Instantaneous 4D micro-particle image velocimetry (µPIV) via multifocal microscopy (MUM).

Multifocal microscopy (MUM), a technique to capture multiple fields of view (FOVs) from distinct axial planes simultaneously and on one camera, was used to perform micro-particle image velocimetry (µPIV) to reconstruct velocity and shear stress fields imposed by a liquid flowing around a cell. A diffraction based multifocal relay was used to capture images from three different planes with 630 nm axial spacing from which the axial positions of the flow-tracing particles were calculated using the image sharpness metric. It was shown that MUM can achieve an accuracy on the calculated velocity of around (0.52 ± 0.19) µm/s. Using fixed cells, MUM imaged the flow perturbations at sub-cellular level, which showed characteristics similar to those observed in the literature. Using live cells as an exemplar, MUM observed the effect of changing cell morphology on the local flow during perfusion. Compared to standard confocal laser scanning microscope, MUM offers a clear advantage in acquisition speed for µPIV (over 300 times faster). This is an important characteristic for rapidly evolving biological systems where there is the necessity to monitor in real time entire volumes to correlate the sample responses to the external forces.

The cell wall regulates dynamics and size of plasma-membrane nanodomains in Arabidopsis.

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport, and cellular signaling. For these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signaling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using subdiffraction-limited Airyscan confocal microscopy. Single particle tracking reveals that these proteins move at different rates within the membrane. Actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics, thereby contributing to the formation and functionality of membrane nanodomains.

Transmission of Squash vein yellowing virus to and From Cucurbit Weeds and Effects on Sweetpotato Whitefly (Hemiptera: Aleyrodidae) Behavior.

Since 2003, growers of Florida watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai] have periodically suffered large losses from a disease caused by Squash vein yellowing virus (SqVYV), which is transmitted by the whitefly Middle East-Asia Minor 1 (MEAM1), formerly Bemisia tabaci (Gennadius) biotype B. Common cucurbit weeds like balsam apple (Momordica charantia L.) and smellmelon [Cucumis melo var. dudaim (L.) Naud.] are natural hosts of SqVYV, and creeping cucumber (Melothria pendula L.) is an experimental host. Study objectives were to compare these weeds and 'Mickylee' watermelon as sources of inoculum for SqVYV via MEAM1 transmission, to determine weed susceptibility to SqVYV, and to evaluate whitefly settling and oviposition behaviors on infected vs. mock-inoculated (inoculated with buffer only) creeping cucumber leaves. We found that the lowest percentage of watermelon recipient plants was infected when balsam apple was used as a source of inoculum. Watermelon was more susceptible to infection than balsam apple or smellmelon. However, all weed species were equally susceptible to SqVYV when inoculated by whitefly. For the first 5 h after release, whiteflies had no preference to settle on infected vs. mock-inoculated creeping cucumber leaves. After 24 h, whiteflies preferred to settle on mock-inoculated leaves, and more eggs were laid on mock-inoculated creeping cucumber leaves than on SqVYV-infected leaves. The transmission experiments (source of inoculum and susceptibility) show these weed species as potential inoculum sources of the virus. The changing settling preference of whiteflies from infected to mock-inoculated plants could lead to rapid spread of virus in the agroecosystem.

A 'pocket guide' to total internal reflection fluorescence.

The phenomenon of total internal reflection fluorescence (TIRF) was placed in the context of optical microscopy by Daniel Axelrod over three decades ago. TIRF microscopy exploits the properties of an evanescent electromagnetic field to optically section sample regions in the close vicinity of the substrate where the field is induced. The first applications in cell biology targeted investigation of phenomena at the basolateral plasma membrane. The most notable application of TIRF is single-molecule experiments, which can provide information on fluctuation distributions and rare events, yielding novel insights on the mechanisms governing the molecular interactions that underpin many fundamental processes within the cell. This short review intends to provide a 'one stop shop' explanation of the electromagnetic theory behind the remarkable properties of the evanescent field, guide the reader through the principles behind building or choosing your own TIRF system and consider how the most popular applications of the method exploit the evanescent field properties.

Simultaneous widefield single molecule orientation and FRET microscopy in cells.

We combine single molecule fluorescence orientation imaging with single-pair fluorescence resonance energy transfer microscopy, using a total internal reflection microscope. We show how angles and FRET efficiencies can be determined for membrane proteins at the single molecule level and provide data from the epidermal growth factor receptor system in cells.

Nuclear translocation of the calcium-binding protein ALG-2 induced by the RNA-binding protein RBM22.

By yeast two-hybrid screening using the calcium-binding protein ALG-2 as bait a new target of ALG-2 was identified, the RNA-binding protein RBM22. In order to confirm these interactions in vivo we prepared fluorescent constructs by using the monomeric red fluorescent protein to label ALG-2 and the enhanced green fluorescent protein to label RBM22. Confocal microscopy of NIH 3T3 cells transfected with either ALG-2 or RBM22 expression constructs encoding fluorescent fusion proteins alone revealed that the majority of ALG-2 was localized in the cytoplasm whereas RBM22 was located in the nucleus. When cells were co-transfected with expression vectors encoding both fusion proteins ALG-2 was found in the nucleus indicating that RBM22 which can shuttle between the cytoplasm and the nucleus may play a role in nuclear translocation of ALG-2. Using zebrafish as a model mRNA homologues of ALG-2 and RBM22 were microinjected into the blastodisc-yolk margin of zebrafish embryos at the 1-cell stage followed by monitoring the fusion proteins during development of the zebrafish. Hereby, we observed that ALG-2 alone evenly distributed within the cell, whereas in the presence of RBM22 the two proteins co-localized within the nucleus. More than 95% of the two proteins co-localized within the same area in the nucleus suggesting a functional interaction between the Ca(2+)-signaling protein ALG-2 and the RNA-binding protein RBM22.

Multidimensional single-molecule imaging in live cells using total-internal-reflection fluorescence microscopy.

We have developed a wide-field total-internal-reflection fluorescence microscope capable of imaging single molecules in live cells, resolved in both wavelength and polarization. We show fluorescence resonance energy transfer between single pairs of fluorescent molecules bound to signaling receptors in the plasma membrane of live cells and demonstrate the importance of polarization discrimination in addition to wavelength separation.

Energy and fat intake in obese and lean children at varying risk of obesity.

OBJECTIVE: This study compared lean children at high risk (HR) and low risk (LR) of obesity and obese children (OB) to assess the relationship between their energy (EI) and fat intake and adiposity. DESIGN: Cross-sectional study of energy and fat intake in children, using 7-day weighed intakes validated by doubly labelled water (DLW) energy expenditure. SUBJECTS: A total of 114 pre-pubertal children, 50 HR (mean+/-s.d., 6.7+/-0.6 y, 25.7+/-4.8 kg, 21.3+/-6.6% body fat), 50 LR (mean+/-s.d., 6.6+/-0.8 y, 23.6+/-3.7 kg, 18.9+/-5.7% body fat) and 14 OB (mean+/-s.d., 6.8+/-1.0 y, 37.7+/-5.3 kg, 34.8+/-5.6% body fat). MEASUREMENTS: Body fatness was measured using deuterium dilution, total energy expenditure (TEE) by DLW and dietary intake using 7-day weighed records. RESULTS: EI was 98% of TEE in LR children, 95% in HR children and 86% in OB children. Although EI was similar in each group (LR, 7.03+/-1.26 MJ/day; HR, 7.30+/-1.46 MJ/day; OB, 7.55+/-1.67 MJ/day), obese +/-4.6%; P<0.05). There was a significant linear trend towards increasing fat intake (percentage energy) with increasing risk of obesity (P<0.05). While HR children were heavier and fatter than LR children (P<0.05), their EI and fat intake were not significantly greater (HR, 73+/-17 g, 37.3+/-4.4%). Dietary fat intake (percentage energy) was weakly but significantly related to body fatness (r(2)=0.05, P=0.02) by step-wise regression. Since energy from fat was the only macronutrient that was a significant predictor of body fatness, results were therefore analysed using quartiles of fat intake (percentage energy) as cut-offs. When grouped in this way children with the lowest intakes were leaner than those with the highest intakes (19.5+/-7.5 vs 24.9+/-9.4% body fatness; P<0.05). There was a significant trend for increasing fatness as fat intake increased (P<0.05). CONCLUSION: Fat intake is related to body fatness in childhood.

Fluorescence lifetime system for microscopy and multiwell plate imaging with a blue picosecond diode laser.

We report a wide-field fluorescence lifetime imaging (FLIM) system that uses a blue picosecond pulsed diode laser as the excitation source. This represents a significant miniaturization and simplification compared with other time-domain FLIM instruments that should accelerate the development of clinical and real-world applications of FLIM. We have demonstrated this instrument in two configurations: a macroimaging setup applied to multiwell plate assays of chemically and biologically interesting fluorophores and a microscope system that has been applied to imaging of tissue sections. The importance of the adjustable repetition rate of this laser source is discussed with respect to noise reduction and precision in the lifetime determination, illustrating a further significant advantage over conventional mode-locked solid-state lasers.

Time-domain whole-field fluorescence lifetime imaging with optical sectioning.

A whole-field time-domain fluorescence lifetime imaging (FLIM) microscope with the capability to perform optical sectioning is described. The excitation source is a mode-locked Ti:Sapphire laser that is regeneratively amplified and frequency doubled to 415 nm. Time-gated fluorescence intensity images at increasing delays after excitation are acquired using a gated microchannel plate image intensifier combined with an intensified CCD camera. By fitting a single or multiple exponential decay to each pixel in the field of view of the time-gated images, 2-D FLIM maps are obtained for each component of the fluorescence lifetime. This FLIM instrument was demonstrated to exhibit a temporal discrimination of better than 10 ps. It has been applied to chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment. Initially, standard fluorescent dyes were studied and then this FLIM microscope was applied to the imaging of biological tissue, successfully contrasting different tissues and different states of tissue using autofluorescence. To demonstrate the potential for real-world applications, the FLIM microscope has been configured using potentially compact, portable and low cost all-solid-state diode-pumped laser technology. Whole-field FLIM with optical sectioning (3D FLIM) has been realized using a structured illumination technique.

Application of the stretched exponential function to fluorescence lifetime imaging.

Conventional analyses of fluorescence lifetime measurements resolve the fluorescence decay profile in terms of discrete exponential components with distinct lifetimes. In complex, heterogeneous biological samples such as tissue, multi-exponential decay functions can appear to provide a better fit to fluorescence decay data than the assumption of a mono-exponential decay, but the assumption of multiple discrete components is essentially arbitrary and is often erroneous. Moreover, interactions, both between fluorophores and with their environment, can result in complex fluorescence decay profiles that represent a continuous distribution of lifetimes. Such continuous distributions have been reported for tryptophan, which is one of the main fluorophores in tissue. This situation is better represented by the stretched-exponential function (StrEF). In this work, we have applied, for the first time to our knowledge, the StrEF to time-domain whole-field fluorescence lifetime imaging (FLIM), yielding both excellent tissue contrast and goodness of fit using data from rat tissue. We note that for many biological samples for which there is no a priori knowledge of multiple discrete exponential fluorescence decay profiles, the StrEF is likely to provide a truer representation of the underlying fluorescence dynamics. Furthermore, fitting to a StrEF significantly decreases the required processing time, compared with a multi-exponential component fit and typically provides improved contrast and signal/noise in the resulting FLIM images. In addition, the stretched-exponential decay model can provide a direct measure of the heterogeneity of the sample, and the resulting heterogeneity map can reveal subtle tissue differences that other models fail to show.

Whole-field five-dimensional fluorescence microscopy combining lifetime and spectral resolution with optical sectioning.

We report a novel whole-field three-dimensional fluorescence lifetime imaging microscope that incoporates multispectral imaging to provide five-dimensional (5-D) fluorescence microscopy. This instrument, which can acquire a 5-D data set in less than a minute, is based on potentially compact and inexpensive diode-pumped solid-state laser technology. We demonstrate that spectral discrimination as well as optical sectioning minimize artifacts in lifetime determination and illustrate how spectral discrimination improves the lifetime contrast of biological tissue.

Imaging patterns of calcium transients during neural induction in Xenopus laevis embryos.

Through the injection of f-aequorin (a calcium-sensitive bioluminescent reporter) into the dorsal micromeres of 8-cell stage Xenopus laevis embryos, and the use of a Photon Imaging Microscope, distinct patterns of calcium signalling were visualised during the gastrulation period. We present results to show that localised domains of elevated calcium were observed exclusively in the anterior dorsal part of the ectoderm, and that these transients increased in number and amplitude between stages 9 to 11, just prior to the onset of neural induction. During this time, however, no increase in cytosolic free calcium was observed in the ventral ectoderm, mesoderm or endoderm. The origin and role of these dorsal calcium-signalling patterns were also investigated. Calcium transients require the presence of functional L-type voltage-sensitive calcium channels. Inhibition of channel activation from stages 8 to 14 with the specific antagonist R(+)BayK 8644 led to a complete inhibition of the calcium transients during gastrulation and resulted in severe defects in the subsequent formation of the anterior nervous system. BayK treatment also led to a reduction in the expression of Zic3 and geminin in whole embryos, and of NCAM in noggin-treated animal caps. The possible role of calcium transients in regulating developmental gene expression is discussed.

On the mechanism of ooplasmic segregation in single-cell zebrafish embryos.

It has been previously shown that localized elevations of free cytosolic calcium are associated with a morphological contraction in the forming blastodisc and animal hemisphere cortex during ooplasmic segregation in zebrafish zygotes. It was subsequently proposed, in a hypothetical model, that these calcium transients might be linked to the contraction of a cortically located actin microfilament network as a potential driving force for segregation. Here, by labeling single-cell embryos during the major phase of segregation with rhodamine-phalloidin, direct evidence is presented to indicate that the surface contraction was generated by an actin-based cortical network. Furthermore, while zygotes incubated with colchicine underwent normal ooplasmic segregation, those incubated with cytochalasin B did not generate a constriction band or segregate to form a blastodisc. During segregation at the single-cell stage, ooplasm simultaneously moved in two directions: toward the blastodisc within the so-called axial streamers, and toward the vegetal pole in the peripheral ooplasm. The velocities of both axial and peripheral streaming movements are reported. By injection of a fluorescein isothiocyanate (FITC)-labeled 2000 kDa dextran into the peripheral ooplasm it was demonstrated that a portion of it feeds into the bases of the extending streamers, which helps to explain the lack of accumulation of ooplasm at the vegetal pole. These new data were incorporated into the original model to link the bipolar ooplasmic movements with the calcium-modulated, actin-mediated contraction of the animal hemisphere cortex as a means of establishing and driving ooplasmic segregation in zebrafish.

Calcium signalling during zebrafish embryonic development.

Calcium signals appear throughout the first 24 hours of zebrafish development. These begin at egg activation, then continue to be generated throughout the subsequent zygote, cleavage, blastula, gastrula, and segmentation periods. They are thus associated with the major phases of pattern formation: cell proliferation, cell differentiation, axis determination, the generation of primary germ layers, the emergence of rudimentary organ systems, and therefore the establishment of the basic vertebrate body plan. When signals need to be transmitted across significant distances they take the form of waves, either intracellular waves when the cell size is large, or later in development when the cell size is reduced, intercellular waves. We will consider both types of calcium signals and their integration into signalling networks, and discuss their possible functions and developmental significance with regard to pattern formation. BioEssays 22:113-123, 2000.

Whole-field optically sectioned fluorescence lifetime imaging.

We describe a novel three-dimensional fluorescence lifetime imaging microscope that exploits structured illumination to achieve whole-field sectioned fluorescence lifetime images with a spatial resolution of a few micrometers.

A wave of free cytosolic calcium traverses zebrafish eggs on activation.

The activation process in a variety of deuterostome and protostome eggs is accompanied by cytosolic calcium transients that usually take the form of either a single or multiple propagating waves. Here we report that the eggs of zebrafish (Danio rerio) are no exception in that they generate a single activation wave that traverses the egg at a velocity of around 9 microm/s. There appears, however, to be no difference between the calcium-mediated activation response of eggs with regard to the presence or absence of sperm in the spawning medium. This leads us to suggest that these eggs are normally activated when they come in contact with their spawning medium and are then subsequently fertilized. The aspermic wave is initiated at the animal pole in the region of the micropyle, appears to propagate mainly through the yolk-free egg cortex, and then terminates at the vegetal pole. As neither sperm nor external calcium is required for the initiation (or propagation) of the activation wave, this suggests that an alternative wave trigger must be involved.

Neurotransmitters, peptides, and neural cell adhesion molecules in the cortices of normal elderly humans and Alzheimer patients: a comparison.

Immunocytochemical techniques was used to compare the proportion of neurons expressing various neurotransmitters (tyrosine hydroxylase, choline acetyltransferase and gamma-aminobutyric acid), neuropeptides (Leu-enkephalin and substance P) and neural cell adhesion molecules (NCAM) in the hippocampus, frontal (area 10) and occipital (area 17) cortices of neurologically normal elderly humans to that of age-matched Alzheimer disease (AD) patients. There was no difference in the proportion of GABAergic and cholinergic cells between the normal and AD groups in all three brain regions studied. However, the catecholaminergic cells in the frontal cortex of the AD patients revealed a significant decrease. The catecholaminergic cells present in the cortex were both neurons and astrocytes, as revealed by a double immunostaining of tyrosine hydroxylase and glial fibrillary acid protein (GFAP). Furthermore, the difference in the proportion of cells expressing Substance P and Leu-enkephalin was minimal between the two groups studied. Although there was little difference in the levels of NCAM in the occipital cortex and hippocampus of the two groups, there were significantly fewer positive NCAM neurons in the frontal cortex of AD than normal aging individuals.

Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation.

Oscillations of cytosolic free calcium levels have been shown to influence gene regulation and cell differentiation in a variety of model systems. Intercellular calcium waves thus present a plausible mechanism for coordinating cellular processes during embryogenesis. Herein we report use of aequorin and a photon imaging microscope to directly observe a rhythmic series of intercellular calcium waves that circumnavigate zebrafish embryos over a 10-h period during gastrulation and axial segmentation. These waves first appeared at about 65% epiboly and continued to arise every 5-10 min up to at least the 16-somite stage. The waves originated from loci of high calcium activity bordering the blastoderm margin. Several initiating loci were active early in the wave series, whereas later a dorsal marginal midline locus predominated. On completion of epiboly, the dorsal locus was incorporated into the developing tail bud and continued to generate calcium waves. The locations and timing at which calcium dynamics are most active appear to correspond closely to embryonic cellular and syncytial sites of known morphogenetic importance. The observations suggest that a panembryonic calcium signaling system operating in a clock-like fashion might play a role during vertebrate axial patterning.