Green Fluorescent Microspheres Research Articles

Advantages of aggregation-induced luminescence microspheres compared with fluorescent microspheres in immunochromatography assay with sandwich format.

Organic fluorescein dye-embedded fluorescent microspheres (FMs) are currently the most established commercially fluorescent markers, and they have been widely used to improve the sensitivity of immunochromatography assay (ICA). However, these FMs have natural defects, such as the aggregation-caused quenching effect and small Stokes shift, which are not conducive to improving the detection performance of ICA. Herein, two green emitted FMs, namely aggregation-induced emission FMs (AIEFMs) and fluorescein isothiocyanate FMs (FITCFMs), were prepared by swelling the AIE luminogens and FITC dyes into the carboxyl group-modified polystyrene microspheres. The average diameters of AIEFMs and FITCFMs were 350 and 450nm, respectively. Compared with FITCFMs, the AIEFMs exhibited stronger fluorescence intensity and a larger Stokes shift. These two FMs were used as the labeling markers of ICA for procalcitonin (PCT) detection with the sandwich format. Among them, AIEFM-ICA showed dynamic linear detection of PCT from 7.6pgmL-1 to 125ngmL-1 with the limit of detection (LOD) at 3.8pgmL-1. These values were remarkably superior to those of FITCFM-ICA (linear range from 61pgmL-1 to 62.5ngmL-1 and LOD value at 60pgmL-1). Furthermore, the average recoveries of the intra- and inter-assays of AIEFM-ICA ranged from 86% to 112%, with coefficients of variation ranging from 1.2% to 8.8%, indicating accuracy and precision for PCT quantitative detection. Additionally, the reliability of the developed AIEFM-ICA was further assessed by analyzing 30 real serum samples from systemic inflammatory response by infectious diseases, and the results showed good agreement with the chemiluminescence immunoassay. In conclusion, compared with traditional FITCFMs, green emitted AIEFMs as a novel fluorescent label, exhibits greater potential to enhance the detection performance of the ICA platform.

The effect of phase modulation in dual-frequency excited standing surface acoustic wave (SSAW) on microparticle manipulation

Standing surface acoustic wave (SSAW) in a microfluidic channel generated from a pair of interdigital transducers (IDTs) has already shown its capability of particle accumulation at the pressure node and its potential in biomedical research. Manipulation of cells or microparticles to the desired position precisely is an important issue of this technology. Using the phase modulation in the dual-frequency excitation, the fundamental and third harmonic of the same IDTs simultaneously with the initial phase difference between them and the power ratio of the fundamental wave to the third harmonic of 85%, the positions of three pressure nodes of the SSAW in the microchannel moves at different rates because of changes to the synthesized waveform and resultant acoustic radiation force. Meanwhile, percentages of microparticles at each pressure node also vary with the initial phase. Experimental measurement of green fluorescent microspheres in a diameter of 4 µm in a 300 µm-microchannel driven by SSAW at 6.2 and 17.7 MHz simultaneously has a good correlation with the numerical prediction (R2 = 0.967 and 0.89 for the position of each pressure node and the percentage of microspheres accumulated at each pressure node in the range of initial phase from 0° to 360°, respectively, where R is the correlation coefficient). This proposed method could provide more tunability for SSAW-based microparticle manipulation with few changes to the setup of lab-on-a-chip system.

A 3D Dual-Particle Tracking Co-Localization Microscope for the Study of DNA Dynamics in Free Solution

Measuring the co-localization of fluorescent constructs is a powerful technique in life science microscopy that can provide critical functional information about coincident events in the cell. Fluorescence coincidence can be used to determine the separation distance of binding targets, binding kinetics, conformational change, or even the orientation of a target construct. Techniques such as FRET and plasmonic resonance energy transfer can act as nanometric “rulers” to provide a separation of two targets, but their instrumentation relies on a slow camera based approach that is not amenable to three-dimensional freely diffusing targets, and they provide no rotational information. Here we present a dual-particle tracking microscope that can simultaneously localize two spectrally distinct targets in three dimensions with a time resolution down to 1 ms. The targets can be tracked with a separation distance up to 400 nm with 20 nm accuracy. And since each target is individually localized, a wealth of data may be extracted; such as, 3D position, 2D rotation, separation distance, and fluorescent lifetime of each target. The approach is a multicolor extension to an existing technique known as, TSUNAMI, Tracking Single-particles Using Nonlinear And Multiplexed Illumination. Just like TSUNAMI, this dual-color extension is capable of tracking within thick and scattering samples up to 200 μm deep. The technique is applied to the measurement of DNA flexing dynamics in free solution. DNA constructs with both red and green fluorescent microspheres labelled at the ends are observed in free diffusion. The trajectories from different lengths of DNA linker constructs are analyzed for binding distance and positional information to elucidate the differences in DNA dynamics during free diffusion.

181 M1 MACROPHAGES AND IRF5 EXACERBATE ATHEROSCLEROTIC DISEASE

Introduction Atherosclerosis is an inflammatory disease that occurs in vascular sites subjected to complex blood flow, e.g. oscillatory (OSS) or low shear stress (LSS), the latter leading to the formation of a macrophage-rich plaque which is vulnerable to rupture and thrombotic events. Monocyte-derived macrophages can be conditioned towards a pro-inflammatory phenotype (M1) or a regulatory one (M2) and may determine atherosclerotic plaque outcome. Interferon Regulatory Factor 5 (IRF5) has been shown to be instrumental for M1 macrophage polarisation. We aimed to establish the occurrence of macrophage polarisation and the expression of IRF5 in murine atherosclerosis. Methods A perivascular shear stress modifying cast was tied around a carotid artery, mimicking LSS and OSS in ApoE−/− mice fed a high fat diet. Homeostatic Ly-6Clo monocyte recruitment to the carotid artery was observed after labelling them in vivo with green fluorescent microspheres. We examined the expression of the macrophage polarisation markers CD206 (M2), iNOS and IRF5 (M1) in the cast-treated carotid artery by Immunohistochemistry and Confocal Immunolocalisation. Results Labelled Ly-6Clo monocytes were more prevalent in the upstream half of LSS-modulated plaques (p=0.0006) in comparison to the downstream half and OSS-modulated lesions. These monocytes co-localised with the expression of iNOS not CD206. In addition, most macrophages in the LSS-induced carotid artery lesions (vulnerable phenotype) expressed iNOS (p=0.006) and IRF5 (p=0.03) compared to OSS-modulated lesions (stable phenotype) while CD206 expression showed no differences. Conclusions Ly-6Clo monocyte recruitment and M1 macrophages were significantly more prevalent in low shear stress-modulated plaques. These differences suggest low shear stress promotes monocyte recruitment and macrophage polarisation towards the pro-inflammatory M1 phenotype.

Heterogeneity of the intrahepatic portal venous blood flow: Impact on hepatocyte transplantation

BackgroundThe poor repopulation rate of the liver by transplanted hepatocytes markedly hampers liver cell therapy, which might be due to a limited sequestration of cells within the hepatic microvasculature. We therefore present intravital fluorescence microscopic data of transplanted hepatocytes immediately after portal venous injection demonstrating their intrahepatic distribution. MethodsMale Wistar rats were transplanted with freshly isolated, rhodamine 123 labelled, primary rat hepatocytes. Cells (106 in 0.5ml) were slowly injected via a catheter in the V. lienalis over 2min. Their distribution in the left lateral liver lobe was visualized simultaneously as well as over the following 30min by intravital fluorescence microscopy. In a second set of animals green fluorescent microspheres exhibiting a size of 15μm were injected and observed identically. For further analyses of portal venous blood flow distribution sodium fluorescein was injected via the V. lienalis as well as via the V. jugularis. ResultsIn vivo imaging allowed the clear detection and observation of hepatocytes flowing into the liver and forming microemboli, which are trapped particularly in small distal portal branches. To a minor extent they were trapped as solitary cells in the periportal zone of sinusoids. Most interestingly, the distribution of cells within the liver was highly heterogeneous, as wide areas of acini were found free of transplanted cells after portal venous injection, while neighbouring areas showed disproportionately high hepatocyte occurrence. To further investigate this phenomenon sodium fluorescein was injected via the V. lienalis instead and an identical heterogeneous distribution pattern with clear anatomical borders defining highly, semi, and non-portal venous perfused liver acini could be observed. In contrast, systemic injection of sodium fluorescein via the V. jugularis in the same animals resulted in a homogenous dispersion within the liver. ConclusionUsing in vivo fluorescence microscopy and exclusive portal venous injection of a fluorescent dye, we provide evidence for the existence of liver areas, differentially supplied by portal venous blood. As a consequence, hepatocytes transplanted via the portal tract are very heterogeneously distributed within the liver. This observation forces us to reconsider our current knowledge on (i) monitoring engrafted cells, (ii) the optimal hepatocyte number to be transplanted, (iii) portal hypertension after cell injection, and last but not least (iv) the optimal transplantation route. Moreover, the established model for in vivo visualization of transplanted hepatocytes allows development of new therapeutic strategies facilitating an improved engraftment of cells.

Development of a novel two color tracer perfusion technique for the hydrodynamic study of aqueous outflow in bovine eyes

Background Elevation of intraocular pressure is usually associated with primary open angle glaucoma and caused by increased outflow resistance. A two-color fluorescent tracer technique was developed to investigate the hydrodynamics of aqueous humor outflow with changing intraocular pressure within the same eye, to better understand the relationship between outflow facility and effective filtration area. Methods Eighteen enucleated bovine eyes were first perfused at 30 mmHg with Dulbecco's phosphate-buffered saline containing 5.5 mmol/L D-glucose. After a stable baseline facility, red fluorescent microspheres (0.5 μm, 0.002% v/v) were exchanged and perfused. Eyes in the one-color control group (n=6) were immediately perfused with fixative. In the experimental group (n=6), eyes were perfused with green tracer after intraocular pressure reduced to 7 mmHg, while in the two-color control group (n=6), eyes were perfused with green tracer with intraocular pressure remaining at 30 mmHg. All 12 eyes were then perfusion-fixed. Outflow facility was continuously recorded in all eyes. Confocal images were taken along the inner wall of the aqueous plexus and the percent of the effective filtration length (PEFL; length of inner wall exhibiting tracer labeling/total length of inner wall) was measured. The relationships between outflow facility and PEFL were analyzed statistically. Results No significant differences were found in baseline facilities (μl·min-1·mmHg-1) among the three groups (the experimental group: 0.93±0.12; the two-color control group: 0.90±0.19; the one-color control group: 0.98±0.13). In the experimental group, the outflow facility was significantly higher at 7 mmHg (4.29±1.01) than that at 30 mmHg (1.90±0.67, P <0.001), which corresponded to a significant increase in the PEFL at 7 mmHg (54.70±8.42) from that at 30 mmHg ((11.76±4.56)%, P <0.001). The PEFL labeled by red fluorescent microspheres in the experimental group ((11.76±4.56)%) showed no significant difference from that of the one-color control group ((13.39±2.19)%, P=0.473) or the two-color control group ((11.49±4.95)%, P=0.930). The PEFL labeled by green fluorescent microspheres in the experimental group ((54.70±8.42)%) was significantly higher than that of the two color control group ((37.34±8.17)%, P=0.010). A positive correlation was found between outflow facility and PEFL(t=0.897, R 2=0.804) in the experimental group. Conclusions Changes in aqueous humor outflow patterns before and after a change in intraocular pressure can be successfully distinguished within the same eye using our newly developed two-color tracer perfusion technique. The PEFL showed positive correlation with the outflow facility.

Remote Postconditioning is More Potent than Classic Postconditioning in Reducing the Infarct Size in Anesthetized Rabbits

Postconditioning confers protection to the heart after a potentially lethal episode of prolonged ischemia. There is evidence that it may also be protective when applied at a distal artery. In the present study, we sought to determine whether remote postconditioning within the heart (local) or outside the heart (distal) is effective in salvaging the ischemic heart in vivo and to compare its effect with that of the classic postconditioning. Twenty seven open chest New Zealand white anesthetized male rabbits were divided into four groups and were exposed to 30 min regional myocardial ischemia (isc), after ligation of a prominent coronary artery, followed by 3 h reperfusion (rep) after releasing the snare. Control group (n = 7) was subjected to no additional interventions, postC group (n = 6) was subjected to four cycles of 1 min isc/1 min rep of the same coronary artery at the beginning of reperfusion, remote local postC group (n = 7) to four cycles of 1 min isc/1 min rep of another coronary artery 30 s before the end of index isc and remote distal postC group (n = 7) to four cycles of 1 min isc/1 min rep of another (carotid) artery again 30 s before the end of index isc. Infarct size (I) and area at risk (R) were delineated with the aid of TTC staining and green fluorescent microspheres respectively and their ratio was expressed in percent (%I/R). Remote local and remote distal postC reduced the % I/R ratio (17.7 +/- 1.7% and 18.4 +/- 1.6%, respectively vs 47.0 +/- 2.5% in the control group, P < 0.01). Classic PostC had an intermediate protective effect (33.1 +/- 1.7%, P < 0.05 vs all the other groups). Remote postconditioning consisted of 1 min isc/1 min rep protects the ischemic rabbit heart in vivo, independently of the site of the remote artery. This intervention seems to confer a stronger protection than the classic postconditioning.

Micro‐pixel light emitting diodes with wavelength‐tunable output

AbstractA novel design and implementation of a single‐chip wavelength tunable light emitting diode (LED) is introduced in this paper, with varying emission spectral characteristics ranging between 470 nm (blue) and 525 nm (green). The emission wavelength of green fluorescent microspheres (525 nm), acting as a colour conversion agent, is chosen to be efficiently excited by a GaN LED of 470 nm. It is selectively‐coated onto designated micrometer‐scaled pixels, which are interconnected via metal lines. As a result, the LED allows separate control of optical output either 470 nm, 525 nm or even a mixture of both wavelengths. Since the pixels are of micron‐scale which are non‐resolvable by the unaided human eye, the output appears optically mixed. This type of device, combining the application of micro‐light‐emitting diode and fluorescent microsphere technologies, offers a colour tunable output suitable for a wide range of applications including such as outdoor panel displays. (© 2008 WILEY‐VCH Verlag GmbH &amp; Co. KGaA, Weinheim)

Fate of fluorescent microspheres in developing Ictalurus punctatus following prolonged immersion

Particulate antigen uptake by the mucosa of developing channel catfish was determined by immersing larvae and fry [2-day post-hatch (dph), 1-, 2-, 3-, 4-, and 8-week post-hatch (wph)] to two forms of fluorescent microspheres (FMS): blue FMS were carboxylated, and green FMS were coated via conjugation with a crude extract of Edwardsiella ictaluri outer membrane protein (OMP). Phagocytosis, destination, and clearance appeared similar for the two types of FMS used. In the older age classes, primary uptake was observed in epithelial cells of the torso, fins, nares and to a lesser extent the gills. Fluorescent microspheres were less frequently observed within mononuclear phagocytes in the epidermis, dermis and underlying connective tissue of the tissue mentioned above. Limited FMS trafficking was observed from 4- to 24-h post-immersion (hpi). Significantly higher numbers of FMS (blue and green)/mm 3 of tissue were observed in the posterior kidney of the 4- and 8-wph age classes and in the anterior kidney and spleen of the 8-wph age class when compared to younger age classes ( p < 0.05). Significantly higher FMS (blue and green)/mm 3 of tissue were observed in the posterior kidney of 4- and 8-wph fish when compared to all other organs ( p < 0.05). The present study indicates that FMS uptake increases with age in channel catfish. The younger age classes may possess an increased ability to exclude particulate antigen, or lack the specific mechanisms that needed to take up particulates in the form of FMS.

Projections of the sexually dimorphic calcitonin gene-related peptide neurons of the preoptic area determined by retrograde tracing in the female rat.

The medial preoptic area of the rat exhibits morphologic sex differences and is implicated in the control of sexually dimorphic behavior and function. Neurons expressing calcitonin gene-related peptide (CGRP) within the anteroventral periventricular (AVPV) and medial preoptic nucleus (MPN) of the medial preoptic area exhibit female-dominant sex differences in number through organizational and activational effects of gonadal steroids. The present study used retrograde tracing experiments to establish the projections of the AVPV and MPN CGRP neurons in the female rat. After the intraperitoneal administration of Fluoro-Gold to female rats (n = 5), we were unable to detect retrograde tracer in any CGRP-immunoreactive cells of the hypothalamus. Intracerebral injections of 50- to 100-nl volumes of Fluoro-Gold into the mediobasal hypothalamus resulted in up to 70% of CGRP neurons in the AVPV and MPN containing retrograde tracer. Similar large volume tracer depositions in the lateral septum, periaqueductal gray, two likely CGRP projection sites, resulted in no labeling of preoptic CGRP neurons. Experiments using small volume (30-nl) injections of Fluoro-Gold and green fluorescent microspheres at multiple sites in the mediobasal hypothalamus (n = 18) revealed that approximately 60% of AVPV and 30% of MPN neurons expressing CGRP were projecting to the region of the tuberal and ventral premammillary nuclei, with a minor projection to the dorsomedial nucleus. These findings demonstrate a major projection of the preoptic CGRP neurons to the posterior hypothalamus in the female rat and support further a functional role for these neurons in the sexually dimorphic regulation of reproductive functioning.

Lucifer yellow, retrograde tracers, and fractal analysis characterise adult ferret retinal ganglion cells.

The dendritic morphology of retinal ganglion cells in the ferret was studied by the intracellular injection of lucifer yellow in fixed tissue. Ganglion cells were identified by the retrograde transport of red or green fluorescent microspheres that had been injected into different target nuclei, usually the lateral geniculate nucleus or superior colliculus. This approach allows the comparison of dendritic morphologies of ganglion cells in the same retina with different central projections and also identifies cells with branching axons. The digitised images of dendritic arbors were analysed quantitatively by a variety of measures. Dendritic complexity was assessed by calculating the fractal dimension of each arbor. The ferret has distinct alpha, beta, and gamma morphological classes of cells similar to those found in the cat. The gamma cell class was morphologically diverse and could be subdivided into "sparse," "loose," and "tight" groups, reflecting increasing dendritic complexity. Whereas the beta cell projection was limited to the lateral geniculate nucleus alone, alpha and gamma cells could project to either or both nuclei. Retinal ganglion cells labelled from the pretectal nuclei formed a morphologically distinct class of retinal ganglion cells. The ipsilateral projection lacked alpha cells and the most complex, "tight" gamma cells. However, ipsilaterally projecting "loose" gamma cells overlapped alpha cells in both soma and dendritic dimensions. Different morphological classes of retinal ganglion cells hence show characteristic axon behaviour both in their decussation at the chiasm and in which targets they innervate. Fractal measures were used to contrast variation within and between these identified classes.

Retrograde labeling of lumbosacral interneurons following injections of red and green fluorescent microspheres into hindlimb motor nuclei of the cat.

In order to map the laminar and segmental positions of lumbosacral interneurons that project to L7 motor nuclei, red and green fluorescent latex microspheres ("beads") were pressure-injected through micropipettes into the deep peroneal or posterior biceps-semitendinosus motoneuron pools of cats. Micropipette tips were positioned by recording the antidromic field potentials evoked by electrical stimulation of the muscle nerves. Projecting interneurons were identified by the presence of retrogradely transported beads. Small bead injections labeled large numbers of neurons. These were observed in all the spinal segments examined, L1-S2, and were most densely concentrated within laminae VI and VII ipsilateral to the injections and lamina VIII contralaterally. In addition, significant numbers of labeled cells were observed in lateral lamina V ipsilaterally and in lamina X. A few cells with bilateral projections were double-labeled following injections of red and green beads on opposite sides of the cord. These were most often observed in midlumbar segments (L3-L5) in medial regions of the gray matter. The results suggest that the intermediate zone (laminae V-VIII and X) of the lumbosacral spinal cord is a major source of interneuronal projections to the L7 ventral horn. This is true for both lateral and medial areas of the intermediate zone, as the fluorescent microspheres labeled neurons in medial regions of the cord largely undetected in previous studies in which other methodologies were employed.

Translocation of Particles by Macrophages from Lung to Lymph Nodes in Rats

The mechanism by which particles deposited in the alveolar region of the lung are translocated to tracheobronchial lymph nodes (TBLN) remains unclear. In the present study, we investigated the ability of rat pulmonary alveolar macrophages (PAM) to contribute to this process. Rats were instilled intratracheally with 5 x 109 fluorescent microspheres about 1.3 μm diameter and groups were killed at 1, 2, 4 and 8 days after instillation. Two-micron plastic sections of the TBLN revealed that approximately 90% of the microspheres in the TBLN were within macrophages at each time point. The distribution of the numbers of microspheres per TBLN macrophage did not change between 1 and 8 days even though the total number of particles in the TBLN increased linearly during this time. Had the microspheres accumulated in the TBLN as free particles, it seems likely that there would have been a shift toward larger numbers of microspheres per macrophage with time. We also instilled pulmonary alveolar macrophages (PAM), approximately half of which were labeled with red fluorescent microspheres and half with green fluorescent microspheres, into rat lungs. After three days the TBLN of the rats contained numerous macrophages containing only one color of microsphere. The use of the two colors of microspheres precluded the possibility that the microspheres were translocated as free particles and were phagocytized in the TBLN by resident macrophages. These results suggest that in the rats, a considerable number of microspheres was translocated from lung to TBLN within PAM.

Phagocytosis of opsonized fluorescent microspheres by human neutrophils: A two-color flow cytometric method for the determination of attachment and ingestion

A highly reproducible two-color fluorescence cytometric method is described for determining the amount of receptor-mediated and of non-specific phagocytosis by human neutrophils of polystyrene microspheres that have been covalently coated with C3b, iC3b, IgG, mixtures of these, BSA or human F(ab′) 2. The method includes a correction for externally bound particles and thus measures net phagocytosis. The method involves the incubation of neutrophils with coated green fluorescent microspheres in buffer alone or with cytochalasin D to inhibit phagocytosis followed by staining the externally bound microspheres with red fluorescent antibodies to the immobolized ligand, and determining the green and red fluorescence in a dual laser fluorescence activated flow cytometer. The red to green fluorescence ratio of the mixtures containing cytochalasin D allows for a correction of the green fluorescence due to externally bound microspheres in the mixtures not containing cytochalasin D. The corrected green intensities thus represent net phagocytosis. The specificity of receptor-mediated phagocytosis was confirmed by inhibition with fluid-phase C3b or iC3b or monoclonal antibodies to the receptors. The method can be applied to the determination of both adherent and suspension phagocytosis and can be used as a general model of the phagocytosis of bacteria by neutrophils.

Migration of neutrophils from lung to tracheobronchial lymph node.

Translocation of particulate antigen deposited in the lung to tracheobronchial lymph nodes (TBLN) is important in the induction of pulmonary immune responses. We have previously shown that alveolar macrophages can contribute to particle translocation to TBLN, but whether neutrophils can also contribute to this process is not known. To determine if neutrophils can carry particles to the TBLN, dog neutrophils were elicited by instillation of red or green fluorescent microspheres into individual lung lobes. Autologous neutrophils that had phagocytized fluorescent microspheres were then instilled into an unexposed lobe of the same dog's lung. After 24 hr, the TBLN of instilled dogs had numerous neutrophils, 99% of which contained either red or green fluorescent microspheres but not both. Use of the two different colored microspheres as labels precluded the possibility that neutrophils had phagocytized the microspheres in the TBLN. In a second experiment, dogs were depleted of peripheral blood neutrophils by injections of hydroxyurea. Hydroxyurea-treated and normal dogs were instilled with fluorescent microspheres and killed after 40 h. Hydroxyurea treatment reduced neutrophil accumulation in the lung by 79% and reduced particle translocation to the TBLN by 80%. Results of these experiments indicate that neutrophils are similar to pulmonary alveolar macrophages (PAM) in their ability to phagocytize particles in the lung and then migrate to the TBLN.

The role of macrophages in particle translocation from lungs to lymph nodes.

Red fluorescent and green fluorescent microspheres were instilled into separate but adjacent areas of dog lung lobes. After 7 days, the tracheobronchial lymph nodes that drained both of the instilled areas contained many macrophages with all red or all green microspheres but rarely both. This indicates that the particles did not translocate passively and that lung macrophages phagocytized the microspheres in the lung and carried them to the tracheobronchial lymph nodes. In addition, two populations of pulmonary alveolar macrophages (PAM's), one that had phagocytized red microspheres in vivo and one that had phagocytized green microspheres, were lavaged from the lungs of dogs, mixed into one population, and instilled back into a previously unexposed lung lobe of the same dogs. As in the first experiment, the tracheobronchial lymph nodes that drained the instilled area contained numerous macrophages with either all red or all green microspheres. This suggested that the instilled PAM's had migrated to the tracheobronchial lymph nodes. Thus, lung macrophages, including PAM's probably play a critical role in the induction of lung immunity and in protection from disease by determining particle translocation.