Use Of Video Microscopy Research Articles

Freeze Frame Reflow to Enhance Yields for Advanced Low-Profile Bottom Terminated Packages

Today, designers are demanding an overall form-factor reduction to save board space, increase functionality, and allocate more circuit board real estate toward end-user applications – all with less space allocated to power management where not just the X-Y shrink but the 3D volumetric shrink is required. Unlike most gull wing style leaded packages, QFNs (Quad Flat Pack No lead packages) also known as BTCs (Bottom Terminated Components) have coplanar terminals below the package body to answer this ever-decreasing form-factor requirement. The final standoff in these QFN style packages are dictated by the solder joint geometry formed. Within this advanced package family also finding a similar trend are power WCSPs (Wafer Chip Scale Packages) where traditional solder spheres are being replaced with low profile interconnects in expanded and asymmetrical geometries. This paper presents factors that influence advanced low standoff package solderability performance with the use of video microscopy and live in situ X-ray reflow. To reflect the QFN board assemblies that are typically designed a series of layouts were implemented across different coupon test boards. Assembled coupon test board were designed by varying 1) through hole via design, 2) solder mask tenting techniques, 3) periphery and center thermal pad printed solder joint volumes, 4) no-clean and water-soluble solder paste flux chemistry all using SAC 305 alloy and type 4.5 powder size. One case study using live in-situ X-ray system will explain the effects of a QFN land pattern layout incorporating vias in the center thermal pad with solder mask tenting over the via openings located on the back of the PCB. In another configuration vias were removed from the center thermal pad and vacuum reflow performed during the liquidus phase of the solder with an explanation of why some of the voids remained. The first and second order results presented within this paper have practical implications and from their observations a novel process evaluation technique coined as Freeze Frame Reflow (FFR) was developed. In the last case study presented, an advanced mrQFN (multi-row QFN) package is investigated for factors affecting yields. FFR is then implemented resulting in an increase in yield further demonstrating the benefits this new & novel technique can offer.

Velocity of cytoplasm streaming in basal and subbasal cells of antheridium as well as internodal cells of pleuridium in Chara vulgaris L. and GA3 influence on it: videomicroscopic observations

The velocity of cytoplasm streaming in an antheridial basal cell and in a subbasal cell as well as in internodal cells of pleuridia carrying antheridia were measured with the use of videomicroscopy. Velocity of streaming proved different depending on a cell type. The most intensive streaming (ca 40 µm/s) was observed in a subbasal cell while in a basal cell it was quite intensive during antheridial filament cells proliferation but falling to half of it during spermatozoid differentiation (ca 20 µm/s and 10 µm/s respectively). In internodal cells of pleuridia the velocity was ca 17 µm/s. GA<sub>3</sub> at the 10<sup>-5</sup>M concentration decreased the velocity of streaming in a basal cell during proliferation of antheridial filament cells and increased it during spermiogenesis. In internodal cells of pleuridia the velocity diminished while in a subbasal cell it rose a little after GA<sub>3</sub> administering. The obtained data suggest that cytoplasm streaming and its reaction to exogenous gibberellin depend on the role of a cell in a multicellulate system; it also depends on a developmental stage.

Characterizing the rotation of non symmetric objects in an optical tweezer

We present an optical tweezer based study of the rotation of microscopic objects with shape asymmetry. Thermal fluctuations and rotations are simultaneously monitored through laser back scattering. The rotation causes a modulation in intensity of the back scattered light incident on a quadrant photo detector. The resulting power spectrum is a modified Lorentzian with additional peaks located at the fundamental rotational frequency of the object and at the integer harmonics. The manifestation of these peaks reveals that the rotations are periodic but with varying angular velocity. We model our experimental results to illustrate the hydrodynamic interplay between the rotor and the surrounding medium that results in the time dependence of the angular speed of the former. Further, we demonstrate the use of video microscopy for characterization of low reflectivity rotors, such as biological cells. We propose through these studies that an analysis of these rotations can provide insights into the role of hydrodynamics at micron levels.

Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives from their characteristic apical bundle of stereocilia, a critical element for detection and transduction of sound energy. OHCs are able to change shape -elongate, shorten and bend- in response to electrical, mechanical and chemical stimulation, a motor response considered crucial for cochlear amplification of acoustic signals. OHC stimulation induces two different motile responses: i) electromotility, a.k.a fast motility, changes in length in the microsecond range derived from electrically-driven conformational changes in motor proteins densely packed in OHC plasma membrane, and ii) slow motility, shape changes in the millisecond to seconds range involving cytoskeletal reorganization. OHC bending is associated with electromotility, and result either from an asymmetric distribution of motor proteins in the lateral plasma membrane, or asymmetric electrical stimulation of those motor proteins (e.g., with an electrical field perpendicular to the long axis of the cells). Mechanical and chemical stimuli induce essentially slow motile responses, even though changes in the ionic conditions of the cells and/or their environment can also stimulate the plasma membrane-embedded motor proteins. Since OHC motile responses are an essential component of the cochlear amplifier, the qualitative and quantitative analysis of these motile responses at acoustic frequencies (roughly from 20 Hz to 20 kHz in humans) is a very important matter in the field of hearing research. The development of new imaging technology combining high-speed videocameras, LED-based illumination systems, and sophisticated image analysis software now provides the ability to perform reliable qualitative and quantitative studies of the motile response of isolated OHCs to an external alternating electrical field (EAEF). This is a simple and non-invasive technique that circumvents most of the limitations of previous approaches. Moreover, the LED-based illumination system provides extreme brightness with insignificant thermal effects on the samples and, because of the use of video microscopy, optical resolution is at least 10-fold higher than with conventional light microscopy techniques. For instance, with the experimental setup described here, changes in cell length of about 20 nm can be routinely and reliably detected at frequencies of 10 kHz, and this resolution can be further improved at lower frequencies. We are confident that this experimental approach will help to extend our understanding of the cellular and molecular mechanisms underlying OHC motility.

Measuring short-range repulsive forces by imaging directed magnetic-particle assembly title

We present the use of video microscopy of magnetic colloidal particles to measure short range repulsive forces between surfaces. The forces between the particles can be directly measured by monitoring the inter-centroid distances between the particles at various magnetic field strengths. Using a chain consisting of a minimum of 35 micron-sized particles, a distance resolution of ∼0.21 nm is achieved. Measurements of the interactions between charged colloids agree well with predicted force interactions calculated using DLVO theory. We also report the first measurements of the force versus distance relationship between very short (<75 bases) oligonucleotide-coated surfaces. The interactions between oligonucleotide-coated surfaces agree well with Milner's polymer brush theory for strands greater than 50 bases but deviate for strands less than 35 bases.

Use of video microscopy in teaching medical histology labs

To address constraints of expanding class size but limited space, our institution considered the use of two alternate teaching technologies; computer-based “virtual microscopy” or small-group-centered video microscopy. We felt that small groups of students interacting with each other around a video microscope system would provide for a more cooperative, learner-centered educational environment that would foster the emergence of ‘student-educators’. We purchased two video microscope systems, each utilized by a group of 4 volunteer students. The remaining students were taught by more traditional means, each student having their own standard microscope. The volunteer students utilizing the video microscopes received grades at the class average while the class average on the NBME subject exam was dramatically higher than previous classes. Student feedback has been extremely positive. However, our most promising findings have been more qualitative. Faculty feel this is a more efficient and un-ambiguous method of teaching, since it is easy to point to, and discuss the relationship between, structures on a video monitor. Secondly, students using the video microscopes spend more time in the laboratory, and examine slides more thoroughly. The volunteer students have taken the initiative in collecting images for the class from their slide sets and disseminating this information via a class web site, demonstrating their development into ‘student educators.’ A third advantage is that the video microscopes can be easily attached to video projectors for review sessions with larger groups of students, providing for a qualitatively dynamic educational experience. In conclusion, we find the use of video microscopes has many advantages over traditional methods and plan to convert the entire lab to this system.

Cilia take charge in L–R asymmetry

Left–right asymmetry is determined by the distinct activities of two populations of cilia in the node of the mouse embryo, according to results from James McGrath, Martina Brueckner (Yale Univesity, New Haven, CT), and colleagues.Previous experiments suggested that ciliary movement at the node generates movement of the extracellular fluid surrounding the node, called nodal flow. Nodal flow is critical for establishing L–R asymmetry and requires the motor protein left–right dynein (lrd). But just what kind of signal could be at work in such a situation was unclear. To find out, Brueckner and her team engineered a mouse expressing GFP-tagged lrd under the control of the wild-type lrd promoter. GFP-lrd is expressed in the central cilia in the node, but is absent from a second population of cilia surrounding those expressing lrd. With the use of videomicroscopy, the team finds that the lrd-expressing cilia are motile, whereas many of the cilia in the surrounding population are nonmotile. Figure Calcium is concentrated on the left side of the node (red, green). Both populations, however, express polycystin-2, a cation channel that acts as a mechanosensor in the kidney, where fluid currents trigger an influx of calcium. When the Yale team looked at calcium signaling in the node, they found that only those cilia to the left of the motile population induce a rise in intracellular calcium. This asymmetric distribution is disrupted in lrd and polycystin-2 mutants, suggesting that nodal flow produced by the central motile cilia induce calcium influx only in cells in the direction of flow. “The whole nodal flow hypothesis has been somewhat of a difficult sell, because a major developmental process—formation of one of the three primary body axes—is happening in the extraembryonic space,” says Brueckner. “But our model suggests that L–R development is entirely dependent on the physical force created by nodal flow, and its inherent simplicity makes it really satisfying.” The next step is to determine how asymmetrically expressed genes like nodal are regulated by calcium signaling. ▪ Reference: McGrath, J., et al. 2003. Cell. 114:61–73. [PubMed]

Angiostatin: a negative regulator of endothelial-dependent vasodilation.

Angiostatin is known to inhibit certain aspects of endothelial function, eg, angiogenesis. Here we investigated the effects of angiostatin on another aspect of endothelial function, vasodilation, and examined mechanisms of inhibition--namely, association of heat-shock protein 90 (hsp90) with endothelial nitric oxide synthase (eNOS) and endothelial generation of nitric oxide (*NO) and superoxide anion (O2-). This avenue of investigation was based on recent reports suggesting that hsp90 modulates NOS production of *NO and O2-. Effects of angiostatin on vasodilation were determined in arterioles with the use of videomicroscopy in response to endothelium- and *NO-dependent vasodilators, acetylcholine (ACh) and vascular endothelial growth factor (VEGF), and an endothelium-independent agonist, papaverine. Association of hsp90 with eNOS was determined in rat aortas and bovine aortic endothelial cells (BAECs). Effects of angiostatin on *NO and O2- generation by BAECs were determined by ozone chemiluminescence and superoxide dismutase (SOD)--inhibitable ferricytochrome c reduction, respectively. Angiostatin impaired vasodilation mediated by ACh and VEGF but not papaverine. Pretreating arterioles with polyethylene glycolated--SOD (PEG-SOD) improved vasodilation to ACh and VEGF. Angiostatin decreased the association of hsp90 with eNOS in aortas and BAEC cultures and increased O2- generation in stimulated BAECs by an Lgamma-nitroargininemethylester (L-NAME)--inhibitable mechanism. These data indicate angiostatin alters endothelial function by allowing eNOS to generate O2- on activation. Such changes in enzyme function begin to explain, in part, why angiostatin is antiangiogenic and impairs endothelium-dependent vasodilation.

Microinjection of DNA into the Nuclei of Human Vascular Smooth Muscle Cells

Background. It is challenging to successfully transfect human vascular cells by conventional techniques. We evaluated the efficiency of transfection of human smooth muscle cells (SMC) using a method of direct nuclear microinjection of DNA constructs.Materials and methods. The nuclei of explanted human saphenous vein SMC were microinjected with the plasmid pCMVβ, containing the lacZ gene for β-galactosidase (β-gal). Efficiency of injection and expression were assessed by histochemical staining for β-gal. Injected SMC were subjected to standard assays of viability and migration.Results. Parameters affecting the conditions of injection were systematically analyzed to achieve optimal transfection efficiency. A vertical injection resulted in a twofold increase in expression of β-gal compared to a horizontal approach. A DNA concentration of 100 ng/μl (390 copies/injection) provided a maximal rate of expression. No further increase in expression was evident at higher concentrations. Maximal expression was achieved with a time of injection of 200–500 ms, an injection pressure of 5–10 psi, and a pipette tip size of 0.6 μm, resulting in an injection volume of 0.03 pl. Cytoplasmic injection did not result in gene expression. The ability of SMC to migrate under videomicroscopy was not altered by the injection process. Optimizing all injection parameters resulted in cell viability >95% and efficiency of injection of 59%.Conclusion. DNA encoding a variety of intracellular proteins can be efficiently microinjected into human vascular SMC. Coupled with the use of videomicroscopy, this technique can allow for the evaluation of genes that might modulate important cellular processes such as proliferation and migration.

Making sense of melanosome dynamics in mouse melanocytes.

Molecular motors drive most if not all organelle movements in Eukaryotic cells. These proteins are thought to bind to the organelle surface and, through the action of their mechanochemical domains, to translocate the organelle along a cytoskeletal track. In the case of the myosin family of molecular motors, the cytoskeletal track is filamentous actin. Microtubules serve as the cytoskeletal track for the kinesins and dyneins. While a considerable amount is known about the motors and tracks responsible for the bi-directional movement of pigment granules in fish and frog melanophores, relatively little is known about how melanosomes in mammalian melanocytes are transported out the cells dendritic arbor, accumulated at the ends of these dendrites, and transferred to keratinocytes. In this short review, we focus on the use of video microscopy to address these questions in mouse melanocytes, and we describe how an analysis of melanosome dynamics within wild type and dilute melanocytes shaped our thinking regarding the role of an unconventional myosin in melanosome transport and distribution.

Selective membrane test for crude oil based model emulsions by use of video-enhanced microscopy

A method for testing water/oil emulsion droplet membranes selectively has been demonstrated. The method uses electric fields to induce attraction, membrane thinning and coalescence between aqueous droplets deposited in an oil continuum. The coalescence process is monitored visually by the use of videomicroscopy. A set of model oils containing indigenous surfactants (asphaltenes) from a crude oil has been studied, and the effects of asphaltene concentration, oil phase aromaticity, aging of oils and interfacial exposure time have been investigated. The strength of the field at the point of coalescence is defined as the critical parameter describing membrane strength. In the current experiments a.c. fields were used and droplet sizes were of the order of 500–600 μm.

Functional analysis by imaging of melanophore pigment dispersion of chimeric receptors constructed by recombinant polymerase chain reaction

Analysis of functional aspects of the molecular structure of proteins often requires a means to selectively alter structure and subsequently analyze function. We have adapted a method of overlap extension polymerase chain reaction (PCR) to generate multiple domain replacements in G-protein coupled receptors. The examples described herein are β 2-adrenergic receptors whose G-protein coupling domains have been replaced by homologous domains of olfactory receptors, but the procedure has also been used to produce constructs with mutations, deletions, and fusions of two complete open reading frames. The chimeric olfactory-adrenergic receptors were assayed by functional expression in clonal lines of Xenopus melanophores. The ability of G-protein coupled second messenger pathways to cause translocation of pigment organelles within melanophores allows the use of video microscopy to assay the function of the chimeric receptors. Digital automation of microscope stage, camera, and image processing allows multiple parallel experiments to be performed. Melanophores allow responses mediated by the Gs, Gq and Gi pathways to be assayed with equal efficiency and the specificity of the coupling between chimera (or receptor) and G-protein subtypes can be rapidly determined.

Use of video microscopy to study the mechanisms of cytotoxicity in vitro in individual living cells

Recent technological innovations have made it possible to determine simultaneously multiple biochemical parameters in individual living cells. This new technique, commonly designated as video microscopy, makes use of fluorescent probes, specific for a certain cellular parameter. Cells are loaded with the probes and placed under a fluorescence microscope. After excitation at a specific wavelength the probes emit light of a longer wavelength. The intensity of the emitted light is proportional to the magnitude of the specific parameter. Emission is recorded with a video camera and the signal transferred to an image processor for extraction of quantitative data from the image, or to improve image quality. An overview of the technique itself and the necessary hardware, the major types of calculations and how to handle cells and probes is presented in this paper.

Ketanserin accelerates wound epithelialization and neovascularization

We investigated the acute effects of topical ketanserin, a 5-HT(2) (serotonin) receptor blocker, on wound epithelialization and vascularization with the use of the hairless mouse ear model. Varying concentrations of Ketanserin (0%, 0.2%, 2.0%, 20% weight/volume) were administered to standardized full-thickness skin wounds on the dorsum of the hairless mouse ear immediately after surgery and daily thereafter. With the use of video microscopy and computer-assisted planimetry, vascularization and epithelialization were traced every third day until the wounds were fully healed. Arteriole diameters at selected sites near the skin wound were measured before wound creation and after wounding. It was concluded that topically administered ketanserin significantly accelerates both the vascular (p < 0.001 at 2% and 20% concentrations) and epithelial (p < 0.001 at 20% concentration) rates of wound healing in full-thickness nonpathologic skin wounds. Vasodilation of terminal arterioles was not a major response to Ketanserin. Faster epithelialization was possibly due to direct effect of ketanserin on epithelial cells.

The hairless mouse ear: an in vivo model for studying wound neovascularization

Microvascular ingrowth into damaged tissue is an essential component of the normal healing process. In fact, wound therapy is often aimed at promoting neovascularization. However, little is known about the mechanisms that regulate microvascular ingrowth into a healing wound. This limited knowledge is largely due to the lack of adequate models in which microvascular ingrowth can be quantitatively analyzed throughout the healing process. To address this deficiency, we developed a model in which a wound was created on the ear of the hairless mouse-a well established model for directly viewing and measuring skin microcirculation. While the animals were under ketamine and xylazine anesthesia, 2.25 mm diameter full-thickness wounds were created on the dorsum of hairless mouse ears down to but not including the cartilage (0.125 mm depth). With the use of video microscopy and computer-assisted digitized planimetry, the precise epithelial and neovascular wound edge was viewed and measured regularly throughout healing. Therefore, this model can provide objective data on wound epithelialization and neovascularization throughout healing. This model was used to examine the effect of topical wound agents on epithelialization and neovascularization. Differential effects by these anti-microbial agents on these two processes were observed, which suggests clinical implications for their use.

Some further observations on the various features of aluminium alloy microstructures that are transferred, bodily, into the oxide coating formed by anodising

When anodised in a sulphuric acid electrolyte, many of the microstructural features of aluminium alloys are transferred into the oxide coating so formed. Even with thick coatings these features can be seen, in depth, with the optical microscope, although certain procedures may be required for optimum clarity. Depending on their oxidation characteristics, some micro-constituents appeared in the oxide unchanged, others were convened to a transparent form, and some disappeared entirely to leave cavities. In most cases, particularly where the features within the oxide had characteristic shapes, they could be related back to the original constituent type as seen in the alloy itself. This identification was aided by the three-dimensional viewing capability that gave a direct appreciation of particle morphology and distribution. Magnesium silicide (Mg 2Si), when anodised, was converted to a transparent substance that retained its original rounded form. It could be specifically stained, notably with the aniline dye Malachite Green. This provided a useful marker for that constituent's three-dimensional distribution within any microstructure, as none of the other particle species were stained by this reagent. Contrast enhancement by the use of video microscopy proved invaluable, particularly for viewing fine precipitates in suspension within the depths of the oxide.

Differential effect of cyclooxygenase inhibitors on absorptive hyperemia.

To test whether the hydrolytic products of digestion could stimulate vasoactive prostaglandin synthesis in the intestine, the muscularis and mucosa of the rat jejunum were suffused with a bicarbonate-buffered Ringer vehicle containing cyclooxygenase inhibitors (meclofenamate or indomethacin; 3 X 10(-5) M). To evoke blood flow changes, either glucose (56 mM), oleic acid (20 or 40 mM), or sodium arachidonate was added to the mucosal vehicle. Bile salt (taurocholic acid, 10 mM) was added to emulsify oleic acid. Blood flow was calculated (BFc) in submucosal arterioles by use of video microscopy. Neither bile salt nor cyclooxygenase inhibitors altered resting BFc. Neither oleic acid concentration nor solution osmolality altered the magnitude of absorptive hyperemia. After 10 min of glucose, BFc increased 36 +/- 6% with vehicle (n = 16) and 39 +/- 8% with meclofenamate (n = 10). After 10 min of oleic acid, BFc increased 21 +/- 5% with vehicle (n = 17) and 34 +/- 6% with inhibitors (n = 17). In animals exposed twice to the same concentration of oleic acid, the paired difference between the vehicle and inhibitors (19 +/- 7%; n = 9) was significant. Arachidonate alone produced no dose-related (0.06-1.9 mM) effect on BFc (n = 41), but arachidonate (0.3 mM) combined with cyclooxygenase inhibitors (6 X 10(-5) M) produced a significant BFc increase of 35 +/- 7% (n = 6). These observations suggest that the absorption of oleic acid, but not glucose, stimulates the synthesis of a vasoactive metabolite of arachidonate. The mechanism for the differential effect of cyclooxygenase inhibitors is unknown but could involve nonprostaglandin metabolites of arachidonate, such as lipoxygenase or cytochrome P-450 products.

Lidocaine constricts or dilates rat arterioles in a dose-dependent manner.

The microvascular effects of varying concentrations of lidocaine were evaluated with the use of videomicroscopy in an in vivo rat cremaster muscle preparation. Animals were anesthetized with chloralose and urethane and breathed room air spontaneously. Mean arterial pressure and heart rate were measured via a carotid artery cannula. The cremaster muscle was suffused with a balanced electrolyte solution and pH, temperature, PO2, PCO2, and osmolarity were controlled. Internal diameters of fourth-order arterioles in the cremaster muscle were measured with an electronic vernier system. In one group of animals (n = 7), arteriolar diameters were measured every 30 s during a 10-min control period, a 10-min period of topical application of lidocaine hydrochloride, and a 10-min recovery period. Lidocaine hydrochloride, 10(0), 10(1), 10(2), 10(3), or 10(4) micrograms X ml-1, produced changes in arteriolar diameters to 88.9 +/- 0.9, 79.0 +/- 1.3, 67.5 +/- 2.4, 60.1 +/- 3.4, and 127.1 +/- 7.2 per cent of control, respectively (P less than 0.001). In a second group of animals (n = 4), fourth-order arteriolar diameters were measured during administration of intravenous lidocaine, 1.2 mg X kg-1 bolus plus 0.3 mg X kg-1 X min-1. Vasoconstriction to 91.3 +/- 0.9% of control was observed (P less than 0.001). These results demonstrate a biphasic dose-dependent response to lidocaine. At lesser concentrations, including those that occur in the plasma of patients during intravenous infusion or nerve blocks, dose-related vasoconstriction occurred. Lidocaine, 10(4) micrograms X ml-1, a concentration similar to that which occurs at the site of injection during infiltration, nerve block, or epidural anesthesia, produced vasodilation.(ABSTRACT TRUNCATED AT 250 WORDS)