, no data are available regarding the dynamics of auxin concentration, cellular localization and the expression of biosynthesis genes during microspore embryogenesis. This work involved the analysis of auxin concentration and cellular accumulation; expression of TAA1 and NIT2 encoding enzymes of two auxin biosynthetic pathways; expression of the PIN1-like efflux carrier; and the effects of inhibition of auxin transport and action by N-1-naphthylphthalamic acid (NPA) and α-(p-chlorophenoxy) isobutyric acid (PCIB) during Brassica napus microspore embryogenesis. The results indicated de novo auxin synthesis after stress-induced microspore reprogramming and embryogenesis initiation, accompanying the first cell divisions. The progressive increase of auxin concentration during progression of embryogenesis correlated with the expression patterns of TAA1 and NIT2 genes of auxin biosynthetic pathways. Auxin was evenly distributed in early embryos, whereas in heart/torpedo embryos auxin was accumulated in apical and basal embryo regions. Auxin efflux carrier PIN1-like gene expression was induced in early multicellular embryos and increased at the globular/torpedo embryo stages. Inhibition of polar auxin transport (PAT) and action, by NPA and PCIB, impaired embryo development, indicating that PAT and auxin action are required for microspore embryo progression. NPA also modified auxin embryo accumulation patterns. These findings indicate that endogenous auxin biosynthesis, action and polar transport are required in stress-induced microspore reprogramming, embryogenesis initiation and progression.

High-contrast fluorescence imaging based on the polarization dependence of the fluorescence enhancement using an optical interference mirror slide.

High-contrast fluorescence imaging using an optical interference mirror (OIM) slide that enhances the fluorescence from a fluorophore located on top of the OIM surface is reported. To enhance the fluorescence and reduce the background light of the OIM, transverse-electric-polarized excitation light was used as incident light, and the transverse-magnetic-polarized fluorescence signal was detected. As a result, an approximate 100-fold improvement in the signal-to-noise ratio was achieved through a 13-fold enhancement of the fluorescence signal and an 8-fold reduction of the background light.

Evaluation of the response to treatment of psoriasis vulgaris with reflectance confocal microscopy.

BACKGROUND/OBJECTIVE: Reflectance confocal microscopy (RCM) is a noninvasive, objective imaging technique that provides in vivo, high-resolution skin imaging. We sought to assess epidermal and dermal changes associated with the psoriasis and its treatment with RCM before the treatment and at weeks 4 and 8 of the treatment. MATERIAL AND METHODS: This is an investigator-blinded, internal-controlled, follow-up study. A total of 25 patients with plaque psoriasis were included in the study. The RCM evaluation criteria were defined on the basis of the histopathological diagnostic criteria for psoriasis. The clinical severity of the psoriasis was evaluated using the Psoriasis Area Severity Index (PASI). RESULTS: The RCM findings which were correlated with the PASI can be used to follow up the patient's response to treatment have been identified as follows: the acanthosis, the number of spongiotic sites, the number of regular/irregular honeycomb-like sites, the number of epidermal inflammatory cells, the number of focal microabscesses, the total epidermal thickness, the number of nonedge dermal papillae, the length of the papillary dermis, the number of dermal inflammatory cells, and the vascularization in the papillary dermis (P < 0.05). CONCLUSION: This is the first study with a large group of patients to perform a noninvasive assessment with RCM of the response of psoriasis to different treatments: phototherapy, systemic and topical treatment. Micrometric and morphometric changes occurring in the psoriatic tissue during the 8-week treatment period were identified by in vivo RCM in a noninvasive manner. RCM is capable of monitoring of treatment response in psoriasis.

Transcriptional consequence and impaired gametogenesis with high-grade aneuploidy in Arabidopsis thaliana.

Aneuploidy features a numerical chromosome variant that the number of chromosomes in the nucleus of a cell is not an exact multiple of the haploid number, which may have an impact on morphology and gene expression. Here we report a tertiary trisomy uncovered by characterizing a T-DNA insertion mutant (aur2-1/+) in the Arabidopsis (Arabidopsis thaliana) AURORA2 locus. Whole-genome analysis with DNA tiling arrays revealed a chromosomal translocation linked to the aur2-1 allele, which collectively accounted for a tertiary trisomy 2. Morphologic, cytogenetic and genetic analyses of aur2-1 progeny showed impaired male and female gametogenesis to various degrees and a tight association of the aur2-1 allele with the tertiary trisomy that was preferentially inherited. Transcriptome analysis showed overlapping and distinct gene expression profiles between primary and tertiary trisomy 2 plants, particularly genes involved in response to stress and various types of external and internal stimuli. Additionally, transcriptome and gene ontology analyses revealed an overrepresentation of nuclear-encoded organelle-related genes functionally involved in plastids, mitochondria and peroxisomes that were differentially expressed in at least three if not all Arabidopsis trisomics. These observations support a previous hypothesis that aneuploid cells have higher energy requirement to overcome the detrimental effects of an unbalanced genome. Moreover, our findings extend the knowledge of the complex nature of the T-DNA insertion event influencing plant genomic integrity by creating high-grade trisomy. Finally, gene expression profiling results provide useful information for future research to compare primary and tertiary trisomics for the effects of aneuploidy on plant cell physiology.

The Arabidopsis thaliana GRF-INTERACTING FACTOR gene family plays an essential role in control of male and female reproductive development.

Reproductive success of angiosperms relies on the precise development of the gynoecium and the anther, because their primary function is to bear and to nurture the embryo sac/female gametophyte and pollen, in which the egg and sperm cells, respectively, are generated. It has been known that the GRF-INTERACTING FACTOR (GIF) transcription co-activator family of Arabidopsis thaliana (Arabidopsis) consists of three members and acts as a positive regulator of cell proliferation. Here, we demonstrate that GIF proteins also play an essential role in development of reproductive organs and generation of the gamete cells. The gif1 gif2 gif3 triple mutant, but not the single or double mutants, failed to establish normal carpel margin meristem (CMM) and its derivative tissues, such as the ovule and the septum, resulting in a split gynoecium and no observable embryo sac. The gif triple mutant also displayed severe structural and functional defects in the anther, producing neither microsporangium nor pollen grains. Therefore, we propose that the GIF family of Arabidopsis is a novel and essential component required for the cell specification maintenance during reproductive organ development and, ultimately, for the reproductive competence.

A mouse model for human deafness DFNB22 reveals that hearing impairment is due to a loss of inner hair cell stimulation.

The gene causative for the human nonsyndromic recessive form of deafness DFNB22 encodes otoancorin, a 120-kDa inner ear-specific protein that is expressed on the surface of the spiral limbus in the cochlea. Gene targeting in ES cells was used to create an EGFP knock-in, otoancorin KO (Otoa(EGFP/EGFP)) mouse. In the Otoa(EGFP/EGFP) mouse, the tectorial membrane (TM), a ribbon-like strip of ECM that is normally anchored by one edge to the spiral limbus and lies over the organ of Corti, retains its general form, and remains in close proximity to the organ of Corti, but is detached from the limbal surface. Measurements of cochlear microphonic potentials, distortion product otoacoustic emissions, and basilar membrane motion indicate that the TM remains functionally attached to the electromotile, sensorimotor outer hair cells of the organ of Corti, and that the amplification and frequency tuning of the basilar membrane responses to sounds are almost normal. The compound action potential masker tuning curves, a measure of the tuning of the sensory inner hair cells, are also sharply tuned, but the thresholds of the compound action potentials, a measure of inner hair cell sensitivity, are significantly elevated. These results indicate that the hearing loss in patients with Otoa mutations is caused by a defect in inner hair cell stimulation, and reveal the limbal attachment of the TM plays a critical role in this process.

Optimization of Entamoeba histolytica culturing in vitro.

Entamoeba histolytica is among the most deadly parasites accounting for the second highest mortality rate among parasitic diseases. Nevertheless, contrary to trypanosomatids, this protozoan in hardly studied by parasitology groups. This astonishing discrepancy is largely due to the remarkable intricate conditions required for parasite proliferation in vitro, particularly whenever large cell numbers are required. The present study was undertaken in order to optimize E. histolytica culturing harvest, using mineral oil layers preventing culture medium-air contact to maintain anaerobic conditions in culture plate wells. 2×10(4) trophozoites were plated on each well in 2.0 mL YI-S-33 medium, supplemented with bovine serum and 700 µL mineral oil. Parasites were daily quantified by light microscopy counting for up to 96 h and trophozoite motility was also assessed. We notice that E. histolytica cultures in 24-well plates reached several-fold higher cell densities, particularly whenever the mineral oil layer was placed on top of the medium surface, blocking the air interface. At least 99% of the parasites were vigorously motile for 72 h in oil-containing wells, whereas only less than 5% displayed significant motility in oil-devoid wells. In order to determine whether such different growth responses were due at least in part to the oxidative stress, we used the reactive oxidant species fluorescent probe dihydroethidium (DHE). The remarkably higher DHE parasite labeling in oil-devoid cultures indicate that oxidative stress reduction can play a significant role in elevated growth rates observed in oil supplemented cultures. Propidium iodide and Trypan blue dye-exclusion assays indicate that parasite necrosis resulted from the stressing conditions. The present study indicates that E. histolytica culturing in oil-sealed wells may comprise a valuable tool for bioactivity of antiparasitic compounds.

Cell's intrinsic biophysical properties play a role in the systematic decrease in time-locking ability of central auditory neurons.

Studies in the vertebrates have shown that the time-locking ability of central auditory neurons decreases progressively along the ascending auditory pathway. This decrease is presumably attributed to a progressive reduction in the fidelity of synaptic transmission and an increase in the influence of synaptic inhibition along the cascade. The extent to which neurons' intrinsic biophysical properties contribute to the change in time-locking ability is unclear. We carried out whole-cell patch clamp recordings from the auditory thalamus of leopard frogs and compared their biophysical properties and time-locking abilities (determined by cell's responses to depolarizing pulse trains applied intracellularly) with those of lower auditory brainstem neurons. We found that frog thalamic neurons were homogeneous, exhibiting uniformly sustained, regular firing patterns, but not having low-threshold transient Ca2+ current which mammal thalamic neurons generally possess. Furthermore, intrinsic biophysical properties of the thalamic neurons are such that the time-locking ability of these neurons was very poor. The homogeneity of thalamic auditory neurons is in contrast to the heterogeneity of lower auditory brainstem neurons, with different phenotypes exhibiting different time-locking abilities and with sustained-regular phenotype consistently showing the worst time-locking ability among all biophysical phenotypes. Auditory nuclei along the ascending auditory pathway showed a progressive increase in the population of sustained-regular phenotype-this corresponded to a systematic decrease in the overall time-locking ability, with neurons in the dorsal medullary nucleus showing the best, and thalamic neurons exhibiting the poorest time-locking ability, whereas neurons in the torus semicircularis displayed intermediate time-locking ability. These results suggest that the biophysical characteristics of single neurons also likely play a role in the change in temporal coding ability along the ascending auditory pathway.

High-resolution full-field optical coherence microscopy using a Mirau interferometer for the quantitative imaging of biological cells.

In this paper quantitative imaging of biological cells using high-resolution full-field optical coherence microscopy (FF-OCM) is reported. The FF-OCM was realized using a swept-source system, a Mirau interferometer, and a CCD camera (a two-dimensional detection unit). A Mirau-interferometric objective lens was used to generate the interferometric signal. The signal was analyzed by a Fourier analysis technique. Optically sectioned amplitude images and a quantitative phase map of biological cells such as onion skin and red blood cells (RBCs) are demonstrated. Further, the refractive index profile of the RBCs is also presented. For the 50× Mirau objective, the experimentally achieved axial and transverse resolution of the present system are 3.8 and 1.2 µm, respectively. The CCD provides parallel detection and measures enface images without X, Y, Z mechanical scanning.

Quantitative cell imaging using single beam phase retrieval method.

Quantitative three-dimensional imaging of cells can provide important information about their morphology as well as their dynamics, which will be useful in studying their behavior under various conditions. There are several microscopic techniques to image unstained, semi-transparent specimens, by converting the phase information into intensity information. But most of the quantitative phase contrast imaging techniques is realized either by using interference of the object wavefront with a known reference beam or using phase shifting interferometry. A two-beam interferometric method is challenging to implement especially with low coherent sources and it also requires a fine adjustment of beams to achieve high contrast fringes. In this letter, the development of a single beam phase retrieval microscopy technique for quantitative phase contrast imaging of cells using multiple intensity samplings of a volume speckle field in the axial direction is described. Single beam illumination with multiple intensity samplings provides fast convergence and a unique solution of the object wavefront. Three-dimensional thickness profiles of different cells such as red blood cells and onion skin cells were reconstructed using this technique with an axial resolution of the order of several nanometers.

Intracellular signaling cascades induced by relaxin in the stimulation of capacitation and acrosome reaction in fresh and frozen-thawed bovine spermatozoa.

Relaxin is one of the 6-kDa peptide hormones, which acts as a pleiotropic endocrine and paracrine factor. Our previous studies revealed that sperm capacitating medium containing relaxin induced capacitation and acrosome reaction (AR) in fresh and frozen-thawed porcine or bovine spermatozoa. However, the intracellular signaling cascades involved with capacitation or AR induced by relaxin was unknown. Therefore, the present study was designed to investigate the intracellular signaling cascades involved with capacitation and AR induced by relaxin in fresh and frozen-thawed bovine spermatozoa. Spermatozoa were incubated in sperm Tyrode's albumin lactate pyruvate (Sp-TALP) medium supplemented with (40 ng ml(-1)) or without relaxin, and subjected to evaluation of chlortetracycline staining pattern, cholesterol efflux, Ca(2+)-influx, intracellular cyclic adenosine monophosphate (cAMP) and protein tyrosine phosphorylation. Capacitation and AR were increased (P<0.05) in both fresh and frozen-thawed spermatozoa incubated with relaxin. Cholesterol effluxes were greater in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa incubated with relaxin than the spermatozoa incubated without relaxin. Ca(2+)-influxes were also significantly stimulated by relaxin in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa. The Sp-TALP medium containing relaxin influenced the generation of intracellular cAMP in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa, and exhibited higher exposure of protein tyrosine phosphorylation in both sperm types than the medium devoid of relaxin. Therefore, the results postulate that relaxin exerts the intracellular signaling cascades involved with capacitation and AR through accelerating the cholesterol efflux, Ca(2+)-influx, intracellular cAMP and protein tyrosine phosphorylation in fresh and frozen-thawed bovine spermatozoa.

A digital heterodyne laser interferometer for studying cochlear mechanics.

Laser interferometry is the technique of choice for studying the smallest displacements of the hearing organ. For low intensity sound stimulation, these displacements may be below 1 nm. This cannot be reliably measured with other presently available techniques in an intact organ of Corti. In a heterodyne interferometer, light is projected against an object of study and motion of the target along the optical axis causes phase and frequency modulations of the back-reflected light. To recover object motion, the reflected light is made to interfere with a reference beam of artificially altered frequency, producing a beating signal. In conventional interferometers, this carrier signal is demodulated with analog electronics. In this paper, we describe a digital implementation of the technique, using direct carrier sampling. In order to obtain the necessary reference signal for demodulation we introduce an additional third light path. Together, this results in lower noise and reduces the cost of the system. Within the hearing organ, different structures may move in different directions. It is therefore necessary to precisely measure the angle of incidence of the laser light, and to precisely localize the anatomical structure where the measurement is performed. Therefore, the interferometer is integrated with a laser scanning confocal microscope that permits us to map crucial morphometric parameters in each experiment. We provide key construction parameters and a detailed performance characterization. We also show that the system accurately measures the diminutive vibrations present in the apical turn of the cochlea during low-level sound stimulation.

Motion-enhanced, differential interference contrast (MEDIC) microscopy of moving vesicles in live cells: VE-DIC updated.

Video-enhanced differential interference contrast microscopy with background subtraction has made visible many structures and processes in living cells. In video-enhanced differential interference contrast, the background image is stored manually by defocusing the microscope before images are acquired. We have updated and improved video-enhanced differential interference contrast by adding automatic generation of the background image as a rolling average of the incoming image stream. Subtraction of this continuously updated 12-bit background image from the incoming 12-bit image stream provides a flat background which allows the contrast of moving objects, such as vesicles, to be strongly enhanced while suppressing stationary features such as the overall cell shape. We call our method MEDIC, for motion-enhanced differential interference contrast. By carrying out background subtraction with 12-bit images, the number of grey levels in the moving vesicles can be maximized and a single look-up table can be applied to the entire image, enhancing the contrast of all vesicles simultaneously. Contrast is increased by as much as a factor of 13. The method is illustrated with raw, background and motion-enhanced differential interference contrast images of moving vesicles within a neurite of a live PC12 cell and a live chick motorneuron.

Biocompatibility of atomic layer-deposited alumina thin films.

Presented in this paper is a study of the biocompatibility of an atomic layer-deposited (ALD) alumina (Al2O3) thin film and an ALD hydrophobic coating on standard glass cover slips. The pure ALD alumina coating exhibited a water contact angle of 55 degrees +/- 5 degrees attributed, in part, to a high concentration of -OH groups on the surface. In contrast, the hydrophobic coating (tridecafluoro-1,1,2,2-tetrahydro-octyl-methyl-bis(dimethylamino)silane) had a water contact angle of 108 degrees +/- 2 degrees. Observations using differential interference contrast microscopy on human coronary artery smooth muscle cells showed normal cell proliferation on both the ALD alumina and hydrophobic coatings when compared to cells grown on control substrates. These observations suggested good biocompatibility over a period of 7 days in vitro. Using a colorimetric assay technique to assess cell viability, the cellular response between the three substrates can be differentiated to show that the ALD alumina coating is more biocompatible and that the hydrophobic coating is less biocompatible when compared to the control. These results suggest that patterning a substrate with hydrophilic and hydrophobic groups can control cell growth. This patterning can further enhance the known advantages of ALD alumina, such as conformality and excellent dielectric properties for bio-micro electro mechanical systems (Bio-MEMS) in sensors, actuators, and microfluidics devices.

High-resolution line-scanning optical coherence microscopy.

An optical coherence microscopy system based on line illumination and detection is demonstrated. The system uses a Linnik-type interferometer illuminated by a broadband Ti:sapphire laser and detected by a high-speed, line-scan CCD camera. This approach is less sensitive to incoherent scattering and sample motion than full-field imaging. Spatial resolutions of approximately 2 microm x approximately 3 microm(transverse x axial) are achieved. The sensitivity of the system is 93 dB with averaging over 30 line scans. En face real time, cellular-level imaging of biological tissues is demonstrated at approximately 2 frames/s.

Biomolecular interaction analysis under electrophoretic flow conditions.

Combining the advantages of electrophoresis with the advantages of biomolecular interaction analysis (BIA) enables the biospecific detection of separated molecules; for example it permits differentiation between a complementary single-stranded DNA and a single nucleotide polymorphism. In order to integrate these two techniques, it is necessary to investigate whether it is possible to detect a biomolecular interaction under electrophoretic flow conditions. To this end a novel detection system was developed for electrophoresis that utilizes a label-free and time-resolved detection technique: reflectometric interference spectroscopy (RIfS). The biological functions of important analytes were investigated using this system. Although RIfS can be used as a postcolumn detector, it is also possible to use it to detect relevant substances under electrophoretic flow conditions. DNA-LNA, biotin-streptavidin and protein-protein interactions were detected using this coupled electrophoresis-RIfS set-up.

Phagocytosis, endosomal/lysosomal system and other cellularaspects of macrophage activation by Canova medication.

Canova is a homeopathic medication with immunomodulatory properties, recommended for diseases where the immune system is depressed. Our research aims to study the activation of mice peritoneal macrophages when submitted to in vivo and in vitro Canova treatment. Morphological parameters and acid phosphatase activity were analyzed using light and transmission electron microscopy. Differential interference contrast microscopy, including serial time acquisition in living cells, was also performed. The results demonstrated a greater spreading ability in Canova treated macrophages, a higher phagocytic activity of non-infective microorganisms (Saccharomyces cerevisiae and Tripanosoma cruzi epimastigotes) and a tendency to lower the phagocytic activity of the infective microorganisms T. cruzi trypomastigotes and Leishmania amazonensis, when compared with control cells. Acid phosphatase activity was analyzed and showed that Canova treatment stimulates an increase of the endosomal/lysosomal system. Treated macrophages that do or do not interact with yeast present a higher number of acid phosphatase marked vesicles compared to control cells. In contrast, the activity of tartrate resistant acid phosphatase (TRAP), is lower in Canova treated macrophages. The net results demonstrate that Canova medication is an effective stimulator of macrophage activity.

Coherent anti-stokes Raman scattering imaging of axonal myelin in live spinal tissues.

We present a vibrational imaging study of axonal myelin under physiological conditions by laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy. We use spinal cord white matter strips that are isolated from guinea pigs and kept alive in oxygen bubbled Krebs' solution. Both forward- and epi-detected CARS are used to probe the parallel axons in the spinal tissue with a high vibrational contrast. With the CARS signal from CH2 vibration, we have measured the ordering degree and the spectral profile of myelin lipids. Via comparison with the ordering degrees of lipids in myelin figures formed of controlled lipid composition, we show that the majority of the myelin membrane is in the liquid ordered phase. By measuring the myelin thickness and axon diameter, the value of g ratio is determined to be 0.68 with forward- and 0.63 with epi-detected CARS. Detailed structures of the node of Ranvier and Schmidt-Lanterman incisure are resolved. We have also visualized the ordering of water molecules between adjacent bilayers inside the myelin. Our observations provide new insights into myelin organization, complementary to the knowledge from light and electron microscopy studies of fixed and dehydrated tissues. In addition, we have demonstrated simultaneous CARS imaging of myelin and two-photon excitation fluorescence imaging of intra- and extraaxonal Ca2+. The current work opens up a new approach to the study of spinal cord injury and demyelinating diseases.