text:
|Publication number
|Publication type
|Application number
|Publication date
|Jul 21, 1987
|Filing date
|Oct 23, 1985
|Priority date
|Oct 23, 1985
|Fee status
|Also published as
|Publication number
|Kenneth J. Hardy, Michael G. Whitesides
|Original Assignee
|Hardy Kenneth J, Whitesides Michael G, Hoefer Scientific Instr
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (4), Non-Patent Citations (4), Referenced by (14), Classifications (12), Legal Events (8)
|External Links: USPTO, USPTO Assignment, Espacenet
This invention relates to an apparatus and method for analyzing filter-bound samples and, more particularly, to a low volume perfusion slit chamber for treating and/or developing nucleotide and protein-bound membranes.
Many current methodologies for the high resolution analyses of proteins, nucleic acids, and polynucleotides immobilize samples on thin filter membranes and subsequently analyze the filters using immunologic or recombinant hybridization technologies.
Several years ago, it was found that singlestranded DNA binds very tightly to membrane filters made of nitrocellulose, and that such filters failed to bind either double-stranded DNA or RNA. This phenomenon can be used, for example, to determine the presence of specific complementary base sequences in single-stranded DNA by binding one type of single-stranded DNA to a nitrocellulose filter and reacting the bound single-stranded DNA with labeled, singlestranded DNA or RNA. Where such base sequences are present, the second type of single-stranded DNA or of RNA binds to the filter-bound single-stranded DNA. The presence of double-stranded DNA or a DNA-RNA complex bound to the nitrocellulose sheet can be detected utilizing the label attached to the second type of single-stranded DNA or RNA. If, for example, a radioactive label is used, the presence of a double-stranded DNA or DNA-RNA complex can be detected by exposing x-ray films with the nitrocellulose sheet and developing the x-ray films.
A wide variety of analytical techniques have since been developed utilizing this basic phenomenon. See, e.g., D. Freifelder, Molecular Biology, pp. 123-124 and 418-425 (1983). For example, when single-stranded polynucleotides (DNA or RNA) are separated electrophoretically in polyacrylamide gels or other media, the separated polynucleotides and polynucleotide fragments can be transferred or "blotted" from the polyacrylamide gels onto nitrocellulose membranes or sheets. The separated single-stranded DNA, bound to the sheet, can now be localized by reacting the bound single-stranded DNA with specific sequences or fragments of single-stranded DNA or RNA that have previously been labeled for detection. Possible labels include fluorescent and other chemical labels, as well as radioactive labels, such as 32 P-phosphate labels and 14 C labels.
Similar technologies have been developed for the localization of electrophoretically separated proteins and/or detection of proteins or samples of genetic material dotted onto filter membranes for screening.
Such methods all require rigid control of reaction temperatures at each step and accurately controlled development times. Because of the complex kinetics of chemical interaction involved, as well as the fact that many of the reagents, such as radioactively labled polynucleotides, are rare and/or difficult to isolate or produce in large quantities, the development reactions must be carried out in minimum effective volumes. This, in turn, requires optimization of the efficiency of the interaction between the filter-bound sample and reagents. In addition, a wide range of reagent types and conditions are routinely used. Often, the filters are variable in size, shape, and type, are fragile, and are difficult to handle.
Filter analyses are usually done (with agitation) in open, large-volume containers or, in the case of nucleic acid analyses, in crude, heat-sealed plastic bags. For most purposes, such arrangements are cumbersome and wasteful of reagents, and are inefficient in terms of both the amounts of reagents required and in the time required to complete a particular reaction. Where the labels used include high specific activity radioisotopes, such systems may also be very hazardous. The operator may be exposed to significant radiation while handling such equipment, which is prone to leakage and spillage of radioactive solutions. It is also difficult, at best, to properly dispose of all radioactive materials used in such systems due to spillage, inconvenience of the operations which must be performed, and the relatively large quantities of radioactive material which must be handled and disposed of; such systems thus also are a potential source of environmental hazard.
The plastic bags used frequently in nucleic acid hybridizations are difficult to work with, are prone to leakage, and require agitation to maximize filter/reagent interactions. Such bags are not reusable, and do not afford any significant protection to the often fragile filters being analyzed. Due to the flexibility of the plastic bags, reagent distribution is often uneven, since the positions of the filters within such plastic bags are not readily controllable. Further, such plastic bags systems require reopening and resealing after every reagent change and are effectively useless for washing steps which require large volumes of wash reagents and agitation of the filters and reagents. While such plastic bag systems allow the exclusion of air bubbles from the filter/reagent interface, and complete immersion of the filters with a relatively small volume of reagent, both plastic bag development systems and development systems utilizing large-volume containers have proven inconvenient, inefficient, non-reproducible, and often unsatisfactory for routine use in the analysis of filter-bound samples.
An apparatus and method for the localization, development, and analysis of filter-bound samples is disclosed. The apparatus of the present invention generally includes two cover plates, at least one of which has a shallow, generally rectangular recess formed on one side thereof. The two cover plates are matable to form a thin treatment chamber to sealingly enclose a filter or membrane sheet on which single-stranded DNA or other substance of interest has been deposited. The apparatus further includes reagent ports for introducing and alternatively withdrawing reagents from the treatment chamber, and valve assemblies to control the reagent flows.
In the preferred apparatus of the invention, a plurality of filter support pegs are affixed to the opposing interior surfaces of the treatment chamber to position the filter or membrane sheet away from the opposing treatment chamber walls, thereby allowing free flow of reagent along each side of the filter or membrane sheet. Typically, the treatment chamber has dimensions of about 15 cm by about 18 cm by about 0.05 cm; the filter support pegs typically extend into the treatment chamber about 0.015 to about 0.018 cm, leaving a gap of about 0.01 cm to 0.02 cm for placement of the filter or membrane sheet. The treatment chamber thus has a very small volume but very efficient solution-filter sheet contact.
Preferably, the cover plates are provided with pairs of interlocking baffles and slots arranged near either end of the treatment chamber to distribute the introduced reagents evenly over the width of the treatment chamber. Preferably, each baffle is located between the corresponding reagent port and the positioned filter or membrane sheet.
The preferred embodiment of the apparatus also includes reagent paths for fluidly communicating the reagents from the valve assemblies to the corresponding reagent ports, clamping assemblies for releasably securing the cover plates together, and heat exchange fins on the outside of the cover plates to ensure rapid and effective temperature equilibration of the apparatus.
The preferred method according to the invention includes placing a filter or membrane sheet with bound DNA or other substances of interest between the cover plates and clamping the apparatus together to enclose the filter or membrane sheet. Sequentially, prehybridization solution, hybridization solution including a radioactively labeled reagent, and wash solutions of increasing stringency are introduced into the reaction chamber, preferably entirely with syringes in cooperation with syringe fittings mounted to the valve assemblies in controllable fluid communication with the reagent ports. The treated, labeled filter or membrane sheet may then be removed for detection of the labels, and hence the substance of interest by known x-ray film exposure and/or other development methods.
The apparatus according to the present invention and the method for using the apparatus eliminate the majority of the problems associated with the analysis of a filterbound sample using reagent-filled plastic bags and other reaction vessels, while improving and facilitating the analysis of filter-bound nucleic acids and proteins. The preferred embodiment of the reusable, fully perfusable reaction slit chamber of the invention is compatable with the entire range of conditions, filters, and reagents used in current immunologic and recombinant analytic technologies. The apparatus minimizes internal fluid volumes, eliminates air bubbles, and allows rapid insertion and removal of filters. In addition, the apparatus and its use dramatically improve the most important parameter in all such analyses, the homogenous, unobstructed filter/reagent reaction at the interface between the filter and the reagent. This optimization is accomplished by the combination of the filter support pegs, which hold the filter membrane in position away from either side of the treatment chamber, the internal bilaminar flow generation system, and the liquid addition channels or reagent paths. Due to the highly efficient filter/reagent interactions and the design of the apparatus according to the invention, significant reductions in the volumes of the reagents required, radioactive wastes, and reaction times required are achieved. With the apparatus and method of the present invention, significant reduction in signal background is routinely accomplished, and the variability of results between successive experiments is minimized.
The apparatus is readily assembled and operated using externally mounted syringe fittings. The filters are positioned and protected reproducibly within the treatment chamber, which can accommodate a wide range of filter types, sizes, and shapes. The apparatus is fully self-contained; sequential addition and removal of reagents are readily accomplished without manipulation of the filter, transfer of the filter from one container to another, or the need for external tubing, pumps, or vacuum equipment. The apparatus is self-cleaning and is decontaminated during filter wash cycles.
With the apparatus and method of the present invention, environmental hazards are minimized. The cover plates of the preferred apparatus effectively shield the operator from beta emissions during handling and incubation, and the radioactive reagents necessary are conveniently added and removed from the preferred perfusion slit chamber without danger of spillage and, in small volumes, ready and convenient for disposal. The external syringe ports of the preferred embodiment facilitate safe and efficient handling of hazardous liquids.
The apparatus and method of the present invention can be used for a wide variety of types of filters and membranes, including both nitrocellulose and nylon based filters, some impregnated with various substances and resins. The apparatus and method of the invention can conveniently be used with a wide variety of analytical techniques, including, but not limited to, DNA hybridization techniques, DNARNA hybridization techniques, autoradiographic techniques, the southern blot technique used with DNA electrophoresis, the northern blot technique used with RNA electrophoresis, and the western blot technique used in immunoassay of proteins. In addition, the apparatus and method provide a convenient technology for genetic disease screening, especially of the type termed "Restriction Fragment Length Polymorphism" (RFLP). RFLP assays are currently being used for detection of Huntingtons Chorea, sickle cell disease, and other diseases. RFLP assays are also being devised as prenatal screening tests, e.g., Tay Sachs disease, phenylketonuria, galactosemia, sex determination, and other factors.
The preferred apparatus according to the invention can be principally manufactured from plexiglass or polysulfone; such plastics are readily machinable and/or moldable, readily withstand the often elevated temperatures required for incubation and reaction, do not interact with any presently used or known reagents for analyzing filter-bound samples, and do not readily adsorb DNA, RNA, or protein. Other plastics having similar properties may be utilized as necessary. For example, silicon rubber is conveniently used for the required O-rings and gaskets, and the valve assemblies can be fabricated using Delrin. The preferred apparatus of the present invention incorporates no metal parts which might react with the reagents used. Such reactions are to be avoided since corroded metal parts interfere with the operation of the apparatus and use of the method; leached metals can also cause degredation of the reagents and samples of interests.
A presently preferred membrane for use with the apparatus and method of the present invention is a treated nylon 66 membrane manufactured by Molecular Separations, Inc.; This filter does not contract and expand significantly when dried or moistened, respectively. Although it is more convenient for the operator to use such filter types, the apparatus and method of the present invention are not so restricted.
The above and other features and objects of the invention, and the operation and advantages of the invention, will be better understood and appreciated by those skilled in the art in view of the description of the preferred embodiment given below in conjunction with the accompanying drawings.
FIG. 1 is a perspective view of the preferred perfusion slit chamber according to the invention, showing schematically the placement of a syringe in phantom.
FIG. 2 is an exploded perspective view of the preferred perfusion slit chamber according to the invention.
FIG. 3 is a cross-sectional view of the preferred perfusion slit chamber along section 3--3 of FIG. 1.
FIG. 4 is a detail view of the preferred perfusion slit chamber showing the membrane support pegs, taken in the region 4--4 of FIG. 3.
FIG. 5 is a cutaway plan view of the preferred perfusion slit chamber showing one set of fluid distribution baffles and, schematically, the fluid flow therethrough, and including in cutaway, a detail of one of the valve assemblies.
FIG. 6 is a perspective view of a clamp for use with the preferred perfusion slit chamber.
The preferred perfusion slit chamber 10, according to the present invention, is shown assembled in perspective view in FIG. 1 and in the exploded perspective view in FIG. 2.
Referring principally to FIGS. 1 and 2, preferred perfusion slit chamber 10 generally includes first and second cover plates 12 and 14, matable to define therebetween a treatment chamber 16. Treatment chamber 16 will be described more fully below in conjunction with the description of FIGS. 3-5.
Perfusion slit chamber 10 also includes, in the preferred embodiment, first and second reagent ports 18 and 20 (FIG. 3), which allow fluid communication between treatment chamber 16 and the exterior of perfusion slit chamber 10. In the preferred embodiment of the invention, the flow of reagents through first and second reagent ports 18, 20 is controlled with valve assemblies 22, 24, which are, in turn, fluidly coupled to first and second reagent ports 18, 20 via first and second reagent paths 26, 28. In the preferred profusion slit chamber 10, first reagent port 18 is located near bottom 30 of perfusion slit chamber 10 and defined through first cover plate 12; second reagent port 20 is defined, near top 32, in second cover plate 14. As will be explained more fully below, it is convenient to form first and second reagent ports 18, 20 in different cover plates 12, 14 to faciliate the construction of reagent paths 26, 28 and the mounting of valve assemblies 22, 24. However, in alternate embodiments of the invention, first and second reagent ports 18, 20 could both be defined in a single cover plate 12 or 14. Similarly, it is convenient in the preferred perfusion slit chamber 10 to define one reagent port 18, 20 near bottom 30 and the other of reagent ports 18, 20 near top 32; however, in alternate embodiments of the apparatus according to the invention, other arrangements are possible and will be apparent to those skilled in the art. The arrangement of reagent ports 18, 20, as shown in FIG. 1, allow the optimum flow of reagents through treatment chamber 16.
For ease of handling and insertion of perfusion slit chamber 10 in a heated and temperature controlled water bath (not shown), it is convenient to mount first and second valve assemblies 22, 24 near top 32 so that valve assemblies 22, 24 can be positioned to extend above the water or fluid level of the heat bath. However, it is not necessary that both the valve assemblies 22, 24 be positioned either near the same end of perfusion slit chamber 10 or near top 32. Similarly, it is convenient to mount first and second valve assemblies 22, 24 on first and second cover plates 12, 14, respectively; however, first and second valve assemblies 22, 24 will be mounted on the same or different cover plates 12, 14 in accordance with the locations of the reagent ports 18, 20. First and second valve assemblies 22, 24 will be explained in greater detail below in conjunction with FIG. 5.
First and second reagent paths 26, 28 are constructed to connect first and second valve assemblies 22, 24 with first and second reagent ports 18, 20, respectively, to allow introduction and removal of reagents from treatment chamber 16.
Preferred perfusion slit chamber 10 also includes a plurality of clamping assemblies 34. In preferred slit chamber 10, four clamping assemblies 34 each include a pair of clamping posts 36 and a clamp 38. Each pair of clamping posts 36 is rigidly fixed to first and second cover plates 12, 14 and arranged to extend outward therefrom approximately colinerally. Clamping posts 36 are conveniently molded or constructed from the same piece of material as cover plates 12, 14 and provide mounting points and fulcrums for clamps 38.
Each clamp 38 includes a pair of hook portions 40 and a clamp portion 42. A perspective view of the clamp 38 is shown in FIG. 6. Each hook portion 40 is shaped to partially encircle one of clamping posts 36; the respective hook portions 40 of a single clamp 38 are arranged to extend from clamp portion 42 in generally mutually parallel spaced apart relation; the distance between hook portions 40 is chosen to allow hook portions 40 to engage simultaneously the corresponding pair of clamping posts 36. Each clamp portion 42 has a generally C-shaped cross-section (clearly shown in FIG. 3) and a length corresponding, in the preferred embodiment, to the particular dimension of cover plates 12, 14. To clamp cover plates 12, 14, hook portions 40 are engaged to the corresponding clamping posts 36 and, in a lever-type motion, clamp portion 42 is advanced toward cover plates 12, 14 so that the opposing sides of the C-shaped cross-section of clamp portion 42 engage opposing sides of cover plates 12, 14. To faciliate the engagement of the opposing sides of clamp portion 42 to opposing sides of cover plates 12, 14, clamp portions 42 are preferably provided with upturned flange portions 44. Flange portions 44 may be shaped as necessary to allow clamp portions 42 to slide onto the opposing surfaces of cover plates 12, 14 and to avoid a conflict with other portions of perfusion slit chamber 10, such as valve assemblies 22, 24. Such a shaped flange portion 44 is indicated as 45 in FIG. 6.
In FIG. 1, four clamping assemblies 34 are shown; however, in alternate embodiments of the invention, fewer clamping assemblies 34 could be used. For example, a pair of clamping assemblies 34 on opposing sides of perfusion slit chamber 10 with clamping posts 36 mounted, for example, on opposing corners of cover plates 12, 14 could satisfactorily close and hold cover plates 12, 14 together.
In the preferred embodiment, perfusion slit chamber 10 includes a plurality of heat exchanger fins 46 on each of first and second cover plates 12, 14. Heat exchanger fins 46 are arranged to extend outward of treatment chamber 16 from cover plates 12, 14 and are conveniently molded or machined from the same piece of material used to construct first and second cover plates 12, 14. The particular arrangement of heat exchanger fins 46 shown in FIG. 1 is convenient, but other arrangements will be apparent to those skilled in the art. The function of heat exchanger fins 46 is to strengthen first and second cover plates 12, 14 while simultaneously allowing rapid equilibrium of the temperature of treatment chamber 16 with the ambient.
Perfusion slit chamber 10 is shown in exploded perspective view in FIG. 2, in which the assembly of perfusion slit chamber 10 is also indicated. Cover plates 12, 14 are mated together over a filter or membrane sheet 48, which is thereby enclosed within treatment chamber 16. As also shown in FIG. 2, the preferred perfusion slit chamber 10 includes a sealing O-ring 50 set into an O-ring groove 52 in a known to those skilled in the art. In the preferred perfusion slit chamber 10, O-ring groove 52 is inscribed into one of cover plates 12, 14; in alternate embodiments, an analogous O-ring groove 52 could be inscribed into each of cover plates 12, 14. Although cover plates 12, 14 are sealingly engaged with O-ring 50 in the preferred embodiment, in alternate embodiments, other types of sealing gaskets and arrangements could be used. Since perfusion slit chamber 10 is operated without significantly elevated pressures or high vacuums, O-ring 50 need not be able to withstand high pressure differentials between the ambient and treatment chamber 16, although O-ring 50 will be sufficient to prevent introduction of contaminants from the ambient into treatment chamber 16 and to substantially prevent the escape of radioactive and other reagents from treatment chamber 16. O-ring 50 can be made of silicon rubber or other suitably inert, pliable material.
In the preferred perfusion slit chamber 10, each cover plate 12, 14 is provided with a generally rectagonal recess 53. In FIG. 2, recess 53 is seen most clearly on second cover plate 14. The opposing recess 53 in cover plate 12 is analogous to that shown in cover plate 14. When first and second cover plates 12, 14 are mated together, recesses 53 cooperate to form treatment chamber 16. In alternate embodiments of the invention, a single recess 53 could be inscribed in one of cover plates 12, 14.
Each cover plate 12, 14 is also provided with a baffle 54 and a slot 56. In FIG. 2, only baffle 54 and slot 56, provided on cover plate 14, are shown. Baffle 54 is located near bottom 30, while slot 56 is located near top 32. Baffle 54 and slot 56 of cover plate 12 are analogous to those shown with cover plate 14; however, baffle 54 on cover plate 12 is located near top 32, and slot 56 on cover plate 12 is located near bottom 30. In this manner, when perfusion slit chamber 10 is assembled, baffle 54 of each cover plate 12 or 14 fits into the corresponding slot 56 of the other cover plate 12, 14. Baffles 54 and slots 56 are located such that one baffle 54 is interposed between each reagent port 18, 20 and the majority of treatment chamber 16. For the purposes of discussion, the reagent of treatment chamber 16, located between baffles 54, will be referred to as filter/filter region 58. Baffles 54 serve to distribute reagents entering treatment chamber 16 through reagent ports 18, 20 in laminar fashion over the entire width of filter region 58, and will be described more fully below in conjunction with the discussion of FIG. 5.
Filter or membrane sheet 48 is, in the assembled perfusion slit chamber 10, located in filter region 58 between baffles 54. To hold filter or membrane sheet 48 away from the walls of cover plate walls 60, 62, cover plates 12, 14 are provided in filter region 58 with a plurality of filter support pegs 64 to allow reagents to flow essentially unimpeded on either side of filter or membrane sheet 48 with each port communicating with both sides of the membrane. Several filter support pegs 64 are shown in detail in FIG. 4. Preferably, in the preferred perfusion slit chamber 10, filter support pegs 64 are positioned such that pairs of support pegs 64 projecting from cover plates 12, 14 oppose each other with a gap between opposing support pegs 64, which is somewhat wider than the width of paper or membrane sheet 48. Arranging opposing pairs of support pegs 64 substantially prevents wadding or creasing or slippage of filter or membrane sheet 48 while the preferred gap between opposing support pegs 64 allows some freedom of movement of sheet filter or membrane sheet 48 and allows sheet 48 to be evenly exposed to reagents in treatment chamber 16. While other placements of opposing pairs of support pegs 64 are contemplated to be within the scope of the invention, it has been empirically found that positioning support pegs 64 such that pairs of support pegs 64 are opposed also leads to more even contact of sheet 48 with reagents in treatment chamber 16, and hence better images and detection of the DNA/DNA, DNA/RNA, and protein complexes on sheet 48.
A cross-sectional view of perfusion slit chamber 10, taken along section 3--3 of FIG. 1, is shown in FIG. 3. FIG. 3 shows the cooperation of recesses 53 to form treatment chamber 16 and filter region 58. FIG. 3 also shows the cooperation of baffles 54 with slots 56.
Also shown in FIG. 3 are reagent paths 26, 28. Reagent path 28 fluidly communicates with treatment chamber 16 via reagent port 20 and with valve assembly 24. The direction of flow of reagent indicated in FIG. 3 through reagent path 28 is for withdrawal of reagent through valve assembly 24, although each valve assembly 22, 24 can be used alternately for introduction and removal of reagent from treatment chamber 16. Reagent path 26 provides for fluid communication from valve assembly 22 to treatment chamber 16 via reagent port 18. Valve assembly 22 is not shown in FIG. 3, but is shown in FIGS. 1 and 5.
Reagent paths 26, 28 are conveniently defined utilizing first and second flow path covers 70, 72. For manufacturing convenience, it is desirable to construct cover plates 12, 14 identically except for baffles 54, slots 56, O-ring groove 52, and reagent ports 18, 20. To this end, cover plates 12, 14 are formed to have Y-shaped channels indicated generally as 76 in FIG. 5. Two legs of the Y-shaped channel 76 lead respectively to the regions in which reagent ports 18, 20 will be formed. The third leg of the "Y" communicates to the corresponding valve assembly 22, 24. Flow path covers 70, 72 are constructed to fit over Y-shaped channels 76 to form the appropriate reagent path 26 or 28. Each of flow path covers 70, 72 has a flow path plug 78, 80, which is inserted in the appropriate leg of the Y-shaped channel 76 to direct flow to reagent port 18 or 20, respectively. Plug 78 is formed to extend from flow path cover 70 to fit in one leg of Y-shaped channel 76 to divert flow to reagent port 18 near bottom 30., Plug 78 is shown in FIG. 5. To form reagent path 28, plug (not shown in FIG. 5) is appended from flow path cover 72 to project into and block one leg of Y-shaped channel 76 between the intersection of the "Y" and bottom 30. Thus, when flow path cover 72 is assembled with cover plate 14, reagent introduced via valve assembly 22 is diverted towards top 32 through reagent port 20. Reagent flow exiting reagent port 20 into treatment chamber 16 is shown in phantom in FIG. 5. Utilizing flow path cover 70, 72, much of the manufacture of cover plates 12, 14 is simplified since substantially identical Y-shaped channels 76 can be formed into each of cover plates 12, 14; the particular reagent path 26 or 28 is then determined by the location of plug 78 or 80 on flow path covers 70 or 72, respectively.
The details of valve assembly 22 are shown in FIG. 5. The details of valve assembly 24 are not separately shown, but are substantially the same as those of valve assembly 22, and the discussion below of valve assembly 22 is equally applicable to valve assembly 24. Valve assembly 22 includes a valve stem 100 and a valve seat 102. Valve seat 102 is conveniently formed in the same piece of material used to form cover plate 12 and is shaped to accommodate valve stem 100. Valve stem 100 is rotatable within valve seat 102 and includes a valve body 104, a valve port 106, a syringe mount 108, and a pair of O-rings 110. Valve body 104 is generally cylindrical and, in the preferred embodiment, grooved around its circumference to accommodate O-rings 110 in a manner well known to those skilled in the art. Valve body 104 also includes an angled valve bore (not shown) to fluidly couple valve port 106 to syringe mount 108. O-rings 110 are positioned above and below valve port 106, and sealingly but rotatably engage valve seat 102 to prevent leakage. Other means of sealingly and rotatably engaging valve stem 100 to valve seat 102 will be apparent to those skilled in the art. Syringe mount 108 is preferably a standard syringe mounting, such as a "LUER LOK" mount of the type which will be well known to those skilled in the art. Other means of coupling a syringe or other reagent delivery device to valve assembly 22 will be apparent to those skilled in the art.
Valve stem 100 is adjustably positioned within valve seat 102 using a positioner 112. Positioner 112 includes a screw 114, a bearing 116, and, defined within an appropriately positioned heat exchanger fin 46, a bearing mount 118. Typically, bearing mount 118 is a slot cut into the appropriately positioned heat exchanger fin 46 for sliding engagement of bearing 116. Screw 114 is threadedly engagable into a matching hole (not shown) in the end of valve stem 100 and passes through bearing 116. Screw 114 can be adjusted to locate valve port 106 at a location corresponding to the appropraite leg of Y-shaped channel 76. In alternate embodiments of the apparatus, screw 114 and bearing 116 can be made as an integral portion of valve stem 100, albeit without adjustability.
Valve stem 100 is conveniently made manually rotatable within valve seat 102 with a tab 120. Tab 120 is fixedly mounted to valve stem 100 to extend to one side therefrom. Other suitable means for rotating valve stem 100 will be apparent to those skilled in the art. In the position of valve stem 100, valve port 106, and tab 120, shown in FIG. 5, valve port 106 is positioned to fluidly communicate with reagent path 26. When valve stem 100 is rotated away from the position shown in FIG. 5, using tab 120, valve port 106 is rotated away from reagent path 26 to control or stop flow of reagent through reagent path 26.
Valve stem 100 is conveniently made from a plastic material such as Delrin, and is sized such that a tight slip fit is formed between valve stem 100 and valve seat 102. Thus, when valve stem 100 is in a position which located valve port 106 away from reagent path 26, the flow of reagent through reagent path 26 is effectively stopped. O-rings 110 assure a leakage-tight fit between valve stem 100 and valve seat 102.
One baffle 54 is also shown in FIG. 5. Baffle 54 includes a plurality of tongues 130 shaped to relatively tightly fit into the corresponding slot 56 and variably spaced to extend across the width of a treatment chamber 16. Generally, the gaps between adjacent tongues 130 increase as the distance from the corresponding reagent port 20 or 22 (reagent port 20, in FIG. 5) increases. That is, the gaps between adjacent tongues 136 are smaller near the center of treatment chamber 16 and wider near its edges. The spacing corresponds approximately to that which would be predicted by Bernoulli's theorum, and is empirically adjusted such that the flow into filter region 53 is approximately constant over the entire width of filter region 53. The gaps between adjacent tongues 130 extend across the entire depth of treatment chamber 16 and the filter region 53, so that reagent is evenly distributed to both sides of filter or membrane sheet 48.
Although the operation of perfusion slit chamber 10 can be inferred from the above description of the preferred apparatus according to the invention, for clarity a description of a typical procedure for using perfusion slit chamber 10 will now be given.
The filter or membrane sheet 48 with the polynucleotide or protein of interest, bound or blotted onto sheet 48, is placed between cover plates 12, 14. If sheet 48 is dry and is of a type which expands upon moistening, it is convenient to premoisten the dry sheet 48 prior to assembly of perfusion slit chamber 10. The disassembled perfusion slit chamber 10 is assembled as indicated in FIG. 2 with sheet 48 between cover plates 12, 14 and filter region 53. Cover plates 12, 14 are clamped with clamping assemblies 34 and, if required by the reagents to be used, perfusion slit chamber 10 is positioned with top 32 up in a water bath of the appropriate temperature. Approximately 25 ml of prehybridization solution (preheated to appropriate temperature) is drawn into a 35 ml capacity syringe, which is, in turn, mounted to, for example, syringe mount 108 of valve assembly 22. A second, empty, closed syringe of similar capacity is mounted to the other of valve assemblies 22, 24. Both valve assemblies 22, 24 are opened and the prehybridization solution is injected into treatment chamber 16. If necessary, the prehybridization solution can be drawn through treatment chamber 16 by withdrawing the plunger of the second syringe. The prehybridization solution is then pushed back and forth from one syringe to the other through treatment chamber 16 until no air bubbles adhere to the membrane. Valve assemblies 22, 24 are then closed and perfusion slit chamber 10 is incubated, as necessary. A prehybridization solution comprising 1.0 M NaCl, 1.0% SDS (sodium dodecyl sulfate) and a nylon-66 sheet 48, incubation at 65� C. for one to three hours has been found to be acceptable.
Following prehybridization treatment of sheet 48, and with perfusion slit chamber 10 positioned such that valve assemblies 22, 24 extend above the surface of the water in the water bath, valve assemblies 22, 24 are opened after the syringes are mounted. The prehybridization solution is removed from treatment chamber 16, again using two syringes, and an appropriate hybridization solution is injected into treatment chamber 16 using a technique similar to that for injection of the prehybridization solution. Any of the wide variety of known hybridization solutions can be used, according to the methodology being performed. A typical hybridization solution comprises approximately 10% dextran sulfate, 1.0 M NaCl, 1% SDS, and 100 mg/μl of denatured heterologous DNA. Additionally, a DNA or RNA species in the hybridization solution will be radioactively labeled with a specific radioactivity of at least 1�108 cpm/μg DNA. The hybridization solution is introduced into perfusion slit chamber 10 and is "washed" from one syringe to the other to ensure removal of all air bubbles from treatment chamber 16 and to maximize contact between the hybridization solution and filter or membrane sheet 48. After closing valve assemblies 22, 24 and removing the syringes, perfusion slit chamber 10 is again incubated at the required temperature for, e.g., 18 to 48 hours. The length of the incubation time depends upon such factors as the specific activity of the probe DNA, the concentration of DNA immobilized on sheet 48, and the frequency of the particular gene sequence of interest. In general, perfusion slit chamber 10, loaded with hybridization solution, should be incubated for one to three times the half-life of the binding reaction between the probe DNA and the immobilized DNA.
After sufficient incubation, syringes are again used to withdraw the hybridization solution from treatment chamber 16 via reagent port 18 (located near bottom 30). This procedure allows essentially spill-proof withdrawal of the radioactive hybridization solution from perfusion slit chamber 10 into the syringe for safe disposal.
The now hybridized sheet 48 is then washed in a manner analogous to the method for introducing prehybridization solution and hybridization solution using successive washes of increasing stringency. For example, a Standard Saline Phosphate EDTA (lX SSPE) solution containing 0.18 M NaCl, 0.01 M NaPO4 buffer (pH 7.4), and 1 mM EDTA (pH 7.4) has been found suitable. The hybridized sheet 48 is washed with successively higher stringency wash solutions. A typical low stringency wash consumes approximately 100 ml of 2X SSPE/1% SDS solution. A typical moderate stringency wash solution consumes about 50 ml of 0.5X SSPE/1% SDS, and a typical high stringency wash solution consumes about 25 ml of 0.1X SSPE/1% SDS. For each wash solution, approximately 35 ml of the appropriate wash solution is introduced into treatment chamber 16 using syringes as for introduction of the prehybridization and hybridization solutions, and repeatedly transferred between syringes. About 10-20 seconds of transfer of the wash solution will remove essentially all unbound radioactivity from sheet 48 and treatment chamber 16. This initial low stringency wash is removed and properly disposed of.
Perfusion slit chamber 10 is again filled with low stringency wash and air bubbles are removed by repeatedly transferring the wash solution between the two syringes. After closing valve assemblies 22, 24, perfusion slit chamber 10 is incubated at, e.g., 65� C. for 30-60 minutes. This procedure is repeated with moderate and high stringency wash solutions. The temperature and salt concentration of the final wash solution should be chosen so that the incubation temperature is about 5�-8� C. below the Tm of the hybrid understudy. Although not ususally necessary, wash solutions can be rapidly screened with a Geiger counter to assess completion of the washing.
Perfusion slit chamber 10 is next disassembled after removal of the final, high stringency wash solution. The treated sheet 48 is removed, blotted, and enclosed, still damp, in, e.g., plastic wrap. In a darkroom, the wrapped sheet 48 is sandwiched between two sheets of x-ray film. The sandwiched sheet 48 is incubated at, typically, -70� C. for one to five days prior to development of the x-ray films.
While the above is a complete description of the preferred embodiment of the invention, other arrangements and equivalents are possible and may be employed without departing from the true spirit and scope of the invention. Many such alternate arrangements and equivalents have been suggested above; others will be apparent to those skilled in the art. Therefore, the description and illustrations should not be construed as limiting the scope of the invention, which is delineated by the appended claim.
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|Omni Blot™ brochure by American BioNuclear.
|Turbo Blot brochure by American BioNuclear.
|Turbo Blot™ brochure by American BioNuclear.
|Citing Patent
|Filing date
|Publication date
|Feb 5, 1987
|May 30, 1989
|Levin Andrew E
|Apparatus for blot screening numerous, small volume, antibody solutions
|Mar 16, 1988
|Mar 13, 1990
|Smyczek Peter J
|Laboratory apparatus
|Dec 14, 1990
|Mar 2, 1993
|Minuth Wilhelm
|Apparatus for cultivating cells
|Oct 5, 1990
|May 17, 1994
|Engstroem Gunnar
|Method and apparatus for studying a reaction pattern of a cell or cell aggregates during perfusion with different media
|Oct 3, 1994
|Nov 12, 1996
|Lifecodes Corp.
|Methods and materials for detecting autoimmune antibodies
|Feb 5, 1996
|Dec 1, 1998
|Advanced Tissue Sciences, Inc.
|Apparatus for the growth and packaging of three dimensional tissue cultures
|Feb 23, 1998
|Feb 1, 2000
|Tam; Joseph Wing On
|Flow through nucleic acid hybridisation device
|Jun 5, 2000
|Apr 1, 2003
|Axon Instruments, Inc.
|Perfusion chamber for electrophysiological testing of oocytes
|Nov 19, 1999
|Jan 20, 2004
|Becton, Dickinson And Company
|Rapid protein identification using antibody mixtures
|Feb 26, 2004
|Aug 26, 2008
|Georgia Tech Research Corporation
|Bioreactor and methods for tissue growth and conditioning
|Feb 26, 2004
|Jan 13, 2005
|Gemmiti Christopher Vinson
|Bioreactor and methods for tissue growth and conditioning
|Oct 31, 2006
|Sep 3, 2009
|Alza Corporation
|Methods of Reducing Alcohol-Induced Dose Dumping for Opioid Sustained Release Oral Dosage Forms
|Jan 12, 1995
|Jul 20, 1995
|The Jockey Club
|Non-invasive sampling method for nucleic acid analysis
|Oct 31, 1997
|May 14, 1998
|Grace Bio-Labs, Inc.
|Cytoperfusion of tissue specimens
|U.S. Classification
|435/287.2, 435/297.1
|International Classification
|Cooperative Classification
|European Classification
|Mar 21, 1986
Owner name: HOEFER SCIENTIFIC INSTRUMENTS, A CORP. OF CA.
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Welcome to St. Augustine's Parish,
Nova Scotia
St. Augustine's Church, Chester
With missions in:
St. Anthony's Church, Hubbards
St. Patrick's Church, New Ross
St. Augustine's Church, Chester
With missions in:
St. Anthony's Church, Hubbards
St. Patrick's Church, New Ross
"Blessed are the Peacemakers, for they shall be called the children of God."
(Matthew 5:9)
4:00 pm • St. Patrick's, New Ross
6:30 pm • St. Augustine's, Chester
9:00 am • St. Augustine's, Chester
11:15 am • St. Anthony's, Hubbards
Wednesdays • 9:00 am • St. Augustine's
Thursdays • 9:00 am • St. Anthony's
Fridays • 9:00 am • St. Augustine's
Sunday, May 1: Food Bank Sunday, St. Anthony's Church
Sunday, May 8: Mothers' Day
Monday, May 9: Guild Annual Meeting, 7:00 pm, Home of Pat Christiansen
Sunday, May 15: First Communion, St. Augustine's, 9:00 am
Sunday, May 22: Last Day for Sunday School
Sunday, June 5: Food Bank Sunday, St. Anthony's Church
Tuesday, June 7: Pastoral Council Meeting, Chester Glebe, 7:00pm.
Monday, June 13: Guild Annual Dinner TBD
Sunday, June 19: Fathers' Day
By appointment, please contact the office
By appointment, At least six months notice must
be given. Contact the office.
St. Augustine's Parish offers religious education and sacramental preparation for children from Primary to grade 8. Classes are held from mid September through to the end of May, in Chester, each Sunday following the 9am Mass. In addition to our Sunday classes, St. Augustine's also offers a home schooled based program, for those parents who have difficulty travelling the distance to Chester. This year, classes will begin on Sunday, September 20th and registration forms will be available at the back of each church the first weekend of September.
For further details please contact Jennifer Paterson, Catechetical Program Coordinator, 902-275-4427 or call St. Augustine's office 902-275-5463
Tel: (902) 275-5463 (Office, Chester Glebe)
Fax: (902) 273-3037
Home • About
St. Augustine's • Schedule •
Responsible Ministry •
Get In Touch • Photo Gallery
"Blessed are the Peacemakers, for they shall be called the children of God."
(Matthew 5:9).

subset: commoncrawl


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Golden Fountain: The Complete Guide to Urine Therapy Paperback – October 1, 1996
The Amazon Book Review
Author interviews, book reviews, editors picks, and more. Read it now
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Top Customer Reviews
Firstly - it works. I don't believe this to be a placebo effect, as I've tried many other remedies for various ills, and none have delivered the amazing results that urine therapy has in just three months. My previously constant urinary tract irritation has disappeared at last, and my body feels much stronger. Recently I took an overseas trip where I was surrounded by people with the flu - starting with all the air hostesses on the way out and culminating with most of my family upon my return. I didn't catch it, which for me is quite amazing, especially considering the stress I was under.
I find it perplexing that some reviewers and 'medical experts' are so dismissive of this therapy - claiming that there is no scientific evidence to prove that it works. This seems to fly in the face of the fact that urine derivatives are used in a host of medicines that are prescribed by 'medical experts', and bought over the pharmaceutical counter by countless 'normal' people who would never entertain the idea of ingesting their own urine. However, horse urine or urine from other people (or derivatives thereof) are deemed (perhaps unknowingly) as perfectly acceptable. Go figure!
My advice to all of you out there who are suffering from chronic ailments that are unresponsive to orthodox therapy is - go and buy this book, read it, and give the therapy a go. After all - what do you have to lose? Even the most rabid 'expert' detractors will admit that it does no harm.Read more ›
Kroon first became interested in urine therapy in India after sustaining a serious injury to his foot. When instead of healing, the foot gradually grew worse, someone suggested that he use urine compresses. Having nothing to lose he decided to give it a try - and it worked! He then set about researching the subject, both in India and the West, and 'The Golden Fountain - The Complete Guide to Urine Therapy' brings us the fruits of his research and his years of practical experience.
Although not a large book it's packed with an enormous amount of fascinating and valuable information, too much to even begin to adequately describe. After a brief Introduction, Kroon takes up the history of Urine therapy in both the West - where it was used to cure innumerable complaints in earlier centuries - and in the East.
Urine, we learn, has anti-bacterial, anti-fungal, anti-viral, and anti-cancer properties. Also, by strengthening the immune system and correcting the imbalance which underlies most disease, it can in many cases help to cure just about anything, and it has other valuable properties too.
A chapter on Applications is included which describes the various ways urine can be used, both internally and externally, to promote health, beauty, longevity, and even sexual potency! Detailed instructions are given on drinking, bathing, gargling, douching, eye and ear drops, sniffing, massage, compresses, rubbings, foot and hip baths, scalp and hair massages, and other applications.Read more ›
I used this therapy to help break my heroin addiction, and now I use it to maintain a positive, healthy lifestyle. There have been no relapses, and cravings I've felt in the past just aren't there this time around. If you have any type of addiction, from food to sex, this is the book for you.
Modern science may be a wonderful thing but take it from me - nothing beats a nice cup of your own urine.
Urine is liquid which has been flowing through your veins in the past few minutes and is filtered of impurities by the liver and passed on to your bladder as antiseptic and has no bacteria in it as does the blood and meat of the beef, pork, chicken and other animal products you eat. Urine has no connection to the fecal material that is processed in a different manner and those who try to connect the two are unable to reason correctly.
Ingesting urine was a very difficult decision but after doing a lot of research I finally took the plunge, so to speak. Within three weeks the athlete's foot condition that I've had for 60 years disappeared (yeah, I tried the oral prescription medicine for six months, and had toenails removed, and that didn't work), pre-cancer conditions have both disappeared and receded (as verified by a dermatologist), burning eyes and night sensitivity to light - a 70+ year condition - are totally gone. There are also many other benefits I could mention but these are just a few, and this is after just two months of using it.
For those who want to upchuck at the thought of drinking their own urine, then why has it been done with significant benefits with no adverse effects for thousands of years? Our Western view of "dirt" has produced a sick, antiseptic, antibiotic world which is producing super disease that the drug companies can no longer keep up with. When you lose your health (which many who read this will eventually do) you will kick the MD's in their wallet and seek real healing modularities of which urine therapy is but one.
This is by far the best book there is on the subject of urine therapy.
Customer Images
Most Recent Customer Reviews
Very informative book. Well written and simple easy read. The introduction by the Author is great as is his experience in Chapter 1. Getting the Taste. Chapter 4. Read morePublished 16 days ago by D. Shultz
Very good condition book, like new! I love this book and had already read a neighbors copy and so I wanted a copy of my own. This practice is working well for me. Read morePublished 10 months ago by Amazon Customer
Excellent! Highly informative. I've used the therapy and found it to be incredibly effective.Published 22 months ago by Nina
Kroon does an excellent job of giving the history of urine therapy and the implementation thereof. His examples and outcomes of using the therapy is also excellent in helping to... Read morePublished on December 1, 2013 by Clyde Chiever
This book contained all the info that we wanted, and was a good value. We gave our book on the subject away, and it was good to find a replacement book.Published on February 6, 2013 by Frankie Clayton
It is a deep and complete text about Urine Therapy. Very well written.
Explains what is essential and gives practical examples.
Prof. Flaminio Levy
This book is well written, with fundamental instructions for the beginner and added knowledge for those who are familiar with the concept. Well done!Published on May 28, 2012 by Sandy Sullivan


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Enjoy country living with city conveniences! Well maintained custom 4BD/3BA home with mountain views. Mostly single level. Bright open floor plan designed for entertaining. Large gourmet kitchen, vaulted ceilings, 2 fireplaces. Spacious master suite. Relax by the beautiful koi pond with waterfall. Bonus space for office or crafts room. With 2.4 +/- acres there's room for pool, animals, or guest house. Peaceful creekside setting. Easy commute access.
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|Fernando Padilla <f...@alum.mit.edu>
|Re: slices hangs if run with Multithreaded=true
|Sat, 13 Dec 2008 01:12:59 GMT
So, do you want me to supply a patch, or will you make the quick code changes to turn off ParallelExecutor ( or at least make it configurable ). Pinaki Poddar wrote: >> Since behind the scenes you are executing various different queries using > multiple >> threads, and they might be touching/using shared resources lower down. > > Good point, and most probably you are right. > >> which is to fix up QueryImpl, not to do aggressive locking (at least in >> the isUnique method), and I >> think it will at least remove the current blocking issue. > > So far, Slice has imposed almost no demand on existing kernel -- which is > battle hardened code. So naturally, I am apprehensive of change in threading > logic in kernel.QueryImpl. Any change of this nature *must* validate itself > against a set of multithreaded tests (with or without Slice). >
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Hi and thanks for this awesome plugin.
I found a small annoyance (not really an issue) when you work with wpml and you use parameters for languages (I mean "www.example.com/post?lang=something").
Since you access the live edit screen via "?editing", when you try to edit a translated page, the lang gets stripped out and you work on the original.
IE: I was trying to work on mypage?lang=it and instead, I found myself editing the original, english version.
I easily solved this small annoyance by manually writing the url:
The plugin just should be able to do so for itself...
Also, 2 considerations:
1. wpml is just one case, so I'm pretty sure this annoyance is potentially bigger
2. not every wp user would correct the url manually, and this could result in a major issue for many
Thanks again


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We analyze the ability of mobile phone-generated accelerometer data to detect high-level (i.e., at the semantic level) indoor lifestyle activities, such as cooking at home and working at the workplace, in practical settings. We design a 2-Tier activity extraction framework (called SAMMPLE) for our purpose. Using this, we evaluate discriminatory power of activity structures along the dimension of statistical features and after a transformation to a sequence of individual locomotive micro-activities (e.g. sitting or standing). Our findings from 152 days of real-life behavioral traces reveal that locomotive signatures achieve an average accuracy of 77.14%, an improvement of 16.37% over directly using statistical features.
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LANVIN Christmas tree video
Video of the LANVIN Christmas tree 2011 st the hotel CLARIDGE London.
Video du sapin de Noel LANVIN 2011 a l hotel CLARIDGE de Londres .
Video of the LANVIN Christmas tree 2011 st the hotel CLARIDGE London.
Video du sapin de Noel LANVIN 2011 a l hotel CLARIDGE de Londres .
HEMEN is one of these brand with a strong legacy, while today stories become more and more rare. The Basques believe that in olden days, during transhumance, the sheperds' wives used to give their h...Read More
JR was presenting yesterday night, at GARLERE PERROTIN in Paris his newest solo exhibition called "DECADE, PORTRAIT D'UNE GÉNÉRATION". The exhibition features a selection of videos, ink on wood pi...Read More
We can now enter into the intimacy and daily life of Imperial Stormtroopers, with the series The Other Side of the photographer JORGE PÉREZ HIGUERA. We see them engage in mundane activities like wash...Read More
Parla's Cuban American heritage is a duality that infuses all of his work. Where the urban wall structures signify fixed geographical-political boundaries, the gestural performance symbolize...Read More
the contemporary artist Yoshitomo NARA has collaborated with THE BIG ISSUE JAPAN magazine for the September 2015 issue "NO WAR" . (more…)...Read More
We already talked to you about her, but we still love her. Rotterdam based artist SEVDALIZA just dropped her first real single. The track, titled "THAT OTHER GIRL" is relaxing, surprising. Sevdali...Read More


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4 Reviews
From your son, I've got it in my cookbook!
Good but a little bland. Will try to spice it up next time.
This is really easy and tasty. I have tried many different Shepherds Pie recipes with limited positive feedback from my family. They really liked this one! I had never thought of using chicken before. :-) I strained the broth from leftover vegetable soup and used those vegetables instead of frozen. I browned a couple chicken breasts with some garlic for the chicken and used my leftover mashed potatoes. Very yummy! Thank-you!
really simple and fast i cant believe it wasnt posted either thank you for posting dee


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"Hi, my name is Coco & I'm a recent transplant to the Bay Area. I'm from South Africa & would love to meet new people :)."
"We moved here from Germany in 2012, and we LOVE it! Looking forward to making new friends and discovering new things, whatever they are!"
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Beauty with an edge.
I exhibit my nudes at 30+ shows per year, click to view my Exhibition Schedule and see my work in person.
More than 1000 of my nudes can be seen on My DreamsOfTheGoddess.com website
My Spirit Work can be seen at My SpiritTakesForm.com website


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Top Definition
Tushana is a small child, only about 4 ft tall who hates people taller than them. Tushanas are typically sulky, grumpy children who have nothing better to do than sulk
She's moody today !
Yeah, she's such a Tushana.
Yeah, she's such a Tushana.
by Harry loves me :D December 01, 2009
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JK Rowling's alter ego Robert Galbraith was in the running for a crime-writing prize but it was won by Mackintosh's first book 'I Let You Go'
JK Rowling's boy wizard came second in a survey of our favourite heroes from kids' books – just behind Winnie the Pooh
The next Prime Minister will be a woman. Will the Tories choose the right one?
Most British adults would turn the clock back to the carefree days of their youth – if they could
The children's detective books have been keeping young ones entertained for decades, but one reader's got an issue with them
Creator of the iconic Peter Rabbit and friends series Beatrix Potter was also popular
The first edition of the Enid Blyton classic was sold along with other Noddy titles which belonged to Diana's sisters
Forget the likes of 'Paddington Bear' - it's ALL about modern classic 'Everyone Poops'
A hidden archive of photographs and drawings by the original illustrator has finally emerged after decades in the dark
Margaret Thatcher and seven royals made the controversial list. Read on for what an outraged Paul Routledge thinks of it all...
From Blackpool to Brighton, we all know where the staycation crowds will be flocking this summer, but Britain's coastline has some relatively unknown gems
The adventurers, whose first story was published in 1942, are fronting a list of 10 fun things to do in the fresh air during the long school holidays
Speedy reader Faith Jackson averaged 464 pages a day of classics by Enid Blyton and Roald Dahl as well as newer favourites like the Harry Potter series
A survey found Roald Dahl's Charlie and the Chocolate Factory crossed generations - and despite the boom in technology, children preferred a paperback to e-books
The Coronation Street, Loose Women and Benidorm star has written a new book of recipes to help grandmas make cooking a fun and healthy activity with the little helpers
Escape to the beautiful Dorset coast for an adventure in the footsteps of the Famous Five- with lashings and lashings of fun
The star blogger turned author shares her passions for the printed word
The drama is inspired by former Labour MP Chris Mullin's novel A Very British Coup, which was filmed (also by Channel 4) back in 1988
After months of meticulous planning and sleepless nights the Mirror gardening columnist' Diarmuid Gavin's creation is ready
I would not be a writer if it were not for public libraries.
Today the Daily Mirror launches a brilliant new campaign – We Love Reading.
TV is Edukashiunal
Cruising along England's canals on a houseboat is the perfect break for the whole family...
A COMPANY director told yesterday how his daughter fell 20ft to her death down a mine shaft as she explored a cave on an Enid Blyton-style adventure.
1. Mount Haven
Britain's TV talent enjoyed a triumphant night at the International Emmys, walking off with half the main awards.
CORRIE beauty Samia Smith reckons having a baby has made her fitter than ever – thanks to exercise and oil.
Bob Hoskins and Helena Bonham Carter have each been nominated for International Emmy Awards, as British TV productions garnered nine nods.
Graham Norton kicked off the TV Baftas tonight - with a gag about Britain's Got Talent winners Spelbound.


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Embargo Policy
Accepted abstracts were made available to the public when the embargo is lifted October 1. Information contained in those abstracts may not be released until the abstracts appear on the AASLD website. Academic institutions, private organizations, and companies with products whose value may be influenced by information contained in an abstract may issue a press release to coincide with the availability of the abstract on the AASLD website.
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Television and video crews must be accompanied by AASLD representatives whenever taping inside The Liver Meeting®. Shooting schedules must be provided to AASLD staff in the press room on the first day of the meeting. Artificial light cannot be used during the presentation. Flash photography at scientific presentations and plenary sessions is not allowed. Handheld audio recorders and still cameras are acceptable, assuming that intellectual property copyrights are respected.
An exhibit or poster may not be photographed or videotaped without the permission of the authorized exhibitor or presenter. Permission of the speaker and moderator must be obtained by the journalist before recording or photographing presentations.
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Events Schedule
Mon22Aug2016Mon05Sep201610:00 am - 5:00 pm Ore Wagon Museum - East Avenue and Fifth Street
Wagon Days headquaters opens
Please stop by between 10:00 am and 5:00 pm for information, hats, t-shirts, posters, buttons and much more. We always love seeing your smiling faces so don't hesitate to come say hello.
Fri02Sep201612-1p.m.Ore Wagon Museum
Cowboy Poets Recite
Join us at the Ore Wagon Museum surrounded by Wagon Day Posters and listen to the vintage Cowboy Poets as they swap the stories that have been thrown around cow camp cook fires for centuries. Regional Western Poets: Brian Dilworth; Mick Halverson; Tommie Patton; Wayne Nelson and Bruce (Looselip) Pinson will fill the Barn with classic Cowboy Poetry as well as contemporary stories authored by these same Poets.
Fri02Sep20165 p.m. - 8 p.m. Downtown Ketchum
Sun Valley Gallery Association hosts a guided Ketchum Art Gallery walk on Friday, September 2nd, 2016. Starts at 5 p.m. Features new work at member galleries which includes Gilman Contemporary, Wood River Fine Arts, Broschofsky, Boloix, Kneeland, Severn, Harvey Art Projects, Friesen and Bellinger Fine Arts.
For more information and a map of the galleries please visit Sun Valley Gallery Association
Fri02Sep20165:30-7:00 p.m.Memory Park Main Street between 5th and 6th
Please join the City of Ketchum and the Wagon Days committee as they honor the 2016 Grand Marshal, Sandy Cady. Memory Park on Main Street between 5th and 6th. Live music, food and drink, open to the public.
Fri02Sep2016Sat03Sep2016Various Times Various Locations
Meandering Musicians Perform
Revisit the musical outburst in Ketchum that occurred centuries ago when these same Lewis Ore Wagons, filled to the brim with heavy lead and silver ore, pulled for weeks by mule teams, struggled into town after completing their treacherous, sometimes deadly trip over the steep mountain trails. Their payload supported the town, creating paydays for miners, teamsters, farriers, and jobs at the train station, the local smelter and especially in the restaurants and saloons that thrived in town.
This weekend, as in those times, local musicians and their stringed instruments are meandering through the streets and select venues in town with music to perpetuate that successful Ore Wagon tradition, "when the Wagons are rolling, the fiddlers are strolling".
Participating Venues
Cornerstone Bar & Grill - Globus Restaurant-Grill at Knob Hill - Kneadery - Perry's Restaurant-Pioneer Saloon - Sun Valley Wine Company
Fri02Sep2016Mon05Sep2016Various Times Located throughout the Valley
Festivals throughout the Wood River Valley bring professional arts, crafts, and antique dealers from all over the West.
Hailey's Antique Market
Roberta McKercher Park and Hailey National Armory, Thursday – shop set up!
Ketchum Antique & Art Show Labor Day 2015
Over 30 Friendly Dealers with Reasonably Priced Antiques – Arts – & Collectibles!!
Something for Every Collector!!
Promoted by Blue Cow Antiques – Dana Jo Cameron – Call 312-4900 or Email - email@example.com
Sat03Sep20161:00 pm
The largest non-motorized parade in the Northwest, the Big Hitch Parade includes over one hundred museum quality buggies,carriages,carts,stage,coaches, and wagons.The six enormous Lewis Ore Wagons, known as the Big Hitch, are the grand finale and are pulled by an authentic 20 mule jerkline.
Please click on map for larger printable version
Sat03Sep201610:30 amFestival Meadows
The Eh-Capa Bareback Riders demonstrate the horse riding and jumping techniques of the Native Americans. They ride without the benefit of saddles or bridles.
Sat03Sep20163:00 pmEast Avenue
Join us on East Avenue for music, food, and a family friendly environment. Stay "in tune" with the weekends western theme, and listen to some good ol' country music, from Amy Clawson & Coltrane. The party begins after the parade, the parade awards are announced at 4 p.m., with live music throughout and headliner Amy Clawson and Coltrane taking the stage at 5 p.m.
Sat03Sep2016Sun04Sep20165 p.m. - 8 p.m. Atkinson's Park
Meet, greet and welcome party
Sat03Sep2016Sun04Sep20168 a.m. - noonKetchum Town Square
Start Your Labor Day Weekend With a Pancake Breakfast!
Join us and start the Labor Day celebration right: Papoose Club invites you to our famous all-you-can-eat annual Pancake Breakfast.
Papoose Club members and friends will be cooking up delicious pancakes, sausage, scrambled eggs, and serving Starbuck's coffee. Held at the Ketchum town square during Wagon Days festivities, Papoose Pancake Breakfast is a locals best loved tradition that dates back to 1977.
Come have breakfast with us on Saturday morning and then walk to Main to watch The Big Hitch Parade at 1:00. Come back again for more breakfast on Sunday and bring friends and family!
Saturday, September 3rd
Sunday, September 4th
8:00 AM to 12:00 PM
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$8 Adults
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$5 Children
Free Under 3
Papoose Club is a local charitable organization that helps fund youth programs in Wood River Valley. The Pancake Breakfast is one of our most important fundraising events, so come support your community by eating breakfast with us.
Papoose Club celebrates over 60 years of the Wood River Valley tradition!
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We wish to thank our generous sponsors for another year of support!
Sat03Sep20168:30 pmSun Valley Resort
Sun Valley Ice Show
Buffet doors will open at 7:30pm.Bleacher doors will open at 8:30pm.
Sun04Sep2016Sunday AfternoonKetchum Town Square
The ride finishes in Ketchum's scenic town square. Events at the finish include an old-fashioned Idaho barbecue with food, music, bike valet and heel-kicking festivities.
Mon05Sep20166:00 pmSun Valley Pavilion
Tickets available at: www.sunvalley.ticketfly.com & the Sun Valley Recreation Office.


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We present a technique of nonlinear image encryption by use of virtual optics. The image to be encrypted is superposed on a random intensity image. And this superposed image propagates through a nonlinear medium and a 4-f system with single phase key. The image is encrypted to a stationary white noise. The decryption process is sensitive to the parameters of the encryption system and the phase key in 4-f system. This sensitivity makes attackers hard to access the phase key. In nonlinear medium, optically-induced potentials, which depend on intensity of optical wave, make the superposition principle frustrated. This nonlinearity based on optically induced potentials highly improves the secrecy level of image encryption. Resistance against attacks based on the phase retrieval technique proves that it has the high secrecy level. This nonlinear image encryption based on optically induced potentials is proposed and demonstrated for the first time.
1. Introduction
Optically-induced potentials are generated by propagating beams that create refractive index changes in the host medium. Phenomena such as plasma filamentation and self-focusing of femtosecond pulses are the typical examples. Digital reconstruction of optically-induced potentials is proposed using nonlinear digital holographic technique [1]. In ref [2], complex field exiting out of a nonlinear medium is recorded using the technique of phase-shifting digital holography [3, 4] and the complex field numerically back-propagates by nonlinear Schrödinger equation. It's a new useful method in image and signal processing.
Image processing based on optical technology has shown great potential in the field of information security. Many optical encryption schemes were proposed in the past decade [5–9]. The most extensively studied optical security scheme [5] proposed by Refregier and Javidi uses the double random phase masks to encode an image into a stationary white noise. This method has been extended to fractional Fourier domain [6] and Fresnel domain [7].
In recent years, virtual optics is widely used in image encryption in which both encryption and decryption were done digitally [10–12]. Namely, virtual optics digital encryption and decryption have become the main practical technique in optical information security. This method can easily control the phase mask and the optical wave property. Compared with traditional optical setup, there is no need to fabricate physical phase mask which means that the phase mask key can be easier to change; the alignment problem of two or more phase masks can be easily tackled.
The conventional double random phase encoding (DRPE) method is susceptible to chosen plaintext attack (CPA) and known plaintext attack (KPA). The CPA firstly done by Carnicer et al. [13] indicated the vulnerability of the conventional DRPE scheme. CPA requires the attacker to control the system to encrypt particular image. Peng et al. proposed KPA on conventional DRPE scheme. With the help of the phase retrieval technique, both the random phase keys can be recovered [14]. Frauel et al. proposed an analytical method to access the exact keys with only two known plaintext images [15]. Liu et al. propose a hybrid heuristic algorithm to improve KPA on conventional DRPE scheme, making KPA more practical and effective [16].
For conventional DRPE scheme, cryptanalysis [14, 15] indicates that linearity is the main weakness of this system. In this paper, we propose and demonstrate nonlinear image encryption based on optically induced potentials for the first time. It makes superposition principle frustrated and results in hiding of plaintext by superimposing a random intensity image on the plaintext. KPA is in vain to this method.
2. Principle of optically-induced-potential image encryption
The schematic of image encryption system is illustrated in Fig. 1 . The image to be encrypted is normalized firstly. Random distributed image which acts as intensity key is superimposed on the plaintext. The intensity key can be expressed as r(x, y), where r(x, y) is an independent random function. The treated plaintext image is uploaded on the spatial light modulator (SLM) in plane P0. The SLM is illuminated by a polarized plane wave and the input field propagates through the nonlinear medium. The field exiting out of the nonlinear medium propagates through a 4-f system with single phase key. Two lenses L1 and L2 with same focal length form the 4-f system. Random phase masks (RPM) M is placed in plane P2. Complex transmittances of the phase mask can be expressed as exp(i2πp(x, y)), where p(x, y) is an independent random function that uniformly distributed in [0, 1]. The complex field in plane P3 is recorded by phase-shifting holography.
Fig. 1
For the propagation in nonlinear medium, we consider the case of scalar wave under paraxial approximation. In this condition, the propagation can be described by the nonlinear Schrödinger equation [1, 2]:17,18]:1, 2] in which diffraction and nonlinear operators act individually in a small propagation distance dz. In fact, the diffraction process and the nonlinear process happen simultaneously. But when the propagation distance dz is very small, this approximation will have very small error.
The complex-amplitude of output field of the nonlinear medium can be obtained by:
The complex field in plane P3 is
In procedure of decryption, with correct phase key and correct parameters of the system, the field in plane P3 back-propagates through the system.
The backward propagation in the nonlinear medium is also an initial-value problem. The output field is the starting point. The amplitude of field in plane P0 can be obtained by:
And the decrypted result could be obtained with an intensity key.
The encryption and decryption procedure could be summed up as follows. The superposition of plaintext and intensity key form input field of the encryption system. The encryption procedure is the forward propagation of the input field. And the decryption procedure is the backward propagation of ciphertext with correct phase key and correct parameters of the system to get the input field in plane P0. With a correct intensity key, correct plaintext could be obtained from the input field. During propagation in nonlinear medium, the wavefront of the intensity key and the plaintext would interact with each other. The output field of nonlinear medium in plane P1 is not the sum of individual output field of the intensity key and the plaintext because of this interaction. This property makes attackers hard to retrieve intensity key.
3. Results and discussions
We take out numerical experiments to verify feasibility and effectiveness of this method. The pixel size on SLM in plane P0 is 10μm*10μm. In this situation, paraxial approximation could be satisfied. The Lena image with 256×256 pixels shown in Fig. 2(a) is used as plaintext. Zeros is padded to extend the size of the image to 512×512 and a random intensity image is superimposed on the plaintext. The length of the photorefractive crystal Fe:LiNbO3 is 20cm and parameters of the crystal are Epv=−27×105V/m, ne=2.20, γeff=30.9pV/m, at the wavelength of λ=514.5nm. The intensity of the treated Lena image is normalized with the background intensity. The optical wave in the crystal is e-polarized. The step length dz is 200μm. After the propagation of the nonlinear crystal, the real-part/image-part of complex-amplitude of the field exiting out of the nonlinear medium in plane P1 is shown in Fig. 2(b)/ (c). The real-part/image part of the ciphertext is shown in Fig. 2(d)/ (e). Decrypted image with correct keys and correct parameters is shown in Fig. 2(f). The correlation coefficient (CC) between the plaintext and the decrypted image is 1.0000 meaning that the image can be decrypted without any distortion. The correlation coefficient is defined as CC(x,y)=cov(x,y)/(σxσy), where cov(x,y) is the cross-covariance of two variables and σxσy is the product of two variables' standard deviations.
Fig. 2
The parameters of the system should also be exactly correct in order to get correct decrypted image when the phase key in plane P2 and the intensity key in plane P0 are exactly known. In decryption procedure, the parameter of nonlinear crystal should be exactly correct, or the optically-induced potentials would go wrong which makes the decrypted result incorrect. When γeff increases by 0.0001%, the decrypted image is shown in Fig. 3(a) . The decrypted image is added with noise. When γeff increased by 0.0005%, the decrypted image is shown in Fig. 3(b), the useful information is totally blurring. When γeff increased by 0.001%, the decrypted image is shown in Fig. 3(c), the decrypted image becomes a random noise. The decrypted result is sensitive to nonlinear parameter γeff.
Fig. 3
In a small propagation distance in the nonlinear medium, there are linear diffraction process and nonlinear process. The decrypted result is also sensitive to the parameter of linear process. When the wavelength λ increases by 0.0001% in linear process while the nonlinear parameters are kept exactly correct, the decrypted image is shown in Fig. 3(d). The decrypted image is added with noise. When λ increased by 0.0005% in linear process, the decrypted image is shown in Fig. 3(e), the useful information is totally blurring. When λ increased by 0.001%, the decrypted image is shown in Fig. 3(f), the decrypted image becomes a random noise. The linear diffraction parameter is also critical to the decrypted result.
To investigate the decryption sensitivity to parameters of the system when the phase key in plane P2 and the intensity key in plane P0 are exactly known, we calculate the dependence of CC on the nonlinear coefficient and wavelength λ in linear diffraction process. We define nonlinear coefficient as:
The dependence of CC on δγ/γ and the dependence of CC on δλ/λ are shown in Fig. 4(a) and Fig. 4(b), respectively. When CC<0.2, the decrypted image cannot be distinguishable. When the deviation of γ is larger than 0.001%, the decrypted image would be undistinguishable. The insets show the zoomed figures. It's clear that the dependence of CC on nonlinear coefficient γ or linear wavelength λ is highly sensitive.
Fig. 4
We investigate decrypted results when a random noise is superimposed on the phase key in plane P2. The random noise can be expressed as Amexp(i2πq(x, y)), where q(x, y) is an independent random function that uniformly distributed in [0, 1], Am is the amplitude of the noise. A random noise whose amplitude Am is 0.00001% to the amplitude of the phase key (the amplitude of the phase key is 1) is superimposed on the phase key in plane P2. The decrypted image with this noise-added phase key is shown in Fig. 5(a) .the decrypted result is added with noise. When the amplitude of the noise increases to 0.0000005,the quality of the decrypted image decreased dramatically(see Fig. 5(b)). When the amplitude of the noise increases to 0.000001, the decrypted image cannot be distinguishable (see Fig. 5(c)). When the amplitude of the noise increases to 0.000005, the decrypted image becomes a noise (see Fig. 5(d)). We also investigate decrypted results in the absence of the nonlinear medium when a random noise is superimposed on the phase key in plane P2. While the parameter of γeff equals zeros (in the absence of nonlinear medium), the encryption system becomes a linear system. Figure 5(e), (f), (g), and (h) respectively show the decrypted images in the absence of the nonlinear medium while random noises with different amplitude of 0.01, 0.05, 0.1, and 0.5 are superimposed on the phase mask M. When the amplitude of the noise increases to 0.1, the decrypted image is still distinguishable (see Fig. 5(g)). The comparison of the decrypted results with noise-added phase key shows that phase encoding in the presence of the nonlinear medium is more sensitive to phase changes than in the absence of the nonlinear medium.
Fig. 5
In order to analyze the nonlinearity of this system, we investigate the situation of superposition of two ciphertexts. For linear system, the output of superposition of two (or more) weighted input functions is the superposition of the weighted individual outputs. In our proposed method, the condition is not satisfied because nonlinear phase change depends on the intensity of optical wave. When the superposition of two plaintexts (ciphertext) becomes a new plaintext (ciphertext), nonlinear phase changes are not the same as they do in the situation of single plaintext (ciphertext). Figure 6(a) and Fig. 6(b) show a pair of ciphertext and corresponding decrypted plaintext. Figure 6(c) and Fig. 6(d) show another pair of ciphertext and corresponding decrypted plaintext. The decrypted result with the sum of the two ciphertexts with weights of 0.5 is shown in Fig. 6(e). In Fig. 6(f), the linear superposition of two plaintexts is shown. Compared with Fig. 6(f), useful information of the plaintext in Fig. 6(e) vanishes. The frustration of superposition principle means the encryption system is nonlinear. Based on this property, hiding of plaintext could be realized by superimposing a random intensity image on the plaintext.
Fig. 6
For known plaintext attack, proposed attacker knows ciphertext (shown in Fig. 7(a) ) and corresponding binary plaintext (shown in Fig. 7(b)). The binary plaintext would be easier to achieve good attack result [14]. Phase retrieval technique is used to calculate the phase key, ignoring the nonlinear crystal and the intensity key. The plaintext retrieved by hybrid input–output algorithm [14] is shown in Fig. 7(c). The attacker uses the retrieved phase key to decrypt another ciphertext (shown in Fig. 7(d)). The decrypted result is shown in Fig. 7(e), while correct plaintext of this ciphertext is shown in Fig. 7(f). The decrypted image with the retrieved phase key doesn't contain any information of the correct plaintext. To access phase key using phase retrieval technique, one should get the correct complex-amplitude in plane P1. In our system, the complex-amplitude of the field in plane P1 is the output field of the nonlinear crystal. The attacker cannot obtain the distribution of complex-amplitude in plane P1 because of the existence of the intensity key and the nonlinear medium. Phase retrieval technique which works in the linear transform systems doesn't work in nonlinear transform systems. In our nonlinear encryption system, the wavefront would be distorted by the intensity of field. The wavefront of the intensity key and the plaintext interact with each other during propagation in nonlinear medium. Phase retrieval technique to recover the phase key will not work. All the attacks based on phase retrieval technique will fail.
Fig. 7
4. Conclusion
In summary, we proposed an optical encryption method with nonlinear medium. The plaintext overlapping with a random intensity key is the input image of the encryption system. The input image propagates through a nonlinear medium and the field exiting out of the nonlinear medium passes a 4-f system with single phase key. Decryption procedure is the backward propagation of ciphertext. The decryption is sensitive to the parameters of the system. The frustration of superposition principle of the encryption system is demonstrated. The system can resist attacks based on phase retrieval technique such as KPA because of its nonlinearity and the random intensity image superposed on the plaintext. The simulation results have shown this encryption method based on optically-induce potentials has high secrecy level.
This work is supported by the National Natural Science Foundation of China (11074311, 10804131, and 10874250), the Fundamental Research Funds for the Central Universities (2009300003161450), and the Guangdong Natural Science Foundation.
References and links
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6. G. Unnikrishnan, J. Joseph, and K. Singh, "Optical encryption by double-random phase encoding in the fractional Fourier domain," Opt. Lett. 25(12), 887–889 (2000). [CrossRef] [PubMed]
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11. H. Kim, D. H. Kim, and Y. Lee, "Encryption of digital hologram of 3-D object by virtual optics," Opt. Express 12(20), 4912–4921 (2004), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-12-20-4912. [CrossRef] [PubMed]
12. X. Wang, D. Zhao, F. Jing, and X. Wei, "Information synthesis (complex amplitude addition and subtraction) and encryption with digital holography and virtual optics," Opt. Express 14(4), 1476–1486 (2006), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-14-4-1476. [CrossRef] [PubMed]
13. A. Carnicer, M. Montes-Usategui, S. Arcos, and I. Juvells, "Vulnerability to chosen-cyphertext attacks of optical encryption schemes based on double random phase keys," Opt. Lett. 30(13), 1644–1646 (2005). [CrossRef] [PubMed]
14. X. Peng, P. Zhang, H. Wei, and B. Yu, "Known-plaintext attack on optical encryption based on double random phase keys," Opt. Lett. 31(8), 1044–1046 (2006). [CrossRef] [PubMed]
15. Y. Frauel, A. Castro, T. J. Naughton, and B. Javidi, "Resistance of the double random phase encryption against various attacks," Opt. Express 15(16), 10253–10265 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-16-102532. [CrossRef] [PubMed]
16. W. Liu, G. Yang, and H. Xie, "A hybrid heuristic algorithm to improve known-plaintext attack on Fourier plane encryption," Opt. Express 17(16), 13928–13938 (2009), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-17-16-13928. [CrossRef] [PubMed]
17. N. V. Kukhtarev, V. B. Markov, S. G. Odulov, M. S. Soskin, and V. L. Vinetskii, "Holographic storage in electrooptic crystals 1. Steady-state," Ferroelectrics 22(1), 949–960 (1978). [CrossRef]
18. W. L. She, C. C. Xu, B. Guo, and W. K. Lee, "Formation of photovoltaic bright spatial soliton in photorefractive LiNbO3 crystal by a defocused laser beam induced by a background laser beam," J. Opt. Soc. Am. B 23(10), 2121–2126 (2006). [CrossRef]


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Tokyo Gov. Naoki Inose told the Tokyo Metropolitan Assembly this week that he went home directly after receiving a large sum of money from a House of Representatives lawmaker last year, but the claim has been found to be false, metropolitan government officials said Thursday.
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7 Reviews
it was good with the bannas and strawberrys but we added 2 heathy treat which was tofu and spinich it may look green but you dont taste the tofu or spinich at all so its heathly and all,you should try it yummmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmm
very good for summer
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I thought this recipie was really good and easy to make.
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I made this in Famliy and Consumer Science and it was delicious! It doesn't look that good at first but if you put die in it, it looks more appetizing.
We have made this recipe in my Family and Consumer Sciences class for four years now. This year we added one banana, a little frozen spinach and a little tofu to make it healthy. This recipe is very good and covers up the taste of spinach. I think the tofu makes it creamier. The color comes out disgusting, but some food coloring helps. I think I will continue to make this recipe and experiment next time by adding other vegetables.