text:
|Publication number
|Publication type
|Application number
|Publication date
|Jun 19, 1990
|Filing date
|Apr 1, 1988
|Priority date
|Apr 1, 1988
|Fee status
|Also published as
|Publication number
|Richard P. Watts, Wylie I. Lee, John W. Vorpahl
|Original Assignee
|Syntex (U.S.A.) Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (12), Non-Patent Citations (2), Referenced by (25), Classifications (15), Legal Events (8)
|External Links: USPTO, USPTO Assignment, Espacenet
1. Field of the Invention
The present invention relates to an apparatus and method for detecting fluorescence or light scatter, which may relate to the presence or absence of an analyte, in a liquid sample. In particular, the invention relates to a dual optical fiber probe in which the spatial configuration of the optical fibers is arranged to define a finite interrogation volume independent of the volume of the liquid sample. The interrogation volume can be visualized as the intersection of the two light cones emanating from the first and second optical fibers having a determined spatial configuration when each optical fiber transmits visible light. The size of the interrogation volume is determined by the spatial orientation and optical parameters of the dual optical fibers, preferably creating an interrogation volume of less than 10-5 cubic centimeters. The invention is particularly useful for clinical laboratory applications such as blood typing.
2. State of the Art
Cells and other particles in the range of 0.05 to 100 microns suspended in a liquid medium have been individually detected by streaming the liquid to provide a fine stream or to create very small droplets so as to provide a detection volume and monitoring of the light scattered by the particles, including Rayleigh and Raman light scattering, or that emitted from the particles such as by fluorescence. The basic principle of light scattering from a small volume is used in flow cytometers. Commercial instruments utilizing light scattering and fluorescence for particle detection are available.
PCT Patent Application Publication No. WO 85/05680, published Dec. 19, 1985, describes an optical system for use in flow cytometers that include multiple optical fibers disposed about and on an orifice plate used to define the flow stream.
Briggs (U.S. Pat. No. 4,564,598) describes principles for the detection of particles that are not confined to small volumes. As described therein, a small detection volume is achieved by illuminating the bulk suspension with a very narrow beam of light, e.g. 10-100 microns, delivered through an optical fiber immersed in the fluid and collecting the light scattered at the tip of the fiber by allowing it to pass back through the fiber past a beam splitter. Because the same fiber is used for illuminating the particles as well as collecting the emitted light, the method is generally useful only where the emitted light is wavelength shifted as in fluorescence. Under these conditions, internal scatter of the fiber can be largely filtered out to permit detection of the signal. However, even when detecting fluorescence, the background scatter limits sensitivity. While in principle multiple wavelengths can be monitored, multiple splitters must be used which further decrease sensitivity and also increases the cost and complexity of the system.
Other devices and apparatus have been described for the detection and measurement of scattered and emitted light from particles. United Kingdom Patent No. 1,506,017 describes a fluorometric system including two optical fibers that transmit and receive electromagnetic radiation to and from the sample-coated surface of a macrobody (typically a sphere on the order of 5-20 millimeters in diameter). The optical fibers are preferably disposed at a small angle, less than thirty degrees, relative to each other. The fibers are not utilized to define a volume in a liquid sample from which the presence or absence of an event will be monitored.
Japanese Patent Application No. 84230306, filed Nov. 2, 1984 (Publication No. 61110033, dated May 28, 1986) describes the use of a plurality of optical fibers to measure particle agglutination.
U.S. Pat. No. 4,564,598 (Briggs), noted previously, describes an optical fiber probe having a single optical fiber, that both supplies the incident radiation and receives the excitation signal, to measure fluorescent signal in a fluid sample. U.S. Pat. No. 4,537,861, (Elings et al) describes an immunoassay technique in which labelled, bound ligand-antiligand complexes are caused to reside in a predetermined spatial pattern that then can be scanned to detect a signal level relative to background signal. European Patent Publication No. 0175545, published Mar. 26, 1986 (Application No. 85306450.9, filed Nov. 11, 1985) describes a method for particle detection that autocorrelates the intensity of an electromagnetic signal over a nonzero interval, the duration of which is short compared to the mean duration of the fluctuations. Additional documents describing particle detection methods are U.S. Pat. No. 4,421,860 (Elings et al) and U.S. Pat. No. 4,560,881 (Briggs) and references described and listed therein.
The present invention provides an apparatus and a method of detecting in a liquid an optical event (i.e. an event that can be observed by an optical fiber), such as fluctuations in electromagnetic radiation (e.g. fluorescence or light scatter), resulting, for example, from the presence of optical event-causing elements, such as an analyte (e.g. particle or other source of light scatter or fluorescence) or fluorescent fluctuation due to temperature changes, without flowing or otherwise confining the liquid to a necessarily restricted shape or volume, while at the same time greatly decreasing background scattering and providing a way to simultaneously or sequentially monitor radiation of multiple wavelengths.
The invention is directed to a dual optical fiber probe adapted for immersion into a liquid sample, comprising a first optical fiber adapted to guide incident electromagnetic radiation (e.g. light), and a second optical fiber adapted to receive and guide scattered or emitted electromagnetic radiation (e.g. scattered or fluorescent light) resulting from the incident electromagnetic radiation, wherein the first and second optical fibers are adapted to be optically coupled and define a finite interrogation volume in a liquid sample independent of the total volume of the liquid sample. Preferably, the interrogation volume defined is less than 10-5 cubic centimeters and most preferably less than 10-6 cubic centimeters. The interrogation volume is formed by spatially arranging the ends of the two optical fibers at the fiber/liquid interface to form an interrogation volume defined by the intersecting light cones of each of the optical fibers when the fibers are energized. The optical fibers are arranged such that light emanating from the first optical fiber is projected at an angle relative to the second optical fiber so that light from the first optical fibers does not substantially enter the second optical fiber in the absence of an optical event-causing element in the interrogation volume. The central longitudinal axes of the optical fibers typically are disposed to approximately intersect and form at their intersection an included angle ranging from about 40° to 140°, and most preferably being about 60°. In addition to the included angle, the distance between the longitudinal axis of each fiber at the liquid/fiber interface, the degree of coplanarity of the fibers, and the core diameter and numerical aperture of each fiber is selected to determine the desired size of the interrogation volume.
The dual fiber probe of the present invention is particularly advantageous in that reflection at the interface of the optical fiber and liquid is not a factor, and the background due to reflection from a cuvette wall is minimized. Additionally, by appropriate choice of the physical and optical parameters of the optical fibers, i.e. numerical aperture, core diameter and separation distance, as well as the angular positioning, the size of the interrogation volume can be adjusted to systems of interest. Moreover, a scattering signal and a fluorescent signal, or two fluorescent signals, can be detected at the output fiber with the aid of a beam splitter and two sets of filters, a filter wheel with two filters or a demultiplexer.
FIG. 1 is a schematic of the overall detection apparatus employing the dual fiber optical probe of the present invention;
FIG. 2 is a sectional view of the dual fiber optical probe of the present invention;
FIG. 3 is an end view of the dual fiber optical probe of FIG. 2;
FIG. 4A is a front view of one half of the probe insert for maintaining the optical fibers in a preferred spatial configuration;
FIG. 4B is a side view of the half of the probe insert of FIG. 4A;
FIG. 5A is a front view of the other half of the probe insert for maintaining the optical fiber in a preferred spatial configuration;
FIG. 5B is a side view of the half of the probe insert of FIG. 5A;
FIG. 6 is an illustration of one configuration of the optical fibers of the dual fiber optical probe showing the optical coupling of the fibers and the interrogation volume; and
FIG. 7 is an illustration of system geometry when the optical fibers are non-planar.
For the purposes of the foregoing and following descriptions, "interrogation volume" means a spatial volume, defined by first and second optical fibers having a determined orientation and physical characteristics, in which an electromagnetic (e.g. optical) event (such as fluorescence or light scattering) resulting from electromagnetic radiation (e.g. light) emanating from one optical fiber will be sensed by the second optical fiber. The interrogation volume can be visualized as the intersection of the two light cones emanating from the first and second optical fibers having a determined spatial configuration when each optical fiber transmits visible light. "Optical coupling" means the circumstance where an optical event (such as fluorescence or light scattering) resulting from electromagnetic radiation (e.g. light) emanating from one optical fiber is sensed by a second optical fiber with the proviso that light emanating from the first optical fiber does not substantially enter the second optical fiber in the absence of an optical event-causing element; the two optical fibers are then considered to be "optically coupled".
With reference to FIGS. 1-5, the dual fiber probe 10 of the present invention utilizes an input optical fiber 12 to transmit electromagnetic radiation (e.g. light) from an electromagnetic radiation source 16, and an output optical fiber 14 to transmit subsequently collected radiation (e.g. scattered or fluorescent light) to a detector 18. Radiation source 16 may be of single wavelength or multiple wavelength, appropriately filtered as required by conventional applications. Wavelengths of between 200-1500 nanometers, preferably 400-900 nanometers, can be conventionally utilized. Depending on the particular wavelength desired lasers may be utilized, and Helium/Neon lasers have been found suitable. The probe 10 itself consists of an elongated, probe housing 20, generally consisting of a hard, cylindrical sheath 21 (e.g. a stainless steel tube) surrounding a molded, probe insert 40 in which are mounted optical fiber 12 and optical fiber 14. Optical fibers 12 and 14 are conventionally clad (the cladding not being shown) on their external, circumferential surfaces and terminate in end faces 22 and 24, that typically will be highly polished.
The ends of optical fibers 12 and 14 are rigidly held in a spaced, angularly disposed configuration by insert 40. Typically, insert 40 is molded in two substantially semi-cylindrical halves 41 and 49. Conventional molding materials can be utilized.
One insert half 41 is formed with a lower portion 42 that has a groove 45 (preferably V-shaped) formed in its bottom surface and angularly disposed grooves 44 in its face. Grooves 44 are formed in face 42 at an appropriate angle to each other such that the longitudinal axes of optical fibers 12 and 14 intersect at the desired angle when placed therein. The upper portion 43 of insert half 41 is formed with holes 46 and 48 to accomodate mating members on second insert half 49 to facilitate alignment of the insert halves 41 and 49 when they are joined. A cylindrical abutment 47 surrounding hole 48 is provided as a spacer means in the completed insert assembly.
Second insert half 49 is formed with a lower portion 50 having a groove 52 (preferably V-shaped) in its bottom surface. Groove 52 aligns with groove 45 when insert halves 41 and 49 are joined to form a completed insert assembly. Portion 50 is complementary to portion 42 and is positioned immediately adjacent portion 42 in the completed insert assembly 40 to capture fibers 12 and 14 in a determined spatial configuration and provide for a liquid/fiber interface formed within grooves 45 and 52. Upper portion 51 is formed with members 53 and 54 that are adapted to mate with holes 46 and 48, respectively in insert half 41. A flange 55 surrounds member 54 and cooperates with abutment 47 to provide accurate spacing in the completed assembly of insert 40.
During manufacture of the probe 10, faces 22 and 24 of the emission and collection ends, respectively, of optical fibers 12 and 14 are highly polished and the ends are placed within grooves 44 and fastened in place, typically with an ultraviolet-cured epoxy. Thereafter, sheath 21 is placed about the insert and back-filled with epoxy and cured to provide a completed unitary assembly in which the optical fiber faces 22 and 24 are spatially configured in the manner desired.
As part of the overall probe system, input end 30 of optical fiber 12 receives light transmitted through a focusing means 28, as for example provided by a conventional lens or lenses, which itself receives light from light source 16. Optical fiber 12 transmits andd guides light to end face 22, and into a liquid sample volume in which the probe is inserted. The resulting light from a particle or solution, which may appear either by scattering or fluorescence within the interrogation volume, is received by output light fiber 14 at end face 24 and transmitted and guided via that optical fiber to output end 38 of fiber 14. The emitted output light conventionally is transmitted through filter means 36 and received within housing detector 32 by a photomultiplier tube 34. Conventional systems (e.g. high gain EMI photomultiplier) can be utilized. Filter means 36 may comprise single or multiple band-pass filters, or various beam splitters, to monitor fluorescent or scattered radiation as appropriate. The signal so generated and received is then processed in accordance with conventional methods.
With reference to FIG. 6, the interrogation volume generally designated V (represented in the drawing in two-dimensions by the solid line) can be visualized as the intersection volume of the respective light cones emanating from the end faces 22 and 24 of optical fibers 12 and 14, respectively, when both the optical fibers are energized (i.e. light is transmitted by both fibers). The size of the interrogation volume is determined from the angle of θ and separation distance d of the longitudinal axes of the input and output optical fibers 12 and 14 at the liquid/fiber interface, as well as the core diameter (D) of each fiber and its numerical aperture (N.A.). With reference to FIG. 6, in general the angle θ will have to be greater than the sum of the arcsines of the numerical apertures of the two fibers, where the numerical aperture is the sine of the vertex angle α of the largest cone of meridional rays that can enter or leave the fiber end multiplied by the refractive index of the medium in which the vertex of the core is located. If angle θ is equal or less than this sum, then the outermost rays define a volume that is infinite or undefined. The angle θ, however, must be less than 180° minus the sum of the arsines of the two numerical apertures such that the rays of one fiber cannot enter the other fiber. For a simple case, it can be assumed that the optical fibers have the same optical and physical characteristics, and are located such that the distance of each fiber face from the point of intersection of the longitudinal axes of the two fibers is equal. A reasonable estimate of the interrogtion volume, V, can be given by the following calculation for angles of θ relatively larger than the sum of the arcsines of the numerical apertures. ##EQU1## where N.A.=numerical aperture
D=diameter of fiber core
d=distance between longitudinal axes at liquid/fiber interface
θ=included angle of intersection of longitudinal axes of fibers at liquid/fiber interface.
This approximated interrogation volume is shown in two dimensions by the dashed lines of FIG. 6.
In a presently preferred embodiment, optical fibers 12 and 14 are formed from a 50/125 all-silica step-index fiber having a core diameter of 50 microns; a cladding diameter of 125 microns and a numerical aperture of 0.22. The angle θ and distance d are preferably 60° and 200 microns, respectively. The 60° orientation presents an interrogation volume that facilitates the detection of a limited number of large, very bright particles in the presence of a large number of smaller, less-bright particles, such as for example will occur in an agglutination assay wherein the aggregates are large and highly fluorescent. Additionally, in a preferred configuration the fibers are substantially coplanar. The interrogation volume defined by the dual fiber probes typically will be less than 10-5 cubic centimeters, preferably less than 10-6 cubic centimeters.
The following table illustrates various probe configurations that are suitable. In each instance, the optical fibers are coplanar. The listed volumes of the table are determined from the foregoing formulae. In any instance, the interrogation volume can be measured by passing particles of increasing concentration through the interrogation volume until one particle will always be found in the interrogation volume, as measured by fluctuation analysis or pulse height analysis. The interrogation volume is then the reciprocal of this particle concentration. In general, the smaller the interrogation volume V, the greater the concentration of particles that can be measured.
TABLE 1______________________________________ Axial CoreNumerical θ Separation Diameter VolumeAperture (deg) (microns) (microns) (cm.sup.3)______________________________________.22 60 150 50 1.6 × 10.sup.-6 60 200 50 2.8 × 10.sup.-6 60 300 50 6.4 × 10.sup.-6 90 150 50 7.5 × 10.sup.-7 90 200 50 1.2 × 10.sup.-6 90 300 50 2.5 × 10.sup.-6.22 60 150 100 4.8 × 10.sup.-6 60 200 100 6.9 × 10.sup.-6 60 300 100 1.3 × 10.sup.-5 90 150 100 2.6 × 10.sup.-6 90 200 100 3.5 × 10.sup.-6 90 300 100 6.0 × 10.sup.-6.11 60 150 50 5.4 × 10.sup.-7 60 200 50 7.8 × 10.sup.-7 60 300 50 1.5 × 10.sup.-6 90 150 50 3.1 × 10.sup.-7 90 200 50 4.2 × 10.sup.-7 90 300 50 7.2 × 10.sup.-7.11 60 150 100 2.2 × 10.sup.-6 60 200 100 2.8 × 10.sup.-6 60 300 100 4.3 × 10.sup.-6 90 150 100 1.5 × 10.sup.-6 90 200 100 1.8 × 10.sup.-6 90 300 100 2.5 × 10.sup.-6.11 40 50 10 2.4 × 10.sup.-8 40 500 10 6.7 × 10.sup.-6 40 1000 10 4.9 × 10.sup.-5 140 50 10 4.8 × 10.sup.-9 140 500 10 3.9 × 10.sup.-7 140 1000 10 2.5 × 10.sup.-6.11 40 50 200 1.3 × 10.sup.-5 40 500 200 6.2 × 10.sup.-5 40 1000 200 1.9 × 10.sup.-4 140 50 200 1.0 × 10.sup.-5 140 500 200 2.1 × 10.sup.-5 140 1000 200 3.8 × 10.sup.-5.7 90 50 10 5.2 × 10.sup.-7 90 500 10 3.6 × 10.sup.-4 90 1000 10 2.8 × 10.sup.-3.7 90 50 200 2.0 × 10.sup.-5 90 500 200 7.4 × 10.sup.-4 90 1000 200 4.1 × 10.sup.-3______________________________________
With reference to FIG. 7, the spatial relationships of the fiber ends 22 and 24 located in probe 20 can be defined by constructing a pair of parallel planes passing through each fiber end and longitudinal axis where the planes are separated by a distance h, the dihedral angle between the fibers (defined by the angle O1 'P'O2 between the plane described by lines O1 P and PP' and the plane described by line O2 P' and PP') is θ. When the ends of the optical fibers are coplanar (i.e. h=0), the dihedral angle becomes the included angle between the intersecting longitudinal axes of the fibers. In the description herein, it will generally be assumed that the fiber ends are substantially coplanar so that the dihedral angle and included angle are substantially the same. In general, the minimum θ that will provide a finite overlap volume will be greater than arcsin A plus arcsin B where A and B are the numerical apertures of the two fibers. Preferably h=0 and θ=40°-140°, preferably 40°-120°, more preferably 40°-80°, usually about 60°.
When h=0, it is preferable for the distances O1 P and O2 P' to be as small as possible by causing the optical fibers to nearly contact each other. Higher values of the distances O1 P and O2 P' are acceptable provided the overlapping volume criterion is met. When h ≠0, the cones will not overlap optimally and thus the intensity of light projected from one fiber that is scattered will be suboptimally collected by the other fiber. Balancing the disadvantage of this reduction in sensitivity is the opportunity provided by this configuration for greater flexibility in selecting the size of the overlapping volume. Thus, as h is increased, the overlapping volume of the intersecting cones can be decreased to any desired value. Obviously, h must not be so large that this volume is reduced to zero. In practice, a compromise will be made between reducing the volume on the one hand and loss of signal intensity as a result of reduction of light collection on the other hand. Moreover, when small volumes are defined by increasing h, the volume will become exceptionally sensitive to the acceptance angle of the fibers. Since the latter varies with refractive index of the fluid, a probe constructed in this way could become overly sensitive to refractive index.
The values of O1 P and O2 P' likewise are dictated by practical considerations such as the ease and reproducibility of probe assembly and light collection efficiency. In general, as O1 P and O2 P' increase, the overlapping volume increases. Selection of minimum values for O1 P and O2 P' will therefore be preferred.
The core diameters of the fibers, that is the portion of each fiber carrying the light, will be the range of 10-200 microns, preferably 25-75 microns. The core diameters may be the same or different, most preferably the same, and it will usually be desirable to minimize the acceptance angle rather than core diameter in order to reduce the overlap volume without loss of light collection efficiency. When the core sizes are different, it will be preferable that the core of the excitation light fiber be smaller. Numerical apertures in air of the optical fibers of 0.09-0.7 can be used. In the type of applications described herein, numerical apertures of 0.11-0.24 can be used, with 0.22 being preferable. The angle θ can be between about 40 to 140 degrees. For clinical applications, 40-80 degrees is suitable, 55-65 degrees more typical and 60 degrees is preferred. Angles of 90° or greater are suitable for simply counting fluorescent particles or creating a more homogeneous interrogation volume in which any fluorescent particle will be detected. The separation distance between the longitudinal axes of the optical fibers at the fiber/liquid interface typically is in the range of about 0.05-1.00 millimeters, preferably 0.2 millimeters.
In general, probes of this invention are used by illuminating one optical fiber at the end distal to the probe with the illuminating light and monitoring scattered or fluorescent light collected by the proximal or probe end of the second optical fiber with a photodetector at the distal end of the optical second fiber. For detection of particles, the probe assembly is immersed in a sample of the fluid to be analyzed and moved relative to the fluid to provide for sequential interrogation of multiple small volumes defined by the interrogation volume. In this way detection of light scattering produced internally in the illuminating fiber 12 is largely excluded from detection, the background signal is correspondingly reduced, and the sensitivity thereby increased. Detection of particles either by conventional light scattering or fluorescence can be carried out by monitoring the fluctuations of the scattered or emitted light, as for example by conventional pulse height analysis or time correlation methods.
In certain cases, it will be desirable to measure both fluorescence and Raleigh light scattering from the same sample. Where it is sufficient for this to be done sequentially, the above described system can be used. Where simultaneous measurement is required, the Raleigh scatter can be measured at the distal end of output fiber 14 and the fluorescence can be measured by collecting the scattered light passing back up input fiber 12 by use of a splitter. For example, one could use means such as described in U.S. Pat. No. 4,564,598 which is incorporated herein by reference. Other conventional methods could be used as well.
When the apparatus described is utilized to irradiate a receiver suspected of containing an event causing element (e.g. analyte), the signal resulting from the irradiation of such an element can be detected and measured in an enhanced manner relative to the background radiation. The selected element can comprise diverse entities depending on the measurement system in question. For example, the element can be a particle which fluoresces or elastically scatters light in response to incident radiation. It may be a particle which absorbs incident radiation such that the presence of the particle is detected by a negative fluctuation from a base value for the overall electromagnetic signal emitted. As a further example, the element or object may be an entity having a characteristic causing it to emit a greater signal than the same entity not having the particular characteristic (e.g. a cell having an abnormality which causes it to emit a larger or more powerful signal than that same cell not having the abnormality).
The subject invention has particular application for determining an analyte in a sample, where the amount of analyte affects an observed pattern of fluorescence fluctuations. The analyte is a member of a specific binding pair consisting of ligand and its homologous receptor. The input and output optical fibers are employed to irradiate a portion of the sample volume and to receive fluorescent light from the sample volume, respectively. One observes a plurality of such volumes, either by observing a single volume over an extended period of time, where particles move in and out of the volume, or scanning a plurality of volumes either simultaneously or successively, or combinations thereof. Thus, the percentage of volumes observed which have a predetermined difference in fluorescence from a defined level can be related to the amount of analyte in the medium.
The fluctuations in fluorescence can be achieved by various combinations of particles and continuous media. For example, the combinations can include particles which fluoresce at constant intensity in a non-fluorescing solution, particles which fluoresce at varying intensity in a non-fluorescing solution, particles which are non-fluorescent in a fluorescent solution and fluorescent particles in a fluorescent solution. Furthermore, the fluorescent fluctuation may be a result of aggregation of particles, non-fluorescent particles becoming fluorescent, or fluorescent particles becoming non-fluorescent. The particles may be comprised of polymers, both naturally occurring or synthetic, natural particles, such as virions and cells, e.g., blood cells and bacteria, or the like. Particle sizes will vary from 0.05 to 100 microns, where synthetic particles will generally be from about 0.1 microns to 10 microns diameter.
By employing the above-described method in a fluorescent assay, a large number of protocols and reagents may be employed. One group of protocols will involve measuring fluorescent particles. This group can be further divided into particles which remain uniformly fluorescent, that is, there are basically two particle populations, fluorescent or non-fluorescent, where fluorescence above a certain level is defined as a positive or negative result. The invention has been found to be particularly advantageous in protocols in which a fluorescing molecule is conjugated directly to an antibody (Ab), which then binds directly to a cell.
A heterogeneous population of fluorescent particles can come about in a number of ways. For example, one can have aggregation or agglutination of particles. The analyte could be a receptor or antibody, which is polyvalent in binding sites. Fluorescent particles could be conjugated with ligand, so that the polyvalent receptor would act as a bridge between particles. In this way, the greater the amount of analyte present in the medium, the larger the number of aggregates which will result. The particle of interest could then be chosen as a particle which is an aggregation of two or more or three or more particles. Furthermore, by appropriate electronic means, one could determine the size of the aggregation, counting not only the total number of particles, but the number of members of each population. As the aggregation increases in size, the fluorescence of the aggregate particle will also increase, but not linearly with the increase in the number of particles in the aggregation.
In a second way for having a heterogeneous population, one could have a non-fluorescent particle, where fluorescent molecules become bound to the particle in proportion to the amount of analyte in the medium or to the number of binding sites on the particle. For example, one could have fluorescent molecules bound to an antiligand. Ligand could be bound to a non-fluorescent particle. The fluorescer conjugated antiligand would be combined with the analyte containing sample, so that the analyte could fill the binding sites of the antiligand, with the remaining binding sites being related to the amount of analyte in the sample. Upon addition of the ligand conjugated particles to the medium, the remaining fluorescent conjugated receptor would bind to the particles, providing for a distribution of particles of varying fluorescence.
A third technique may also be illustrated by employing an aggregation. In this technique, non-fluorescent particles are employed, and the continuous phase is made fluorescent. Thus, when the aggregation is present in the sample volume, there will be a substantial diminution in the observed fluorescence. These particles, while non-fluorescent should also be substantially opaque to excitation of fluorescent light. Thus, they will create a substantial shadow, inhibiting the detection of fluorescence in a volume substantially greater than the volume of the aggregation.
A fourth way of obtaining a heterogeneous population of fluorescent particles is to allow a fluorescent tag to label non-fluorescent particles. For example, non-fluorescent particles may be cells which have a plurality of antigens on the cell surface, there being a number of each antigen present. By employing fluorescer-labeled-antibodies to specific surface antigens, a specific set of non-fluorescent cells will become fluorescent. The detection of the presence of such cells is a method of cell identification, e.g. red blood cell (RBC) grouping and typing. For example, in the A, B, O system, if the fluorescent tag were conjugated to anti-A antibody, binding would occur and there would be a greater increase in cell fluorescence if the sample contained the A antigen of type A or type AB blood than if the analyte contained blood types B or O. Alternatively, cells can also be labeled by employing a fluorescent cell-incorporation dye such as those described in European Patent Publication No. 176,252, published Apr. 2, 1986, which is incorporated herein by reference.
In addition to antibodies, certain lectins are known to bind in varying degrees to RBC surface antigens, and are convenient receptors for use in fluorometric assays.
Usually, there will be a distribution of levels of fluorescence, although in some situations it will be feasible to substantially saturate the available binding sites on the cell surface, so as to approximate only two populations, non-fluorescent cells and cells of substantially uniform fluorescence.
While not presently preferred, typing red blood cells (RBCs) or identifying red blood cell (RBC) antigens or the antibodies thereto can be effective by using the RBCs as fluorescence quenchers in an assay employing fluorescent particles to provide a detectable signal. Substances which bind to RBC antigens, normally antibodies or lectins (hereinafter "receptors") are conjugated to fluorescent particles. A solution of particle-conjugates is combined with red blood cells, e.g., whole blood, with an appropriate buffer. If an antigen is present on the RBCs that has a binding or determinate site specific for the receptor, the conjugated particles will bind to the RBCs which act as fluorescence quenchers.
Also, the determination of the presence of antibodies to a RBC antigen can be made. Three different techniques may be used. In one, fluorescently labeled antibody compete with antibodies in the plsma or serum sample for antigen sites on test RBCs of a known group, with the observed cellular fluorescence decreasing with increasing amounts of antibodies against the specific antigen in the sample. Alternatively, the test RBCs may be fluorescently stained and, when combined with serum, the specific antibodies, if present, will agglutinate the fluorescent cells. In a third method, the fluorescent bead may be conjugated with the surface antigen of interest and antibodies present in the sample act as a bridge between RBCs of known type and the antigen conjugated fluorescent particles. In this situation, decreasing fluorescence would indicate the presence of the antibodies.
High extinction coefficients for the fluorescer are desirable and should be greatly in excess of 10,000 cm-1 M-1 and preferably in excess of 100,000 cm-1 M-1. The fluorescer should also have a high quantum yield, preferably between 0.3 and 1.0.
In addition, it is desirable that the fluorescer have a large Stokes shift, preferably greater than 20 nm, more preferably greater than 30 nm. That is, it is preferred that the fluorescer have a substantial spread or difference in wavelengths between the absorption and emission maxima.
One group of fluorescers having a number of the desirable properties are the xanthene dyes, which include the fluoresceins derived from 3,6-dihydroxy-9-phenylxanthhydrol and rosamines and rhodamines, derived from 3,6-diamino-9-phenylxanthene. The rhodamines and fluoresceins have a 9-O-carboxyphenyl group, and are derivatives of 9-O-carboxy-phenylxanthene.
These compounds are commercially available with or without substituents on the phenyl group.
Another group of fluorescent compounds are the naphthylamines, having an amino group in the alpha or beta position, usually alpha position. Included among the naphthylamino compounds are 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate and 2-p-toluidinyl-6-naphthalene sulfonate. Other fluorescers of interest include coumarins, e.g., unbelliferone, and rare earth chelates, e.g., Tb, Eu, etc. Descriptions of fluorescers can be found in Brand, et al., Ann. Rev. Biochem., 41, 843-868 (1972) and Stryer, Science, 162, 526 (1968). Still another group of fluorescers are the squarate dyes described in Euopean Patent Publication No. 176,252 noted previously.
Appropriate particles are combined with the fluorescer using standard techniques to provide fluorescent beads or microspheres. Fluorescent particles are commercially available. The fluorescent beads may be varied widely as to size and composition. The beads will normally be made of an inert material and include a plurality of fluorescent chromophoric functionalities. The beads will have a sufficient concentration of fluorescent functionalities to provide for a large signal per bead. Various organic polymers may be employed for the bead, e.g., polystyrene, polymethacrylate or the like or inorganic polymers, e.g., glass or combinations thereof. The particular choice of the polymeric composition is primarily one of convenience.
Conjugated to the fluorescent beads either covalently or non-covalently are receptors which may be antibodies, including monoclonal antibodies, or lectins, that bind either specifically or differentially to specific RBC surface antigens or antigens having the determinant site(s) of such RBC surface antigens or other antigens of interest.
The receptors are absorbed to the fluorescent bead using standard techniques extensively described in the literature. Alternatively, the receptors may be covalently bound by conventional techniques.
The sequential or simultaneous measurement of scattered and fluorescent light in conjunction with the present apparatus permits a particularly facile method of detecting the presence of an analyte suspected of being in a fluid sample by detecting a first event (e.g. agglutination) associated with the presence of the analyte and a second event (e.g. absence of agglutination) associated with an interfering element that potentially can interfer with the detection of the first event. Other applications include cell differentiation, (such as with double fluorescent labeled cells) aerosol detection and measurement, tracer flow controls, and the like, wherein a multitude of exiting wavelengths is utilized by beam splitting or use of a multitude of output fibers.
The present invention contemplates equivalent multi-fiber probes, e.g. tri-fiber optic probe having three optical fibers, wherein the third optical fiber is introduced at the apex of the V-groove of the dual fiber probe, such that all three fibers have a common interrogation volume. The tri-fiber probe could be used in the manner described herein with two detector units, each having a filter for specified wavelengths, associated with two of the optical fibers and the light source associated with the third optical fiber.
The dual fiber probe of the present invention is useful in a method of determining the presence or absence of an analyte that involves adding a fluorescently tagged receptor for an analyte to a fluid sample that is suspected of containing the analyte and possibly an interfering substance. Next, the combined sample/receptor is processed to remove the suspected interfering substance, such as by washing or the like. Then the processed sample/receptor is treated with an event actuator (e.g. an agglutination reagent) and irradiated. The fluorescent signal at the emitted wavelength and the scattered signal at the incident wavelength are measured and compared to their respective background values. With analyte positive samples in the absence of an interfering substance, either originally absent or effectively removed by processing of the sample, an increase in the fluorescent particle signal heights from background indicates the presence of the analyte. Analyte negative samples are evidenced by fluorescent particles heights substantially the same as background and increased light scattering particle signal heights relative to background. Improperly processed samples (i.e. those in which the interfering substance has not been effectively removed) are evidenced by the absence of fluorescent particle signal heights in excess of background and a suppression of light scatter particle signal heights relative to background.
In the following examples, the dual fiber probe is formed in the manner described herein from 50/125 all silica step-index fiber having a core diameter of 50 microns; a cladding diameter of 125 microns and a numerical aperture of 0.22. The angle θ and distance d are 60° and 200 microns, respectively, and the fibers are coplanar.
The dual fiber probe is employed in a method for screening for antibodies in a patient's blood. In this method, test cells bearing various surface antigens are stained with fluorescent dye. After mixing with the patient's plasma, the test cells are thoroughly washed and combined with anti-human immunoglobulin antiserum. Typically, when antibodies are present, this reagent will agglutinate the test cells. The present device permits detection of the agglutinated cells with the dual fiber probe while illuminating with 633 nm light. Using a particle signal height discriminator (i.e. event height discriminator), the magnitude of the light pulses detected by the photodetector at the distal end of the second fiber signals an agglutination event.
Failure to properly wash the test cells can lead to residual immunoglobulins in the solution that may block the agglutination by the anti-human immunoglobulins. The present device makes it possible to provide a control to assure that the test cells are properly washed. For this purpose, unstained check cells that were coated with human IgG were added to the washed test cells. Only if washing of the test cells is complete will anti-human immunoglobulin aggregate the check cells.
The assay was carried out by first monitoring the fluorescence particle signal heights using a filter between the second fiber and the photodetector to remove 633 nm excitation light. The filter was then changed to a 633 nm band pass filter in order to monitor 633 nm pulses from light scattered by agglutinated check cells. With antibody positive samples, increased fluorescent particle signal heights showed that there was test cell agglutination.
With antibody negative samples, no fluorescent pulses in excess of background were observed showing that agglutination did not occur. Confirmation that the failure of the test cells to agglutinate was not due to poor washing was shown by the ocurrence of increased light scattering particle signal heights in excess of background at 633 nm. Deliberate failure to properly wash led to a suppression of the light scatter particle signal heights relative to background level.
The sensitivity of the dual fiber probe is demonstrated by measurement of the fluorescent response to various solutions of fluorescent dye, 2-(p-diethylamino-m-hydroxyphenyl)-4-(4-diethylimmonio-2-hydroxy-2,5-cyclo-hexadienylidene)-3-oxo-1-cyclobutenolate (DEAS), in 10-3 M β cyclodextrin:
______________________________________ Dye ConcentrationAverage Fluorescence (M)______________________________________13 2 × 10.sup.-924 4 × 10.sup.-933 5 × 10.sup.-946 7 × 10.sup.-959 9 × 10.sup.-965 1 × 10.sup.-8151 2.5 × 10.sup.-8180 3 × 10.sup.-8227 4 × 10.sup.-8281 5.5 × 10.sup.-8329 7 × 10.sup.-8______________________________________
Fluorescent response is shown to increase with increasing dye level.
The following example illustrates the application of the dual fiber probe of this invention for detecting different populations of fluorescing objects (i.e. particles). A population of small latex beads (0.71 microns) were dyed with a squaraine dye (DEAS) at 1×fluorescence and a population of larger latex beads (1.1 micron) were dyed in a similar manner to provide a bead of 4×fluorescence (i.e. the fluorescence of the larger beads are approximately four times that of the smaller beads).
A peak height detector was utilized to determine a sample distribution of number of events versus fluorescence at peak height. That distribution was analyzed to provide an index (i.e. agglutination index) that is the sum of the number of events in each fluorescence level times the difference in fluorescence between that level and the fluorescence cutoff level (set at a multiple of the average fluorescence [av] of the distribution). Differences in the agglutination index at any cutoff indicate an increased fluorescence level due to the presence of the larger bead.
Representative results are shown below:
______________________________________ Agglutination IndexCutoff Run 1 Run 2______________________________________Composition 1:Isotonic buffer containing 0.71micron dyed latex bead at 2.5 ×10.sup.6 beads/mlav* 1.7 880 771av* 1.9 338 273av* 2.0 208 153av* 2.1 208 153av* 2.2 125 86Composition 2:Isotonic buffer containing 0.71micron dyed latex bead at 2.4 ×10.sup.6 beads/ml and 1.1 micron dyedlatex bead at 0.15 × 10.sup.6 beads/mlav* 1.7 2012 2145av* 1.9 1221 1368av* 2.0 950 1095av* 2.1 950 1095av* 2.2 739 885______________________________________
The greater agglutination index of Composition 2 as compared to that of Composition 1 illustrates the detection of the larger beads.
The dual fiber probe and the method described in Example 3 was utilized with the following protocols to illustrate the use of the dual fiber probe in blood typing applications.
The protocols for the various assays are as follows:
10 μl of dyed whole blood (50 μM DEAS; 95% DMSO, 5% H2 O) is mixed with 10 μl of Reagent (Anti-A: 149 mg/ml Celltech cell line 3D3 in buffered saline; Anti-B: 110 mg/ml Celltech cell line NB1 in buffered saline) for about 21/2 minutes, then diluted with 500 μl of buffered saline solution. The fluorescence of the resulting solution is read in the manner described in Example 3.
50 μl of undyed whole blood is mixed with 10 μl of dyed (tetrabutyl squarate: 110-4 μM is dimethylacetamide, 75 μl per ml of 30% red cell suspension in buffered saline, 2% Pluronics environment) A1 or B cells, as the case may be, and mixed for about 21/2 minutes, then diluted with 50 μl of buffered saline. The fluorescence of the resulting solution is read in the manner described in Example 3.
10 μl of dyed (DEAS) whole blood is mixed with 10 μl of Reagent (Rh: 0.9 mg/ml IgG [reduced and alkylated], 2% PVP, 2% BSA in tris buffer; Rh control: same excluding IgG) for about 21/2 minutes, then diluted with about 100 μl of buffered saline. After about 40 seconds of additional mixing, 450 μl of isotonic buffered saline is added and the resulting solution is read in the manner described in Example 3.
The results for A negative and B positive samples are summarized below with cutoffs determined for αA, αB and Forward Control at 1.7 times the average fluorescence, for A1 cells and B cells at 2 times the average Fluorescence and for Rh, Rh control at 1.8 times the average fluorescence.
__________________________________________________________________________ Forward RhαA αB Control A.sub.1 Cell B Cell Control Rh__________________________________________________________________________A negative 5867 0 0 0 12158 0 14B positive 15 12296 140 11248 15 7 11908__________________________________________________________________________
While the foregoing invention has been described with reference to the drawings, the Examples and the presently preferred embodiments, they are intended to be illustrative and not to limit the scope of the invention herein claimed. Various modifications or changes to the methods and apparatus described herein will be apparent to those skilled in the art and are intended to be included and encompassed by the claims appended hereto.
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|U.S. Classification
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|International Classification
|Cooperative Classification
|European Classification
|Jun 9, 1988
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text:
The other shoe drops from last week's intra-Colorado House GOP infighting, Denver Post's Anthony Cotton:
Rep. Kevin Priola, R-Henderson, said Monday he is resigning as Republican minority whip in the wake of an internal party squabble last week…
During a debate last Thursday on dueling amendments to the Student Success Act, which will provide funding to K-12 schools, Priola backed Democratic co-sponsor Rep. Millie Hamner, D-Dillon, over fellow Republican Rep. Jim Wilson, R-Salida. The move drew the immediate ire of a number of Republicans, who alleged Priola wasn't acting in keeping with his role as whip.
Within hours, the Republican caucus held a meeting, with Rep. Chris Holbert, R-Parker, leading an effort to remove Priola. The attempt eventually failed, but it was clear Priola was on shaky ground within the party.
As we discussed last week, there is a great deal of frustration building among conservative Republicans in the Colorado General Assembly. After stoking outrage to a fever pitch during last year's successful recall campaigns against Democrats in the Senate, conservatives have suffered a wave of setbacks in 2014–failing to pack committee hearings for stillborn repeal measures, ridicule after showing up to hearing unprepared to debate their own bills or call witnesses, and widespread criticism of unpopular legislation introduced by Republican legislators like this year's total abortion ban bill. It seems that frustration boiled over last week, when Rep. Kevin Priola supported a Democratic amendment to the Student Success Act over an amendment offered by fellow Republican Rep. Jim Wilson.
The swift retribution campaign against Priola headed by Rep. Chris Holbert ended embarrassingly when caucus leadership declared the move out of order, but we're not at all surprised to see Priola resign from House leadership today. At this point, the caucus would have been weakened further if Priola had not resigned from leadership, even though Priola is not erratic enough to pull a Kathleen Curry and disaffiliate from the GOP altogether. It's expected that the candidate named to replace Priola in last week's dustup, Rep. Polly Lawrence, will be the next minority whip.
With hard right Republican legislative candidates continuing to do well in the primary process, conservatives in the legislature may yet feel empowered to throw their weight around. Don't look for anything to change there until the June primary–or maybe until after November.

subset: commoncrawl


text:
AED Bookstore
AED has partnered with Powell's Bookstore to provide the best prices on more than 200 suggested titles for eating disorders professionals and the general public. For each book purchased through the AED Bookstore, AED will receive a 7.5% dividend. So, your purchases are helping support AED operations!
See the books below and check out more books on eating disorders at AED's Bookstore Partner, Powell's.
AED Featured Titles
Proceeds from the below books benefit the AED:
Kathy Kater has generously pledged to donate all profits from her latest book, Healthy Bodies: Teaching Kids What They Need to Know, to the AED. Promoting health instead of size, this book offers a comprehensive, primary prevention curriculum for addressing body image, eating, fitness, and weight concerns in today's challenging environment.
Aimee Liu has generously pledged to donate proceeds from her latest book, Restoring Our Bodies, Reclaiming Our Lives, to the AED Scholarship Fund.
Books for eating disorders professionals and the general public:
A Trick of the Light, by Lois Metzger
Stop Eating Your Heart Out, by Meryl Hershey Beck
Overcoming Binge Eating for Dummies, by Jennie J. Kramer and Marjorie Nolan Cohn
End Emotional Eating, by Jennifer Taitz
Woman in the Mirror: How to Stop Confusing What You Look Like with Who You Are, by Cindy Bulik
Decoding Anorexia: How Breakthroughs in Science Offer Hope for Eating Disorders, by Carrie Arnold
Night Eating Syndrome, by Jenny Lundgren, Kelly Alison, Albert Stunkard
Your Dieting Daughter (2nd edition), by Carolyn Costin
Food to Eat, by Lori Lieberman and Cate Sangster
Ed Said, U Said: Eating Disorder Translator, by June Alexander
Almost Anorexic, by Jenny Thomas and Jenni Schaefer
Overcoming Binge Eating (2nd edition), by Christopher Fairburn
Animal Models of Eating Disorders, by Nicole Avena
Check out more books on eating disorders on Powell's:
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- Last Updated: 17 March 2016


text:
COMMUNICATION with completely paralysed people may be possible by simply looking into their eyes, scientists have found.
New research has shown that patients who are otherwise unable to communicate can quickly answer yes or no questions with a simple system that measures the size of their pupils.
Using just a laptop and a camera, the researchers were able to measure changes in pupil size that happen automatically when someone does mental arithmetic.
The technique required no specialist equipment or training: the study involved showed healthy test subjects answers to yes or no questions along side a maths problem, who were asked to solve the problem only if the correct answer was showing.
The mental strain of the calculation naturally caused an increase in pupil size, which the researchers could successfully measure and translate into accurate answers to questions such as: "Are you 20 years old?".
The technique was also tested on seven patients who suffered from locked-in syndrome, having suffered brain damage as a result of strokes. In many cases the researchers successfully used pupil size alone to discern their responses to the same kinds of questions.
As well as helping with basic communication for all kinds of patients suffering from severe physical impairments, the researchers also hope it could be used to assess the mental state of patients when doctors are unsure if they are conscious or not.
"We find it remarkable that the system worked almost perfectly in all healthy observers and then could be transferred directly from them to the patients, with no need for training or parameter adjustment," said lead researcher, Wolfgang Einhäuser of Philipps-Universität Marburg in Germany.
"For patients with altered state of consciousness—those who are in a coma or other unresponsive state—any communication is a big step forward."


text:
Dear All,
I am a 45 year old. I passed my CBT and theory test last year and have just passed my Mod 1 but I have a problem (amongst many too disturbing to detail on the web!) -- I struggle getting up to speed -- being knocked about above 50mph is really disconcerting for me.
I know this is all about experience and confidence but frankly when I'm out on my bicycle I don't particularly enjoy fast downhills due to "wind". But I really enjoy being out on the bike (motorised and pedal based).
I think I am going to struggle with mod2 when it comes to zooming up the dual carriageway at 60+ mph to prove that I can "maintain progress". I have absolutely no problem with the concept and I know that it isn't going to change because it doesn't suit me; so what advice please on getting that speed up?
My personal view is:
* Get a very faired and screened up bike for the mod2 -- but all the training schools in the area lack these bikes to train on.
* Buy a suitable bike ahead of mod2 -- am I likely to get insurance?
I know I sound like an old granny but any views / advice would be appreciated, I'm keen to "progress". I have been taking my YBR out and trying to increase speed gradually but I think it will only get me so far.
Cheers, Chris


text:
Petrol pump prices edge higher
The cost of filling up at the pumps has edged up over the last month, with diesel drivers getting a worse deal than those using petrol, according to figures from the AA.
The average price of petrol in the UK has risen from 133.35p a litre in mid-May to 134.61p in mid-June, while diesel has gone up from 138.17p a litre to 139.16p.
Northern Ireland has the most expensive petrol, at an average of 135.8p a litre, with London having the cheapest, at 134.61p.
Northern Ireland also has the dearest diesel (139.8p a litre) with London and south-west England having the least expensive (139.1p).
The AA said the slight rise in average petrol prices nationally represented "something of a lull" after the 8-10p swings in prices over the last 12 months. But it warned that this year retailers have on average been "creaming up to £1 a tank extra off diesel car drivers and up to £1.40 a tank extra off diesel van owners".
The AA continued: "At present, the 1p-a-litre premium that fuel stations are generally adding to the cost of diesel adds 5,500 miles to the break-even point for a new car buyer who chooses diesel instead of petrol. Diesel cars typically cost £1,500 more but the saving from better fuel efficiency should eventually recoup that."
AA president Edmund King said: "To be fair, there is often much greater variation in the price of diesel among retailers in a town than with petrol. However, on average, the profit margin on diesel is consistently at least a penny higher than with petrol. The clear message to diesel drivers is to take advantage of the greater range of prices locally. Some forecourts are more diesel-friendly than others."
Meanwhile, the AA highlighted HM Revenue and Customs' figures showing that UK petrol sales rose last month to 1.56 billion litres - a 49 million litre increase on the April 2013 figure. This followed record low figures for February and March this year.
Government tax receipts from petrol sales recovered to £904 million in May 2013 - up £28 million on the month before and £107 million higher than the year low in March.
Diesel sales, which include haulier and other commercial use, fell back from 2.29 billion litres in April to just under 2.26 billion in May. Overall, petrol and diesel sales in May were the highest so far this year at just under 3.82 billion litres, bringing in fuel duty receipts of more than £2.21 billion.


text: The Warrior Woman wiki last edited by lokiofmidgaard on 03/03/17 12:05PM View full history OriginJulia Koenig, a German woman living in Germany in the 1930's hypnotized an American soldier into telling her the formula to the Super Soldier Serum that created Captain America. Reproducing it, she was accidentally transformed into a Nazi powerhouse.Hitler saw the advantage of a strong Nazi woman and asked her to become his own sick twisted idea of a Nazi superhero, the Warrior Woman. Hitler also coerced her into marrying her male opposite, Wilhelm Lohmer, the original Master Man. The dictator envisioned Warrior Woman and Master Man breeding a new, superhuman Aryan race. However, Koenig found Lohmer repulsive, a testament to everything she hated about men. Their marriage was interrupted by Captain America and his allies, the Invaders. Warrior Woman and Master Man fought the Invaders on other occasions, sometimes at their leader's insistence, and other times in concert with others, like Lady Lotus' Super-Axis.At the end of World War II, as the Third Reich's defeat was imminent, Baron Wolfgang Von Strucker arranged for Koenig and her "husband," to be placed in suspended animation in order to keep the dream of the Reich alive in the future. Warrior Woman was revived, and Axl Nacht, a new Master Man, sought to prove himself as her more ideal mate than Lohmer. The couple managed to escape after a conflict with Namor, the Sub-Mariner, Captain America, and Jim Hammond, the android Human Torch.Warrior Woman, Julia Koenig, is currently at large. She has no known connection to "Warrior Woman" of the Fearless DefendersCreationWarrior Woman was created by Roy Thomas and Jim Mooney in 1977 and first appeared in The Invaders # 16.


text:
Sanford Hospice offers grief support, training
WORTHINGTON -- Sanford Hospice is offering a grief support group open to anyone who has experienced a loss through death.
The group will meet from 4:30 to 6:30 p.m. Wednesdays beginning Sept. 12 at Sanford Worthington Medical Center.
Additionally, a training class is also being offered for anyone interested in volunteering for Sanford Hospice in the Jackson, Windom or Worthington areas. The class will be from 9 a.m. to 4 p.m. Aug. 28 at Sunset Cottage, Worthington.
Pre-register for the group or the class by calling 1-866-655-1017.


text:
Free Your Mind and the Rest WILL Follow!
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The 'Truth' About Tobacco and Other Adorable Lies
"Warning: No beagles, monkeys, nor rodents Ever contracted lung cancer from systematically exposing them to tobacco products, so just go on and have a nice day." Wouldn't this message from our Surgeon General be a refreshing way to open a … Continue reading
Life Whispering 101
They call me… "The Life Whisperer" (Okay, no they don't… but you can if you want to.) I want to show YOU how to be your very own Life Whisperer! There is much talk of experiencing an epiphanous "Aha … Continue reading
Got Love?
"That's it! I'm done!" I remember saying these words, (originally with more emphasis on superlatives.) I meant it, I was no longer going to pursue any sort of romantic relationships… ever. I wasn't broken hearted or sad, I was just … Continue reading
Are You Done Waiting To Be Happy?
Happiness is… A Warm Gun or a Charley Brown episode? Depends… do we believe that 'if such and such wasn't here' life would be grand or do we believe that it is only found in over simplified concepts through animated … Continue reading
How to Let Go of Worry
Have you ever woken up and had that feeling?… you know, the one … Continue reading
The Key to Unlocking Your Wealth
I Found The Key! I have spent many a year trying to figure out "What the hell is wrong with me?" Try as I might I never seemed to find a way to get more of the 4 Squares into … Continue reading
How To Improve Your Memory
"Remember that guy?… you know, the one with the dark curly hair that we met at the pier when we were on spring break in 81?" …uuuhhh… No. Who care about something so long ago? Right? Truth?… I could hardly remember what … Continue reading
My Fab 5 Anti-Aging Secrets
This is gonna sound Blasphemous! My Mama told me something that hit me like a ton of bricks… I will never forget it. Recently I went to visit her after many years of being separated by many miles of highway. … Continue reading


text:
As expected the Ultimate Fighting Championship kicks off the new year with a UFC 125 event on Jan. 1.
Officials today formally announced the event, dubbed "UFC 125: Resolution," with co-headliners of lightweight champ Frankie Edgar vs. Gray Maynard and heavyweights Shane Carwin vs. Roy Nelson.
Four other fights are official for the card, which airs on pay-per-view from the MGM Grand Garden Arena in Las Vegas.
This past week MMAjunkie.com (www.mmajunkie.com) passed along a report from the "UFC Ultimate Insider" Web series confirming the co-headliners, as well as a lightweight main-card bout between Clay Guida and Takankori Gomi.
As previously reported, the event also features middleweights Chris Leben vs. Brian Stann, welterweights Nate Diaz vs. Dong Hyun Kim, and light heavyweights Brandon Vera vs. Thiago Silva.
"We always put a huge card together in Vegas to kick off the New Year," UFC President Dana White stated. "This is going to be a killer start to 2011."
Tickets for the event go on sale later this week. (Stay tuned for details.)
Edgar and Maynard first met at UFC Fight Night 13 in 2008, and it was the current challenger who came out on top. In that fight, Maynard delivered Edgar his lone career defeat.
However, Edgar bounced back with a pair of decision wins over Hermes Franca and former champ Sean Sherk, as well as a submission win over Matt Veach, and was awarded a title shot against then-champion B.J. Penn. Despite seemingly insurmountable odds against him, Edgar defeated Penn in a hotly contested UFC 112 decision and then did it again in more dominant fashion in August during a rematch at UFC 118.
Maynard, meanwhile, followed up his win over Edgar with a five consecutive decision victories over a murderers row of opponents in Rich Clementi, Jim Miller, Roger Huerta, Nate Diaz and Kenny Florian. Despite his undefeated record, the Michigan State University wrestler was passed over in favor of Edgar for the first shot at Penn.
The latest UFC 125 card now includes:
- Champ Frankie Edgar vs. Gray Maynard (for lightweight title)
- Shane Carwin vs. Roy Nelson
- Takanori Gomi vs. Clay Guida
- Chris Leben vs. Brian Stann
- Nate Diaz vs. Dong Hyun Kim
- Thiago Silva vs. Brandon Vera
- Phil Baroni vs. Brad Tavares*
- Antonio McKee vs. Jacob Volkmann*
* – Not officially announced
For more on UFC 125, stay tuned to the UFC Rumors section of MMAjunkie.com.
(Pictured: Gray Maynard)


text:
Issue No. 07 - July (1988 vol. 14)
pp: 1049-1053
DOI Bookmark: http://doi.ieeecomputersociety.org/10.1109/32.42744

Software/hardware systems are considered which can be decomposed into a finite number of modules. It is assumed that control of the system is transferred among the modules according to a Markov process. Each module has an associated reliability which gives the probability that the module will operate correctly when called and will transfer control successfully when finished. The system will eventually either fail or complete its task successfully and enter a terminal state. The reliability of the system is studied in terms of the module reliabilities and the transition probabilities. Improved methods of predicting system reliability, allocating module reliability, and determining module sensitivity are developed. Special branching and sequential systems are studied in detail.

software reliability; branching systems; Markov process; probability; module reliabilities; transition probabilities; sequential systems; Markov processes; probability; software reliability
K. Siegrist, "Reliability of Systems with Markov Transfer of Control", IEEE Transactions on Software Engineering, vol. 14, no. , pp. 1049-1053, July 1988, doi:10.1109/32.42744


text:
Wondering how to give this season and still give back? I was recently emailed about this interesting program called GoodSearch.
(NB: this is not an endorsement of any kind).
From what I can gather, when you shop through their website a percentage of your purchase, at no cost to you, goes to the charity or non-profit org of your choice.
And even more interesting, a staff member at GoodSearch told me that, "… [her]11 year old nephew is fatally allergic to peanuts and so much of [her] family GoodShops and GoodSearches to benefit a [food allergy non-profit]."
She says: "It would be wonderful if you could let your readers know that they too can benefit any other organization helping people with allergies via GoodShop and GoodSearch!"
Pretty cool, right?
Here's is more info about how GoodSearch works and how you can start getting and giving right away!


text:
Read More
Choowap Phu Chaisai Mountain Phu Chaisai Mountain
Location: Hotels In Chiangrai
Phu Chaisai resort and spa offers 50 guest cottages with private terrace overlooking the panoramic views of the hills and mountains. The resort provides Superior, Deluxe, Executive, Honeymoon Suites and One bedroom Pool Villa. Room amenities are such as television sets, electronic alarm clocks, air conditioning, hair dryer, coffee and tea, direct dial phone and private bathroom .
Phu Chaisai Resort and Spa is the perfect place Top Resort for Travellers who want to get back to nature in the peace and quiet of their own private rooms set amid lush tropical gardens. read more..
Superior Cottage : These rustic bamboo Superior Cottages are the original Phu Chaisai bamboo cottages, 30-45 sqm, which provide more space with the panoramic view of the entire valley as a backdrop. With option of king or twin beds, full amenities and a private covered extending balcony, guests can spend most of their time breathing the fresh air and enjoy the scenery.
Deluxe Cottages : more spacious than the Superior Cottages to provide you more comfort during your stay. Each room, 40-55 sqm , also provides a panoramic view of the entire valley. Moreover this room is comprised with more facilities as private indoor bathtub besides its own private covered balcony where you can enjoy reading a good book during the day and private stargazing time at night.
Executive Suite Cottage : Offering more personal space, the Executive Suite cottage can easily accommodate up to 3 guests. With a large day-bed in the room, guests can decide if they want to relax indoor or absorb the surroundings from the outdoor balcony.
Honeymoon Suite : Our 2 honeymoon suite cottages are nestled among trees for your own privacy in different layout and style which you will never want to leave your cottage.
Thai Pool Villa : This spacious Pool Villa, suitable for small families or guests looking for larger space, boasts a traditional Thai architecture with its own private plunge pool. It also features a dining and sitting area with day-bed, large outdoor bathtub and shower.
One Bedrooms Pool Villa : Scattered amongst the upper hills in a remote and secluded location, far from the busy area of the resort, this 1-Bedroom Pool Villa offers separate sleeping and living areas and has a private plunge pool. Nicely designed and spacious, guests can enjoy comfort rivalling those they get from their own home.
Two Bedrooms Villa : Beautifully designed with a blooming lotus pond, this villa has two separate bedrooms with their own en-suite bathrooms and a shared common area consisting of a sitting room, outdoor dining area and a small pantry. Guests can take advantage of their spacious accommodation by choosing to dine in and enjoy their present company.
Three Bedrooms Pool Villa : This villa is the largest accommodation present in Phu Chaisai. Breathtaking in their design, this 3-Bedroom Pool Villa is the epitome of privacy and exclusivity. An octagon sala, encircled by a blooming lotus pond, awaits guests' arrival. This super-spacious compound houses its own kitchen, sitting and dining area, private plunge pool and three separate bedrooms with en-suite bathrooms.
disabled facilities
executive floor
family room
meeting facilities
safety box in room
smoking room
car park
Sport and Recreation
bicycle rental
coffee shop
poolside bar
golf (on site)
fitness room
indoor pool
kids club
outdoor pool
pool (kids)
squash courts
steam room
tennis courts
24hr room service
car transfer
business center
laundry service
pets allowed
Wi-Fi public areas
LAN (free)
LAN (charges)
Wi-Fi (free)
room service
Wi-Fi public ares (charges)
baby cot
from 1 reviews
13 January 2014
ที่พักดูดีมาก เหมาะสมสำหรับราคา ทุกอย่างโดยรวมถือว่าคุ้มค่า


text:
Bihar minister Parween Amanullah was on Thursday attacked and held up for some time by a mob in Vaishali district which was agitating over the midday meal tragedy in the state.
Amanullah, who is the minister for social welfare, was on her way to Muzaffarpur from Patna when the protesters, who had blockaded National Highway 77 near Rai Birendra College, about 5km from Hajipur town, pelted her car with stones, forcing it to halt.
"I was stopped by the mob for nearly 15 minutes before police came and escorted my car to safety... Stones hit my car while I was sitting inside," Amanullah told PTI.
The minister said she cancelled her programme and returned to Patna after the incident.
Vaishali SP Suresh Chaudhary confirmed the incident.
The Bihar government has been under attack by opposition parties over the midday meal tragedy in Saran district which has claimed the lives of 23 children.
RJD, BJP and CPI had separately observed bandh in Saran on Wednesday.
Opposition parties have blamed the state government for the delay in rushing the victims to hospital after they were taken ill upon eating the meal.


text:
Hillsboro News
Here are the community birthdays and anniversaries for the next two weeks in January: Happy birthday to Levi Stevens 15; Kelcy Whitaker, Jonas McDaniel 16; Kenny Messer 17; Joe Nelson 19; Felicia Weaver, Travis Davis 20; Richard Franklin 21; Greg Prevett, Frances Magdefrau 22; Korby Kennedy 23; George Kennedy 25; Lesia Glick 27; Derek Hudson, Justin Miller, Sandy Anderson 28.
Happy anniversary to Mr. and Mrs. Bob Reinier 20.
If you have a birthday or anniversary to add to the community list please call 319-931-1115 or email firstname.lastname@example.com with the name and date and if it is a birthday or anniversary.
The next Hillsboro City Council meeting will be Monday, Jan. 14, at 7 p.m. at the Hillsboro Community Center.
If you would like to speak at any city council meeting please contact Nancy at City Hall the Thursday prior to the meeting you wish to speak at so she can put you on the agenda. City Hall's number is 319-253-4465.
A group of bakers met Saturday, Dec. 15 for the Hillsboro Library's second annual cookie exchange.
Library Director Casey Leffel started the group off with a little word scamble game. As the game progressed and in the spirit of Christmas, players began giving each other hints to some of the more challenging scrambles.
Laughter soon followed as the hints became more like charades. Because we all know it wouldn't be fair if you just give someone an answer. The group may have come up with a new game — "word scramble charades."
If there was an award for most creative cookie shared, Casey's mouse cookies would have taken the the prize. You will have to stop in and ask him how he made them.
New on the shelf at the Hillsboro Public Library:
Adult fiction: "Next Always" by Nora Roberts, "NYPD Red" by James Patterson, "Racketeer" by John Grisham and "50 Shades of Grey" by E. L. James.
Junior fiction: "Eighth Grade Is Making Me Sick" by Jennifer L. Holm.
Young adult fiction: "Choke" by Diana Lopez.


text:
Scene 13
Alexandria. Cleopatra's palace.
(Cleopatra; Charmian; Iras; Mardian)
Terrified, Cleopatra sends Mardian to Antony to falsely report that she has killed herself, while she takes refuge in her monument. (12 lines)
Enter Cleopatra, Charmian, Iras, Mardian.
Help me, my women! O, he's more mad
Than Telamon for his shield; the boar of Thessaly
Was never so emboss'd.
To th' monument!
There lock yourself, and send him word you are dead.
The soul and body rive not more in parting
Than greatness going off.
To th' monument!
Mardian, go tell him I have slain myself;
Say that the last I spoke was "Antony,"
And word it, prithee, piteously. Hence, Mardian,
And bring me how he takes my death. To th' monument!


text: So maybe you don't have any pet peeves when it comes to ebay but I have a couple at least. One of my biggest pet peeves about ebay is sellers doing pre-sales, it bugs me to no end but I live with it. What is it that bugs you about some sellers on ebay? Anyone?


text:
Residential properties in county show 7 to 8 percent decrease in market value
The trend is a downward one. According to the annual report released this week by the Dakota County Assessing Services, the majority of property market values in Dakota County showed a decrease this year. The information was presented this week to the Dakota County Board of Commissioners.
It showed that most residential properties values have decreased between 7 percent and 8 percent for 2010. Apartments are experiencing an aggregate reduction of 7 percent, while commercial properties have decreased 6 percent. Estimated values for agricultural land decreased 25 percent. Taxable agricultural land values, however, which Dakota county received from the Minnesota Department of Revenue, were unchanged for 2010.
County-wide taxable market values, including new construction, decreased about 6.6 percent - to $38.3 billion. The total net tax capacity has decreased 6.5 percent for the 2010 assessment, taxes payable in 2011 - $450,765.130.
The city of Hastings has 6,050 residential homesteads. For a property owner with a median property value of $188,000 in 2009, the property value showed a decrease of about 8 percent - or to $173,000.
The median value is the value of the middle items when all items are arranged by size.
Valuation notices for 2010 market values were mailed March 2. Approximately 148,000 notices were sent to manufactured home, real and personal property owners.
The Assessing Services Department has scheduled its open booking meetings and Board of Appeal and Equalization calendar for this year. The information is available on the county's website at www.co.dakota.mn.us.


text:
Donna Nook seals to be protected against storms
Wildlife wardens are preparing to protect seals pups from high tides, after dozens died because of a tidal surge two years ago.
The pups died because the surge separated them from their mothers at Donna Nook, one of the largest grey seal reserves in the UK.
Rachel Shaw, from Lincolnshire Wildlife Trust, said this year's pups may need to be allowed to escape into sand dunes if the UK's predicted storms cause a similar emergency.
Visitors are being asked to stay away from the reserve this week.
05 Dec 2013
- From the section Lincolnshire


text:
Arriving from the Grid two years after the release of TRON: LEGACY is Hot Toys' long awaited 1:6th scale SAM FLYNN WITH LIGHT CYCLE. The film divided critics but visually served its fans something that they would eagerly want more from. With Hot Toys' Kevin Flynn 1/6th scale figure last year, fans didn't know they would wait this long for a follow-up but I'm sure I speak for all that it was well worth it.
The 1/6th scale Sam Flynn is not your usual Hot Toys release as the figure isn't very poseable. The figure is pre-posed to saddle the included Light Cycle and to look awesome doing it. We'll get into more about why they went this route a little later. The piece is currently sold out from Sideshow's website where it was priced at $299.99, but you might have some luck with the WAIT LIST.
Packaging for Sam Flynn with Light Cycle is a heavy duty box with handle, much like what you'd find when you purchase snowboarding boots. The front display shows off the side view of Sam on his light cycle with a light trail wrapping around the sides and back. Toy credits can also be found on the back of this elegant package. Once the box top is opened, you'll find the plastic trays containing Sam and black foam for his beloved Light Cycle within.
You will find two hand-painted portraits for Sam Flynn in this set, both are of Garrett Hedlund's portrayal of Sam Flynn but one with a helmet and one without. I'm having a bit of trouble seeing Garrett's likeness in the standard portrait but the one in the helmet looks great. For this piece, there actually isn't a need for the standard portrait since he'll always have the helmet when riding but it's always great to have options.
Also included is an extra left leg which can be swapped out to create the look of Sam in breaking position. It's a nice way to change up the display if you decide to do this. Another bonus is an extra right hand that can hold his identity disc while riding, just like he did in the film.
There are two Identity Discs included with Sam. One to attach to his back and one that is "lit" (or painted to be lit) for him to hold. And of course included is the 1/6th scale Light Cycle with LED lights and various opening flaps for the air-brakes and the like. It's quite an impressive looking beast with amazing light up capabilities, including a light up panel.
Included already are button cell batteries for Sam Flynn's costume and helmet; not included are 3A batteries for the Light Cycle. Once everything is put in and working, it's an absolute stunning display piece. A switch is located on the helmet to illuminate the emblem on the top of the helmet as well as illuminate the inside of the helmet. The switch on Sam's back will activate the glow in his costume. For the Lightcycle, the switch below the engine chamber allows two options: the first mode simply lights up the entire bike while the second option activates all the lights as well as activating the spinning engine. It makes quite a bit of noise but it's actually great since this is a cycle after all.
The lighting for this piece isn't as blue as I would want to see, but for photos you take, you can adjust it a bit to give the hue you prefer. But how else do I describe how cool Sam Flynn with Light Cycle is? The whole suit lights up along with the bike! Now we know why we didn't get a fully articulated Sam Flynn figure since it would actually prevent it from looking film accurate, but I would love for an actual poseable Sam Flynn (and Clu, and Quorra, and Rinzler, etc) at some point.
The price may seem a bit steep at $300, especially when getting a limited poseable figure, but honestly now, if you have a Light Cycle, you wouldn't want to display it without a rider now would you? Hot Toys has scored another winner and I hope the sales from this will influence them to continue with the rest of the cast.
Review and Photography by David Yeh
Review Smaple Courtesy of Hot Toys


text:
This product is no longer available.
You can try these similar gifts instead.
80th Birthday Gift Basket
Includes a Happy 80th Birthday picture frame, 1932 Music CD, a 1932 DVD, 80th coffee mug, a 1932 Remember When Booklet and a Happy 80th Birthday Magnifying Ruler, a 1932 greeting card and a year trivia magnet.


text:
Dewayne Wise has been busy this week. The guy who didn't make the Yankees out of spring training got credit for a catch he didn't make earlier in the week and now he's getting the final two outs against the White Sox.
Here is the recap and graph from the rest of Friday night's game.


text:
After years with the same logo, Toys R Us has made some minor changes to their logo. The Star in the middle has changed and is now seen inside the R. The quotation marks around it are no longer. The type looks a little thicker as well. Overall I think this is an improvement over the old logo.
Your toughts?
Read more here.


text:
Tag: Ambient Temperature
How to Collect Energy Data Compliant to EN50001
Using a Data Logger to Collect and Analyze Current and Voltage Data The EN50001 standard requires high energy-consuming industries to implement energy-saving measures and, when required, to install energy management systems. Energy consumption data therefore requires users to record data, which can be performed using Delphin Message series data loggers. Due to their LAN interfaces, … Continued


text:
With the 10-inch D-Link DSM-210 Wireless Internet Photo Frame, you can display your holiday photos from memory cards as well as from photosharing sites like Flickr, Picasa, Facebook and Photobucket.
The D-Link Wireless Internet Photo Frame, which allows you to display and share photos as well as Internet content, is now compatible with Photobucket, a popular standalone photo and video-sharing site.
Users can manage digital photos and also stream Internet content such as news, weather, sports, trivia, and Really Simple Syndication (RSS) feeds directly to the 10-inch D-Link DSM-210 without the need to connect with a PC.
New users can sign up for a free Photobucket account at http://photobucket.com/ to upload and share photos, create group albums, design scrapbooks and even slideshows. Users can then manage their D-Link Internet Photo Frame, including adding their Photobucket account, at internetphotoframe.com.
Source: D-Link


text:
Documents & Forms
Constitutions and Bylaws of Governing Councils
Interfraternity Council bylaws
Panhellenic Council bylaws
Panhellenic Council Recruitment Rules
Multicultural Greek Council bylaws
2012-2017 Strategic Plan
Scholastic Record Release
Must be filled out by all new members.
New Member Release
Must be completed and verified by a professional member of the Center for Student Leadership, Involvement, & Service staff before potential new members can participate in any fraternity/sorority activities (excluding recruitment activities).
Panhellenic Continuous Open Bidding MRAA
Must be completed when a woman joins a chapter outside of the formal recruitment period held in the fall semester.
Hosting Dances
Complete the following forms if you're interested in hosting a dance. Start by completing the RUFAS and Police Services forms. These two need to be submitted at least 30 days in advance of the event. After your event is confirmed with Conference and Event Services, start filling out the information in the 2012-2013 Dance Packet. Any events taking place on-campus with ticket sales need to go though the Campus Box Office. The Box Office Work Order Agreement Form should be filled out with the Box Office staff located on the 1st Floor of the Student Union.


text:
SolarWinds Server & Application Monitor allows you to monitor your hybrid IT applications and systems. Monitor Microsoft® Office 365 Exchange side by side on your on-premises Exchange server and other applications. Check, track, and report on O365 user/shared mailbox growth, users over quota, archived mailboxes, inactive mailboxes, mobile device, mailbox groups, and more. You can also monitor Microsoft portal access to help ensure you're meeting your SLAs.Advertisement
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12 Ways to Die on an Asteroid
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Delphi ASIO & VST Packages
With these packages for Delphi the user can easily create VST plugins or ASIO applications within minutes. The included algorithms for filters and dynamics help to built effects without much knowledge of digital signal processing.29 weekly downloads
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MacMPEG2Decoder (aka MM2D) decodes MPEG2 video into a variety of formats, normally quicktime based, on a Macintosh (Classic & OSX).
MovieGoer Point of Sale
I created this project becuase I have noticed that it is impossible to locate decent and capable software for movie theaters. I belive that small movie theaters should have a fair chance at the market just like the "big boys" of the movie theaters.
Notebook Mini
This is my first project written in Delphi. Please do not scold. Later I will add the source code, software and detailed description.
OTP Enterprise Service Bus
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Restaurant POS (Point-of-Sale)
Since Floreant POS is seemingly dead, lets create an active group of developers who want to make it easier use. Under Development: *Expand Printer Support *Expand Modifier Levels *Customization Menu *Plugin support *Simple Menu Editor


text:
For the first time in a long time, the boys will not be playing in the regional tournament. They were defeated by the host school, Danville, Wednesday night 48-41.
LC got some horrible calls from the refs. Some unbelievable fouls were called totally changing the game.
Congrats to Coach Jackon's and the Patriots. They finished the season 16-10.
Thursday, February 25, 2010
Boys Fall in Opening Round
Posted by BS at 11:12 AM
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text:
Artist description:
No description entered.
Java Draw Created Jun 04, 2011 460 x 640px Unless noted Copyright 2011 SPIDER 08.
We encourage you to submit a comment and let the artist know what you think of this drawing. Please log in or join to post a comment.
Post a comment
very sweet image:) 5/5 and collection! Thank you so much for your comments and support, Cheryl! :3
Ooooooooooooooooooooo What a neat drawing -- I love how you did the shadign on this one! Now I NEED some icecream! LOLOL
So true and So are you :)) can I eat it now. 5♥s
yea , tahts tue sweet as ice cream:) 5
I agree. :) Now can I eat the ice cream cone? hee hee 5/5
RMD certainly is cool and this ice cream looks so sweet and yummy!
i so need chocolate


text:
A Living Portrait
/ Masses / Tempus De / Drumlins
Saturday, Apr 28, 2012 8:00 PM CDT
(8:00 PM Doors)
- Sunday, Apr 29, 2012 12:00 AM CDT
Stage Bar, Chicago, IL
21 years and over
Purchase Tickets
Additional Information
A LIVING PORTRAIT (ex Raise The Red Lantern)
We are four best friends who love playing music together. We just finished creating our debut EP "Sea Bears". Its available for download on Bandcamp for however much you'd like to pay. We love all of you, see you soon.
After a year of looking for a full-time bassist, finally in mid-2010 Chicago band Masses added a third member to its already existent guitar/vocals and drums setup. Since then, the group has established their 'heavy indie' sound as the summation of those three pieces - part angular attack, a la the '90s indie rock sound of Quicksand; a slice of murky sonic drones and galloping palm mutes not unlike Fucking Champs; and a winking homage to guitar heroes like Iron Maiden. Distortion and unassumed melody are premiums here, folks. Expect to feel some rumbling bass and snare blats and maybe see a string or two break in the process, who knows.
With each member having distinct musical backgrounds and influences, Tempus De focuses on writing music that blends together ambient and melodic layers with technical groove-rhythms. We strive to challenge ourselves musically, and to ultimately write something that is our own.
Carved from the landscape and piled upon the horizon, Drumlins collectively gaze from the foot of the lake searching for the primordial ooze of their creation. From the apex of a great glacial kame a sound was born; soaked in the elixirs that helped create it, weathered by smoke and time.