text:
|Publication number
|Publication type
|Application number
|Publication date
|Nov 24, 1987
|Filing date
|Dec 3, 1986
|Priority date
|Dec 3, 1986
|Fee status
|Also published as
|Publication number
|John G. Linner, Stephen Livesey
|Original Assignee
|The Board Of Regents, The University Of Texas
|Export Citation
|BiBTeX, EndNote, RefMan
|Non-Patent Citations (22), Referenced by (41), Classifications (16), Legal Events (8)
|External Links: USPTO, USPTO Assignment, Espacenet
1. Field of the Invention
This invention relates to apparatus and method for the ultrarapid cooling of biological samples. Ultrarapid cooling is a preparatory step to the cryopreparation of biological samples in apparatus such as that described in commonly assigned U.S. Pat. Nos. 4,510,169 and 4,567,847 issued to John G. Linner and commonly referred to as "The Linner Process" or "The Linner Apparatus". It is well known in the medical arts that to examine biological samples and determine the cellular structure and function thereof, the samples must be "fixed" with minimal alteration of ultrastructural integrity prior to the application of nearly all analytical methodologies. The apparatus of this invention can be used to ultrarapidly cool biological samples without the formation of resolvable ice crystals so that the ultrastructural integrity of the sample is not altered.
The terms "biological samples", "tissue samples", and "biological tissue" are used throughout this disclosure to refer to samples that can be ultrarapidly cooled by the method and apparatus of this invention. The terms are used interchangeably and are not intended as a limitation on the functional capability of the method or apparatus disclosed herein. The terms should be understood to include small tissue samples appropriate for microscopic examination and larger tissue masses such as corneas which are appropriate for transplantation The terms should be understood to include any material composed of one or more cells, either individual or in complex with any matrix or in association with any chemical; and to include any biological or organic material and any cellular subportion, product or by-product thereof. The terms should be further understood to include without limitation sperm, eggs, embryos, blood components and other cellular components. The contemplated utility of the apparatus of this invention is not limited to specific types or sizes of tissue, rather it should be understood to refer to any tissue made up from cells. The apparatus of this invention can be designed or adapted to any size, shape or type of cellular tissue. Therefore, the terms "tissue" and "tissue samples" are used interchangeably and are not limiting on the uses to which the method and apparatus of this invention can be placed.
Although the method and apparatus of this invention are preferably used as a preliminary step in the cryopreparation of biological samples for ultrastructural analysis, i.e. electron microscopy, it should be understood that this is not intended as a limitation on the utility of the apparatus and method of this invention. To the contrary, the ultrarapid cooling method and apparatus of this invention have demonstrated utility in any area in which the ultrastructure of cellular components is desirably maintained in an unaltered state. Examples of such utility include, but are not limited to, electron microscopy, tissue preservation, tissue and organ transplants and various analytical and diagnostic methodologies. Therefore, although the method and apparatus of this invention are typically described in relationship to electron microscopy this should be understood not to be a limiting factor on the utility of the invention.
Although the examination of tissue by use of various microscopes or related magnifying apparatus has been practiced for many years, there has been an inherent problem in preparing tissue for use with contemporary high resolution analytical microscopes, such as the STEM electron microscopes, which permit the examination of sample constituents via X-ray analysis at powers of from 500X to 500,000X with point to point resolution of 2 to 3 Angstrom units.
It is difficult to interpret the results of tissue analysis while concomitantly assessing the extent of various artifacts produced during the tissue preparation processes. It is thus essential that artifacts be avoided wherever possible. The term "artifact" refers to a product of artificial character due to extraneous agency. Another problem results from physical shrinkage of the tissue sample itself, which results in alteration of ultrastructure and massive rearrangement of infrastructural resolution.
During the so-called "Golden Age of Morphology" the predominant underlying goal in qualitative and quantitative microscopy has been an aesthetically pleasing image. This goal is readily attainable with the fixation methods and apparatus which are currently available. However, it has become essential that the aesthetically pleasing image, which is produced by the preparation process, also yield a tissue sample which accurately reflects the true condition of tissue in the living organism, i.e., approaching the "living state." This is the problem which is addressed and solved by The Linner Apparatus and Process. One essential step in the preparation process is the cryopreparation or cryofixation procedure (as opposed to the freezing procedure). The cryopreparation method and apparatus of this invention results in the preparation of tissue samples which are readily usable in known magnification and analytical apparatus.
In currently known cryopreparation and freeze-drying devices and methods, problems and limitations are encountered in attempts to rapidly cool the tissue sample without physically harming the sample. If the temperature decrease in the sample to its full depth does not take place at a sufficiently rapid rate, artifacts appear, the ultrastructural integrity of the sample may be damaged and the sample will not appear in its "living state." The prior art has therefore attempted to achieve a rapid rate of temperature decrease to the full depth of the sample, in order to minimize such damage.
Although the primary thrust of this application is in the preparation of tissue samples for analysis by current magnification apparatus, the invention is not intended to be so limited. More specifically, the "preparation" of tissue should be understood to refer to preparation of tissue for analysis as well as the cryofixation of tissue in anticipation of transplantation, modification, in vitro or in vivo cellular growth, fertilization, animated suspension or the more typical resin impregnation, setting, infiltration and analysis. The apparatus of this invention can be used to prepare tissue for any medical or analytical procedure without the ultrastructural damage previously thought to be inevitable in cryopreparation.
The apparatus of this invention is to be distinguished from contemporary freeze-drying apparatus. Freeze-drying is a technique which is well known in the art together with the equipment necessary to implement such freeze-drying. See, for example, U.S. Pat. No. 4,232,453. Although in certain freeze-drying techniques liquid nitrogen is used as a cooling medium, the tissue or sample itself does not attain such temperature. Freeze-drying normally contemplates sample temperatures of -50� C. to -80� C. In contrast, the ultrarapid cooling step of the cryopreparation process of the Linner Process and Apparatus contemplate sample temperatures of -196� C. or less. Therefore, for purposes of this application the terms "cryopreparation" and "cryofixation" are used in distinction to conventional "freeze-drying" technology (-50� C. to -80� C.).
2. Description of the Related Art
The most common prior art method for preparation of tissue samples for analysis is by means of chemical fixation and organic solvent dehydration. Inherent in prior art processes is the concomitant artifact creation, sample shrinkage and resultant damage to and modification of tissue characteristics. These tissue characteristic modifications, whether in the form of artifacts or the like, require interpretation by the individual or apparatus analyzing or evaluating the sample. This introduces, in many instances, an unsatisfactory risk of error.
Chemical fixation is a well known technique and has served the analytical biologist well for many years and undoubtedly will continue to do so in certain limited applications. However, as the use of tissue sample analysis becomes more complex and the use of such analysis becomes more widespread, alternatives to chemical fixation are demanded. This is especially true as advances are being made in the magnification and analytical apparatus which are available. It is necessary that tissue preparation methods and the apparatus necessary to prepare tissue samples be equally advanced as the analytical tools, i.e., electron microscopes, which are being used to analyze the samples. Obviously, if the technology for tissue sample preparation is behind the technology of microscopy then the advanced microscopes cannot be used to full advantage by the morphologist or other tissue examiner.
Similarly, it is essential that cryopreparation methods and apparatus develop concurrently with other medical technology, i.e., surgical transplant techniques, bio-engineering and biogenetics. In short, cryopreparation is an essential intermediate step in evolving processes using or analyzing cells or tissue. If cryopreparation apparatus does not evolve then the thrust of medical technology into unexplained and unexplored medical arts will be blunted. The apparatus of this invention represents the cryopreparation breakthrough that will permit research into the use and preparation of biological tissue to keep pace with other advances in medical technology. The ultrarapid cooling apparatus of this invention provides the mechanism for eliminating the problems associated with available cryofixation apparatus.
The most common alternative to chemical fixation and organic solvent dehydration is freeze-drying cryofixed samples. Freeze-drying following cryofixation is a well documented and well known technique for tissue preservation. It has several advantages. Freeze-drying results in a near-instantaneous arrest of cellular metabolism. There is also a stabilization and retention of soluble cell constituents through elimination of solvent contact with the sample. These are significant advantages to cryofixation freeze-drying that have resulted in a great deal of research in attempting to apply cryofixation and freeze-drying techniques to known tissue preparation processes.
Unfortunately, freeze-drying technology inherently possesses a number of disadvantages relevant to tissue preparation methodologies. The primary disadvantage in currently available freeze-drying techniques and apparatus is the inherent formation of ice crystals. As can be readily appreciated, the formation of ice crystals destroys the ultrastructural integrity of the tissue sample being reviewed. The image is distorted and the cytoplasm becomes reticulated. The formation of ice crystals in the sample can also result in a change in pH within microcompartments of the tissue (eutectic formation) which possibly can result in abnormal tertiary conformation of macromolecules. There is also the possibility that proteins will denature and precipitate. These are but a few of the disadvantages which are inherent in the freeze-drying process.
This general topic is discussed in some detail together with other prior art methods in an article entitled "Freezing and Drying of Biological Tissues for Electron Microscopy", Louis Terracio and Karl G. Schwabe, published in The Journal of Histochemistry and Cytochemistry, Volume 29, No. 9 at pp. 1021-1028 (1981). Problems associated with artifact formation are described in "Understanding the Artefact Problem in Freeze-Fracture Replication: A Review", The Royal Microscopial Society, (1982) at pp. 103-123.
A general principle found applicable to freezing techniques, which has demonstrated utility in the preparation of tissue samples, is that as the cooling rate increases, tissue fluids can be "vitrified" without the separation of water to extracellular spaces. The term "vitrified+ or "vitrification" refers to the cryopreparation of tissue samples without the formation of resolvable ice crystals within the cellular structure. It has been postulated that regardless of the rate of cooling, ice crystals may still be formed, but as the cooling rates increase the size of the intracellular ice crystals decreases. The small size or absence of ice crystals at high freeze rates is of course a substantial advantage in morphology retention as this results in minimal artifact creation and minimal ultrastructural alteration or damage during tissue dehydration. The apparatus of this invention provides the ultrarapid cooling of tissue samples to the vitreous phase in less than one second. The ultrarapid cooling according to the present invention is followed by dehydration of the tissue sample while in the state of reduced partial pressure of water vapor without substantial ultrastructural damage to the tissue cells.
Historically, the criteria by which the techniques for rapid supercooling have been judged was not the cooling rate of the system but simply the temperature of the environment in which the tissue was frozen. Thus, the term rapid supercooling has been applied to any system in which the supercooling agent has a temperature of -150� C. or below. The effectiveness of a cooling system, however, is dependent upon the rate at which heat is removed from the sample. Heat transfer is dependent not only on the temperature of the freezing system but also on its physical and thermal characteristics, as well as the size and thermal characteristics of the tissue.
The most commonly used technique for rapid supercooling is to immerse or "quench" the sample in a fluid cooling bath. The most commonly used fluids for quenching are liquid nitrogen, isopentane, propane and fluorocarbons such as Freon 12 and Freon 22. Although liquid nitrogen is generally regarded as an ideal quenching fluid due to its low temperature (-196� C.), there are inherent disadvantages in the use of liquid nitrogen due to the occurrence of tissue surface film boiling caused at least in part by the low heat of vaporization of liquid nitrogen. Film boiling is a characteristic of liquid nitrogen that inhibits the heat transfer rate by actually insulating the sample.
An alternative method for rapid supercooling is applying the tissue sample to the polished surface of a cryogenic surface such as a chilled metal block. This typically involves opposing the tissue sample to a polished flat metal surface by pressing it firmly against the surface of the metal. Silver and copper are typically used as the polished metal blocks. This method is designed to take advantage of the high thermal conductivities and heat capacities of these metals when cooled to liquid nitrogen or liquid helium temperatures. The critical step in chilling on the surface of a metal is making firm contact with the dry, chilled metal surface with no rotational, translational or rebounding motion. Certain commercially available apparatus having known utility in the medical arts address and provide "bounce-free" freezing. Credit for the development of this apparatus is generally accorded to Dr. Alan Boyne of the University of Maryland School of Medicine.
The Boyne apparatus and method included one or more copper bars partially submerged in a container filled with liquid nitrogen at -196� C. The end of the copper bar was a mirror-finished smooth cryogenic surface, and the thermal conductivity of copper enabled the surface to be maintained at about -196� C. Cold nitrogen gas from vaporization of the liquid nitrogen, which escaped past the end of the copper bar, helped to reduce the contaminants on the cryogenic surface. A tissue sample was then dropped by gravity against the surface. To reduce the bounce of the sample against the surface, the Boyne sample delivery assembly employed a weight dampening system utilizing glycerol to absorb the impact. Each copper bar must be cleaned after slamming a sample. The drawbacks of the Boyne apparatus included problems of hydrocarbon contamination and condensation on the cryogenic surface, inability to eliminate all bounce or vibration between the sample and surface, undesirable precooling of the sample with escaping nitrogen gas, and delays for cleaning and regenerating the cryogenic surface of the copper bars between each sequential sample. The Boyne method and apparatus thus could not reliably provide tissue samples with good ice crystal-free zones nor was it capable of properly vitrifying the samples beyond a depth of 10 to 15 microns.
Further development of freezing tissue samples against a metal block has been credited to Jacques Escaig of Paris, France. The method and apparatus of Escaig is described in "Control of Different Parameters For Optimal Freezing Conditions", Jacques Escaig, published in Science of Biological Specimen Preparation, at pp. 117-122 (1984). The Escaig apparatus also is disclosed in Swiss Patent No. 614,532, French Patent No. 2,337,878 and German Patent No. 2,700,196. Escaig provided several significant features not shown in earlier methods or devices for vitrifying tissue against a metal block. The Escaig method and apparatus cool a copper block with liquid helium, rather than liquid nitrogen, in order to increase the cooling rate of a tissue sample or specimen. Escaig disclosed that the average thickness of ice crystal-free zones in the tissue sample were much larger when the copper block was cooled by liquid helium rather than liquid nitrogen. Additionally, Escaig pointed out that the factors influencing the freezing process, independent of the tissue sample itself, are thermal contact between the specimen and metal block, condition of the block surface, specimen slipping, sample holder bounce and sample holder contact strength. Escaig obtained some control of these factors by utilizing a vacuum pump to keep the metal block under vacuum of 1�10-3 Torr. until just before slamming the specimen against the block, in order to reduce contamination on the block surface. Escaig also utilized an electromagnet to bring the specimen in contact with the block in order to improve mechanical contact between the specimen and the block.
According to the Escaig apparatus and method, a copper block is enclosed in a vacuum chamber. The chamber is then evacuated to approximately 1�10-3 Mbar with an external vacuum pump system. After a vacuum is reached in the chamber, Escaig employed an external nonreusable cryogen source--liquid helium pumped from a reservoir--to cool the copper block. The liquid helium is transmitted to a passage adjacent to the block through a conduit in the vacuum chamber. When the block is cooled to the desired temperature, Escaig used an electropneumatic system to open a stem insert in the liquid helium passage for releasing cold helium gas for several seconds into the vacuum chamber itself. The cold helium gas admitted into the vacuum chamber is obtained from vaporization of the liquid helium which was used to cool the metal block. The cold helium gas raises the pressure inside the vacuum chamber. When atmospheric pressure is reached inside the vacuum chamber, a shutter providing access to the vacuum chamber is spring biased to open the chamber and activiate downward movement of a sample delivery assembly, which plunges or slams the tissue sample through the shutter opening and against the block. Opening of the shutter also triggers closing of the stem insert to stop the release of cold helium gas into the chamber.
Several problems have been encountered in the Escaig apparatus. Because Escaig used cold helium gas to bring the vacuum chamber up to atmospheric pressure, a tissue sample precools for approximately 15 milliseconds as it descends through a layer of cold helium gas at atmospheric pressure prior to plunging or slamming against the metal block. The precooling effect of the Escaig device is undesirable due to unwanted effects, such as ice crystal formation, on the physiology of the tissue sample. Additionally, movement of liquid helium and helium gas through the vacuum chamber inevitably resulted in vibration of the metal block, which was undesirable because it reduced or prevented good mechanical contact between the specimen and block. Escaig's use of nonreusable liquid helium to cool the block further has proved to be expensive, somewhat unsafe and cumbersome due to the necessity of recooling the entire liquid helium system between each tissue sample. The end result was extremely slow turnover time for regenerating the copper block between each sample. Additionally, cleaning the block in the Escaig apparatus proved difficult because of condensation on the block forming after a sample was slammed against the block. To remove the condensation, pressurized nitrogen gas and hot air could be applied against the block. However, removal of the block for repolishing or other cleaning required disassembly of the vacuum chamber itself. Even if the block was not removed, the problems in regenerating the block surface resulted in turnover time between samples which is commercially unacceptable. It was therefore not possible to use the Escaig device if a large number of sequential samples was desired.
The cryopreparation apparatus and method according to the present invention solves the problems inherent in the prior art including the Escaig apparatus and method. The present invention addresses the problems in the prior art of slow turnover time between the vitrification of tissue samples, by enabling several samples to be vitrified sequentially. The present invention also solves the problems in the prior art caused by use of nonreusable liquid helium or other coolant, undesirable precooling of the tissue sample before contact with the cryogenic surface, cleaning and reheating the cryogenic surface between each sample, and removal of the cryogenic surface from the apparatus.
The apparatus and method of the present invention provide for ultrarapid cooling or vitrification of tissue samples by slamming or plunging the samples against a chilled cryogenic surface. The apparatus contemplates chilling of the tissue sample against a mirror-finished smooth cryogenic surface. The cryogenic surface is enclosed in an ultra-high, hydrocarbon free vacuum chamber. In the preferred embodiment, introduction of noncondensable dry helium gas at room temperature into the chamber just prior to plunging or slamming the sample enables the chamber pressure to be raised and a shutter providing access into the chamber to be opened without undesirable condensation on the cryogenic surface, or dehydration and precooling of the tissue sample during its downward movement through the shutter opening towards the cryogenic surface. An external dry helium source is used to supply dry helium to the chamber.
In the preferred embodiment, rapid regeneration of the cryogenic surface between successive samples is enabled by the use of a thermal break and a heating unit proximate to the surface. The thermal break is positioned generally between the cryogenic surface and a helium cryopump or multiple-stage refrigerator. The thermal break, when evacuated, controllably separates the cooling of the helium cryopump from the cryogenic surface. A heating unit is electrically activated to heat the cryogenic surface for cleaning and removing condensation between tissue samples, while the thermal break is evacuated. Before a sample is slammed against the cryogenic surface, dry helium gas is introduced into the thermal break, and the cryogenic surface is rapidly re-cooled to the desired temperature, preferably between -270� C. and -200� C. Thus, the present invention enables sequential vitrification of tissue samples against the cryogenic surface without delays for regeneration and recooling inherent in the prior art. The present invention also provides a cryogen-independent recirculating refrigerant cycle for cooling the cryogenic surface rather than an external cryogen source such as liquid nitrogen or liquid hydrogen as used in the prior art.
Another advantage of the apparatus of this invention is the ability to cryoprepare tissue without overt disruption or destruction of the morphological characteristics of the ultrastructure of tissue cells. The cryopreparation method and apparatus of the present invention provide for ultrarapid cooling of tissue samples so that the tissue then may be dehydrated while maintained in the solid, vitreous phase without creating unnecessary artifacts which restrict interpretation by conventional analytical apparatus.
The cryopreparation process of the present invention has demonstrated an extraordinary application in the transplanting of corneal tissue. Prior to this invention attempts to transplant corneas which involved a necessary freezing or freeze-drying of the corneas after removal from the donor invariably resulted in a clouded cornea upon transplanting. The clouding remained for from as little as several days to the life of the transplanted cornea. This physical condition of the transplanted cornea was caused by ice crystal formation in the cornea itself and concomitant damage to the stroma. Use of the apparatus of this invention has enabled ophthalmologists to cryoprepare corneas and to then transplant those corneas to recipients with virtually negligible clouding or crystal formation. The ability to so transplant corneas represents an exceptional advantage to the process of this invention as well as a medical breakthrough in corneal transplant surgery.
FIG. 1 is a side view, partially in section, of the preferred embodiment of the present invention.
FIG. 2 is a cross-sectional view of the preferred embodiment of the present invention, taken along section line 2--2 of FIG. 1.
FIG. 3 is a cross-sectional view of the preferred embodiment of the present invention, taken along section line 3--3 of FIG. 1.
FIG. 4 is an exploded perspective view of the thermal break as used in the preferred embodiment of the present invention.
In the apparatus of this invention it is a fundamental prerequisite that the desired tissue is obtained. Tissue samples are collected by a variety of means, i.e., surgical extraction, withdrawn blood samples, binders and any of a variety of other techniques which are well known and conventional. The particular method of obtaining the biological sample is not limiting on the apparatus of this invention. However, the preparation of the tissue sample in the apparatus of this invention is enhanced if the tissue sample is processed as soon after excising as is possible.
The preparation of the tissue sample takes place immediately as it is received. The tissue sample cannot be retained in a fixative, i.e., formaldehyde, or another biologically active stabilizing solution, in an attempt to maintain the sample during shipping, storage or other necessary operations. It is also critical that the sample not be routinely frozen or otherwise physically modified prior to preparation according to the method of this invention. The sample may later be physically sectioned or otherwise physically prepared for long-term storage in apparatus or use with various currently available commercial analytical apparatus.
In one application of this invention a tissue sample is prepared for analysis. The preferred optimum biological sample for preparation in the apparatus of this invention is a fresh one cubic millimeter biopsy sample. This sample must be vitrified as soon as possible. By vitrifying or vitrification it is intended to make reference to a process which results in cryofixation of the sample which is different from "frozen." In the process of vitrifying, the cooling apparatus which is used renders the sample in the vitreous phase such that soluble and insoluble moities contained in the tissue sample are not disturbed, translated, or altered nor are they concentrated (as eutectics). By definition, a vitrified liquid will shatter when undergoing a shear stress, e.g., window glass. The vitreous phase involves the conversion of liquid water into an amorphous or "glass" phase. This is accomplished by rapidly supercooling the tissue sample by opposing it "bounce-free" onto the highly polished (mirror-like) condensate-free cryogenic surface preferably at between -270� C. and -200� C. It is preferred that such rapid supercooling be completed in less than one second.
Depending on the anticipated time lag between supercooling of the sample and dehydration of the sample, the sample may be stored submerged in a liquid nitrogen dewar at -196� C. Once the sample has been dried and embedded properly it may be stored virtually indefinitely without cytoplasmic reticulation or other forms of cellular catabolism which will cause modifications and crystal lattice transitions resulting in undesirable artifacts which render the tissue uninterpretable as analytical data.
In the preferred embodiment of the present invention, the major components of the cryopreparation apparatus include sample delivery assembly 10, upper vacuum chamber 11, lower vacuum chamber 13, thermal break 12, and cryogenic surface 38. Cryogenic surface 38 is enclosed within upper vacuum chamber 11, and preferably may be easily removed from the chamber for replacement, reconditioning, or repolishing of the surface. In the preferred embodiment, thermal break 12 is positioned generally between upper vacuum chamber 11 and lower vacuum chamber 13. It should be understood, however, that the thermal break may be positioned in any manner for controllably separating the cryogenic surface from the cooling of a helium cryopump or multiple-stage refrigerator. The chambers are evacuated to reach a hydrocarbon free vacuum and cooled to the desired temperature with the cryogenic surface cooled to preferably between -270� C. and -200� C. One or more tissue samples are then slammed or plunged against the cryogenic surface 38. The cryogenic surface is then cleaned and heated to room temperature while lower vacuum chamber 13 is maintained at the desired cool temperature. The thermal break establishes an insulating region between the cryogenic surface and the helium cryopump or multiple stage regrigerator cooling the lower vacuum chamber. After the cryogenic surface is cleaned, the thermal break is refilled with dry helium gas under pressure, facilitating rapid recooling of the cryogenic surface to between -270� C. and -200� C.
Sample delivery assembly 10 comprises plunger 19 provided with bias means shown as spring 20 extending from within plunger housing 22. Plunger 19 is cooperatively aligned and moveable into and out of housing 22. The spring or bias means serves to recoil the plunger from cryogenic surface 38 after a specimen or tissue sample is plunged against the cryogenic surface. The plunger 19 is further provided with a magnetic sample holder 24. Sample holder 24 includes a magnetic disk to improve mechanical contact between the specimen and the cryogenic surface, and includes mounting means for the tissue sample. Sample holder 24 further includes elastic material such as a foam rubber cushion or other suitable compressible and resilient material to insure that the specimen or tissue sample, when mounted on the sample holder and plunged against the cryogenic surface, will avoid undesirable specimen compression and deformation. The sample delivery assembly 10 further includes support structure 25 cooperatively engaged to plunger housing 22 to position the assembly directly above upper vacuum chamber 11. The plunger 19 is electromagnetically or electropneumatically actuated to move the tissue sample downward against the cryogenic surface when shutter 31 is opened. Other means for actuating the plunger 19, however, are contemplated within this invention.
Upper vacuum chamber 11 is provided with upper end plate 32 and side walls 39, and is preferably composed of stainless steel and constructed so that it may be vacuum sealed. Shutter 31, preferably composed of stainless steel, is located proximate to upper end plate 32 and is moveable to open cooperatively with the downward movement of plunger 19. Shutter 31 is positioned by annular surround 30. Shutter 31 is spring biased to open when the pressure on the inside of the shutter in upper vacuum chamber 11 reaches the pressure outside the shutter, which generally is atmospheric pressure. The opening of shutter 31 activates electromagnetic switches 33 or other switch means. One electromagnetic switch activates downward movement of plunger 19, and the tissue sample is slammed or plunged against the cryogenic surface 38. The second electromagnetic switch shuts off dry helium gas input into the upper vacuum chamber. Although electromagnetic switches are shown in the preferred embodiment, it is contemplated under the present invention that other means such as electropneumatic means or hydraulic means may be used to activate downward movement of the plunger and control dry helium gas input to the upper vacuum chamber. Upper vacuum chamber 11 is further provided with radiation shields 40 enclosed within side walls 39 of the upper vacuum chamber to reduce the heat transfered by radiation to the upper vacuum chamber.
The cryogenic surface 38 located in upper vacuum chamber 11 may be composed of copper, gold, silver, diamond or sapphire or combinations thereof or other suitable means having high heat conductivity at low temperatures and providing a mirror-finished flat smooth surface for contact with tissue samples. The preferred cryogenic surface is copper with a sapphire outer portion. The cryogenic surface is the outer layer over a substrate of materials positioned directly adjacent to thermal break 12. Attached to the cryogenic surface is heating element or cartridge 42, which is provided for heating the cryogenic surface in order to remove condensation and clean the cryogenic surface between successive tissue samples. The heating element or cartridge is connected to electrical connector 44 and a heater input preferably having a temperature regulator. The heating element or cartridge thus enables regeneration of the cryogenic surface for rapid turnover of samples. Attached to heating element or cartridge 42 is upper end plate 45 of the thermal break, which preferably is composed of copper or other suitable material having high heat conductivity at low temperatures. Thermal damper 41 may be positioned between cryogenic surface 38 and heating element 42, if it is desired to reduce temperature fluctuations. The cryogenic surface 38, heating element or cartridge 42, and thermal damper 41 are removably fixed to upper end plate 45; and in the preferred embodiment are removable as a unit through the shutter opening, by loosening and removing screws 40 and 40A. Thermocouple 43 and electrical connector 44 are functionally connected to the cryogenic surface for sensing the temperature of the surface, for example, by use of a silicon dioxide resistor or a germanium chip. Optionally, mannometer thermometer 71 may be used to sense the temperature of the cryogenic surface.
Thermal break 12 includes side walls 46 preferably composed of stainless steel and ceramic walls 48 bonded to the wall separating the upper and lower vacuum chambers. Upper end plate 45 and lower end plate 49 preferably composed of copper or other highly conductive material at low temperature are provided at each end of thermal break 12. Vanes 47, comprised of copper or other suitable material having high heat conductivity at low temperature, extend between upper end plate 45 and lower end plate 49 of the thermal break. The vanes connected to the upper and lower end plates, respectively, are not interconnected, but improve the efficiency for removing heat from the cryogenic surface to nearly 70% when dry helium gas is reintroduced into the thermal break and the cryogenic surface is cooled. The vanes as used in the thermal break increase the efficiency of cooling (between lower end plate 49 and upper end plate 45 of the thermal break) by convection rather than by conduction.
The lower vacuum chamber 13 includes side walls 70, radiation shields 72 within the side walls to reduce the radiant heat transfer to the lower vacuum chamber, and lower end plate 77. In the preferred embodiment, a cryopump or multiple stage refrigerator operating on the Gifford-McMahon principle and using gaseous helium as the coolant is utilized. Preferably, the refrigerator second stage 73 is enclosed within lower vacuum chamber 13. The refrigerator second stage 73 is further connected to refrigerator first stage 76 enclosed by side walls 74 and upper end plate 75.
An external vacuum pump is utilized to create a vacuum in the upper and lower vacuum chambers and thermal break. The vacuum pump is connected via upper chamber vacuum inlet 51 and upper chamber vacuum valve 52, thermal break vacuum inlet 53 and thermal break vacuum valve 54, and lower chamber vacuum inlet 55 and lower chamber vacuum valve 56. The vacuum pump preferably is a vibration isolated turbo molecular vacuum pump or vane pump or equivalent device for creating a vacuum of at least 10-3 Mbar in the upper vacuum chamber and lower vacuum chamber. The vacuum pump employed can be any of a variety of commercially available vacuum pumping apparatus. It is essential that the pump, whether or not it is a turbomolecular pump, yield a hydrocarbon-free vacuum. However, the helium cryopump or multiple stage refrigerator is used to further reduce the pressure in the upper and lower vacuum chambers to preferably about 10-8 Mbar.
The upper vacuum chamber is provided with upper chamber dry helium inlet 58 and upper chamber dry helium inlet valve 59. Additionally, thermal break 12 is provided with thermal break dry helium inlet 60 and thermal break dry helium inlet valve 61. An external dry helium source with controls is used to regulate the supply of dry helium to the upper vacuum chamber and thermal break.
The operation of the present invention in the preferred embodiment is set forth below. Initially upper chamber vacuum valve 52 and lower chamber vacuum valve 56 are opened. Thermal break vacuum valve 54 is closed. The upper and lower vacuum chambers are evacuated with an external vacuum pump system to reach a vacuum of 10-3 Mbar. The vacuum in the upper vacuum chamber helps to prevent undesirable hydrocarbon deposits on the cryogenic surface 38 and undesirable condensation on the cryogenic surface while it is cooled. An external vacuum pump is used to create a vacuum of about 1�10-3 Mbar surrounding the cryopump or multiple stage refrigerator. The external vacuum pump system used helps to prevent condensation in the lower chamber and improve the cooling capability of the cryopump or multiple stage refrigerator. To achieve a hydrocarbon-free vacuum in the upper chamber, however, a lower vacuum pressure is desired. Accordingly, the helium cryopump is utilized to reach a vacuum of approximately 1�, 10-8 Mbar. The external dry helium source also is activiated to fill thermal break 12 with dry helium gas or other thermally conductive gas.
In the preferred embodiment, the cooling of the cryogenic surface is achieved by a helium cryopump or multiple stage refrigerator using a closed helium gas cycle operating on the Gifford-McMahon principle. The Gifford-McMahon refrigerator is a modification of the Stirling-cycle refrigerator. For lower temperatures of between -270� C. and -200� C. as required by the present invention, a refrigerator having two or more stages is preferred. Although the preferred embodiment discloses the use of a two-stage refrigerator operating on the Gifford-McMahon principle, it will be understood that other cryopumps or refrigerators are within the scope of the present invention. In the preferred embodiment, the second stage of the refrigerator is enclosed within the lower vacuum chamber 13, and controllably cools the cryogenic surface when the thermal break is filled with dry helium gas.
Once temperature of the cryogenic surface has reached equilibrium, preferably between -270� C. and -200� C., upper chamber vacuum valve 52 is closed. At this time both the upper and lower chambers are at a vacuum of preferably about 1�10-8 Mbar. A tissue sample or specimen is then mounted on sample holder 24. According to the present invention, more than one tissue sample may be slammed or plunged against the cryogenic surface at one time, depending on the particular sample holder utilized and the sample turnover desired.
In the preferred embodiment, prior to slamming the sample against the cryogenic surface, upper vacuum chamber dry helium inlet valve 59 is opened to allow dry helium gas at room temperature to enter the upper vacuum chamber and raise the pressure inside the upper vacuum chamber to atmospheric pressure. The use of dry helium gas at room temperature prevents pre-cooling of the specimen during movement towards cryogenic surface 38. Helium gas is a non-condensable gas and thus will not condense on the cryogenic surface. It should be understood that any other noncondensable gas may be used, preferably at room temperature, to raise the pressure inside upper vacuum chamber without undesirable condensation on the cryogenic surface or precooling the tissue sample. When the upper vacuum chamber reaches atmospheric pressure, wherein the pressure inside of shutter 31 equals that outside of the shutter, a spring moves the shutter to an open position and triggers a switch to activate downward movement of the plunger and tissue sample towards the cryogenic surface. The shutter also triggers a switch to close the upper chamber dry helium inlet valve 59. The switches may be electromagnetically or electropneumatically activated or operated by other means triggered by the opening of shutter 31. The shutter spring is biased to open when the pressure inside the upper vacuum chamber reaches the outside pressure, generally at atmospheric pressure. The plunger descends into the upper vacuum chamber and the tissue sample is slammed or plunged against the cryogenic surface. In the present invention, the cooling rate of the tissue sample or specimen is in excess of 10,000� C./second to a depth of 30-70 microns in the sample, thereby achieving amorphous or microcrystalline ice to this depth. At the time of application of the sample, the cryogenic surface preferably is at a temperature of -270� C. to -200� C.
After the sample is plunged or slammed against the cryogenic surface, the sample holder is removed from the sample delivery assembly and placed a in liquid nitrogen bath at -196� C. Optionally, at this point the upper vacuum chamber is filled with liquid nitrogen and the sample is removed from the upper vacuum chamber while continuously enclosed in the liquid nitrogen at -196� C. for storage. The use of liquid nitrogen in the chamber reduces the handling of the sample between the chamber and a liquid nitrogen Dewar for storage and maintains the cool temperature of the sample after it is removed from the sample holder.
The thermal break 12 is then evacuated by an external vacuum pump system to reach a vacuum of preferably about 1�10-3 Mbar when thermal break vacuum valve 54 is opened. The vacuum in the thermal break functionally separates the cryogenic surface from the cooling of the cryopump. Heating element 42 is then activiated to raise the temperature of the cryogenic surface. It is preferred that the shutter be closed during heating of the surface to reduce the amount of condensation and/or contaminants deposited on the cryogenic surface and it is most preferred that the shutter be biased closed immediately upon return or recoil of the plunger to its resting position so that the cryogenic surface is maintained under vacuum during heating. When the cryogenic surface reaches the desired temperature, preferably room temperature, the cryogenic surface may be cleaned, preferably with acetone and freon. By functionally separating the cryogenic surface from the cryopump or multiple stage refrigerator, it is possible to regenerate or heat and clean the cryogenic surface at room temperature, then recool the cryogenic surface without lengthy delay. In the past, the turnaround time between each sequential tissue sample has been several hours or more, due to the time for heating the block and reactivating the entire cooling system. In the present invention, the turnaround regeneration time is reduced to between 10 and 15 minutes.
After the cryogenic surface is cleaned, shutter 31 is closed and the upper vacuum chamber is reevacuated by by the external vacuum pump to preferably about 1�10-3 MBar when upper chamber vacuum valve 52 is opened. When a vacuum of 1�10-3 Mbar is obtained in the upper vacuum chamber, heating element 42 is switched off and thermal break vacuum valve 54 is closed. The upper chamber and lower chamber are reconnected to achieve the hydrocarbon-free ultravacuum in the upper chamber. Dry helium gas is reintroduced into the thermal break via thermal break dry helium inlet 60 and thermal break dry helium inlet valve 61. This restores the functional relationship of the cryopump or multiple stage refrigerator to cool the cryogenic surface to the desired temperature. The cryopump then cools the block and obtain a vacuum of preferably about 1�10-8 Mbar in the upper and lower chambers and the cycle is repeated.
According to the present invention it is possible to vitrify a large number of tissue samples sequentially after regenerating the cryogenic surface between each sample. The cryogenic surface is heated to preferably room temperature, cleaned and rapidly recooled between each tissue sample. During cooling, the cryogenic surface is in an ultra-high hydrocarbon-free vacuum. Condensation on the cryogenic surface and/or contamination of the cryogenic surface does not occur prior to application of the tissue sample. Precooling of the sample and condensation on the cryogenic surface is prevented by introducing room temperature dry helium gas, or other non-condensable gas at room temperature, into the upper vacuum chamber for controlling the opening of the shutter sealing the upper chamber and slamming or plunging the tissue sample against the cryogenic surface.
The present invention employs a recirculating liquid helium cryopump, shown in the preferred embodiment as a two-stage refrigerator operating on the Gifford-McMahon principle to cool the cryogenic surface rather than an external nonreusable cryogen source such as liquid helium or liquid nitrogen, as used in the prior art. Helium pumps manufactured by Leybold-Heraeus Vacuum Products provide examples of the cryopumps used in the present invention. The present invention also eliminates problems of vibration to the cryogenic surface due to movement of liquid helium and helium gas proximate to the surface before slamming. Additionally, the present invention incorporates a thermal break as a modification of a helium cryopump, and incorporates a heating unit for rapid regeneration of the cryogenic surface. These features make cryopreparation considerably more economically productive because tissue samples may be processed sequentially while maintaining the cryogenic surface free from contamination.
Although the preferred embodiment of this invention has been described hereinabove in some detail, it should be appreciated that a variety of embodiments will be readily available to a person designing such cryopreparation apparatus for a specific end use. The description of the apparatus of this invention is not intended to be limiting on this invention, but is merely illustrative of the preferred embodiment of this invention. Other apparatus and components which incorporate modifications or changes to that which has been described herein are equally included within this application.
|Boyne, Alan F., "A Gentle, Bounce-Free Assembly for Quick-Freezing Tissues for Electron Microscopy: Application to Isolated Torpedine Ray Electrocyte Stacks", Journal of Neuroscience Methods, 1 (1979), pp. 353-364.
|Boyne, Alan F., A Gentle, Bounce Free Assembly for Quick Freezing Tissues for Electron Microscopy: Application to Isolated Torpedine Ray Electrocyte Stacks , Journal of Neuroscience Methods, 1 (1979), pp. 353 364.
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|Coulter, H. David, Freezing and Drying of Biological Tissues with a Toggle Link Helium Freezer and an Improved Freeze Drying Apparatus: Application to Neuropeptide Immunocytochemistry , Journal of Electron Microscopy Technique, 4 (1986), at pp. 315 328.
|Escaig, Jacques, "Control of Different Parameters for Optimal Freezing Conditions", Science of Biological Specimen Preparation, pp. 117-122.
|Escaig, Jacques, "New Instruments which Facilitate Rapid Freezing at 83K and 6K", Journal of Microscopy, Jun. 1982, at pp. 221-229.
|Escaig, Jacques, Control of Different Parameters for Optimal Freezing Conditions , Science of Biological Specimen Preparation, pp. 117 122.
|Escaig, Jacques, New Instruments which Facilitate Rapid Freezing at 83K and 6K , Journal of Microscopy, Jun. 1982, at pp. 221 229.
|Handley, Dean A., et al., "The Design and Use of a Simple Device for Rapid Quench-Freezing of Biological Samples", Journal of Microscopy, Mar. 1981, at pp. 273-282.
|Handley, Dean A., et al., The Design and Use of a Simple Device for Rapid Quench Freezing of Biological Samples , Journal of Microscopy, Mar. 1981, at pp. 273 282.
|Med Vac Inc., Cryopress (brochure).
|Med-Vac Inc., "Cryopress" (brochure).
|Moor, H. et al., "The Influence of High Pressure Freezing on Mammalian Nerve Tissue", Cell and Tissue Research, (1980), at pp. 201-216.
|Moor, H. et al., The Influence of High Pressure Freezing on Mammalian Nerve Tissue , Cell and Tissue Research, (1980), at pp. 201 216.
|Polaron Instruments Inc., "The Slammer" (brochure).
|Polaron Instruments Inc., The Slammer (brochure).
|Quick Freezing Devices, "Quick Freezing by Bounce-Free Delivery" (brochure).
|Quick Freezing Devices, Quick Freezing by Bounce Free Delivery (brochure).
|Reichert Jung, Cryofract 190 Cryoblock (brochure).
|Reichert Jung, Cryovacublock Instruction Manual.
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|Aug 22, 1989
|Inframetrics, Incorporated
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|Oct 23, 1990
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|Apr 12, 1989
|Dec 25, 1990
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|Feb 26, 1991
|The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration
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|Dec 15, 1988
|Sep 3, 1991
|Board Of Regents, The University Of Texas
|Mar 22, 1990
|Oct 15, 1991
|Stetson Norman B
|Miniature integral Stirling cryocooler
|Mar 13, 1990
|Oct 13, 1992
|Board Of Regents University Of Texas System
|Method and apparatus for cryopreparing biological tissue
|Feb 2, 1993
|Aug 9, 1994
|Lifecell Corporation
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|Nov 15, 1994
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|Mar 25, 1997
|Cryolife, Inc.
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|May 27, 1997
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|Colorado State University Research Foundation
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|Life Cell Corporation
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|Jun 5, 1995
|May 4, 1999
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|Sep 26, 2008
|Aug 16, 2016
|Cornell University
|System and method for increased cooling rates in rapid cooling of small biological samples
|Jun 22, 2009
|Dec 27, 2016
|Giorgio Frossati
|Holder for a sample to be cooled to a low temperature in a vacuum space and 3He—4He dilution refrigerator adapted to accommodate such a holder
|Nov 24, 2003
|Jun 3, 2004
|Hopkins Richard A.
|Cardiac valve replacement
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|Sep 22, 2005
|Asml Netherlands B.V.
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|Oct 5, 2004
|Apr 6, 2006
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|Apparatus for freezing a biological sample
|Jul 7, 2005
|Sep 21, 2006
|Lifecell Corporation, A Texas Corporation
|Method for processing and preserving collagen-based tissues for transplantation
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|May 21, 2009
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|Ultra-rapid freezing device and method
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|System and method for increased cooling rates in rapid cooling of small biological samples
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|Jun 16, 2011
|Giorgio Frossati
|Holder for a sample to be cooled to a low temperature in a vacuum space and 3He-4He dilution refrigerator adapted to accommodate such a holder
|Oct 26, 2012
|Oct 2, 2014
|Merck Sharp & Dohme Corp.
|Methods of preparing lyophilized spherical-shaped pellets of biological materials
|Jul 11, 2013
|Nov 6, 2013
|Surface analysis system with cryopump
|Dec 13, 2000
|Jun 20, 2002
|Friedrich Hoffmann
|Vitrification of cornea, employs tissues of rear cornea lamellae including the stroma, Descemet membrane and endothelium
|Oct 18, 1988
|Apr 25, 1990
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|Board Of Regents The University Of Texas System
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|U.S. Classification
|62/349, 62/100, 118/50.1, 62/55.5, 34/92, 422/131
|International Classification
|Cooperative Classification
|European Classification
|Dec 3, 1986
Owner name: BOARD OF REGENTS, THE, THE UNIVERSITY OF TEXAS 201
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:LINNER, JOHN G.;LIVESEY, STEPHEN;REEL/FRAME:004642/0119
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text:
The Fabulous Baker Brothers
Tom Herbert & Henry Herbert
This book is available for download with iBooks on your Mac or iOS device, and with iTunes on your computer. Books can be read with iBooks on your Mac or iOS device.
Tom and Henry Herbert - The Fabulous Baker Brothers - are fifth generation bakers with a passion for food in all its forms. Tom is a talented master baker whose famous Hobbs House Bakery sits just next door to his younger brother Henry's butchery. Together our young brothers work side by side making the amazing bread and delicious meaty accompaniments and fillings that have made their businesses so successful. Here, in this brand new cook book to accompany the hit Channel 4 show, The Fabulous Baker Brothers share with us mouthwatering oven-based recipes that unlock a world of gorgeous homemade breads, pastries, pies, cakes and confectionary. With carefully chosen ingredients and some easily-mastered techniques - this is healthy, wholesome, beautiful food that doesn't cost the earth to make. Fully illustrated throughout with photographs of the boys, their shops and Cotswold surrounds, and of course their stunning produce, this cook book gets to the fundamental heart of British good food as two of the country's most respected and successful artisans teach us how to bake like professionals in our own homes.
- £16.99
- Category: Food & Drink
- Published: 19 January 2012
- Publisher: Headline
- Print Length: 252 Pages
- Language: English

subset: commoncrawl


text:
A/N: Anyone ever move over Christmas holiday? It's ever so much fun. Maybe not as much "fun" as drowning sorrows with Sirius, but almost. Sigh. Travelling from the West to the East, I'll admit that I'm not looking forward to exchanging blue skies for grey clouds and snow, but I am looking forward to posting the sequel to 'Once in a Blue Moon' next Friday!
Everything and the Moon
Perhaps there was such a thing as being too much of a gentleman. Rubbing the back of his neck, sore from sleeping in a chair, Remus Lupin heard a soft snore and turned his head. Sprawled in the chair opposite, his drinking companion of the last several nights clutched an empty bottle of firewhiskey like a teddy bear. After days of keeping his problems to himself, Remus had finally confessed the ultimatum he'd given Tonks. He'd explained how she'd rushed headlong into danger and lied about her activities to keep him safe. Remembering Sirius' reaction to the end of the story, he huffed in reluctant amusement.
"Are you mental? The girl you're over the bloody moon about tells you to take her, she's yours, and you say 'No'?"
Defensively, Remus muttered after downing another shot, "I wanted the emotional commitment more than the physical."
The drink Sirius had just taken was promptly sprayed out. "Ruddy hell, you can't be that thick!" Propping an elbow on the table between them, he leaned forward to say, "Birds like Tonks don't go laying down for a bloke unless they care."
"Caring's not enough."
Pouring more whiskey, Sirius laughed shortly. "Merlin, that's ironic, coming from you. I remember Dorcas snivelling to Lily about the same exact thing."
Thinking about last night's drunken conversation, Remus admitted that only now did he see his long ago girlfriend's point. She'd wanted him to commit to being her partner, but he'd been unable to. As much as he'd cared about Dorcas, love and marriage were more than he'd been capable of giving. A thought struck that made him feel even more wretched. What if Tonks couldn't give him what he needed?
A whimpering sound brought his attention back to Sirius. Still asleep, the wizard muttered 'Cami' and gave another forlorn whine.
Rising slowly from his seat, Remus grimaced from the pain in his head and the realization that he wasn't the only one currently unlucky in love. The only affection Padfoot got was as Snuffles. The woman his mate wanted thought he was a dog- no pun intended.
Shuffling like an old man kept his head from pounding unbearably. By the time he'd weaved his way to the lavatory, the wizard couldn't decide which need was more pressing. His body was insisting Nauseous no More and Morning After potions be ingested while simultaneously demanding the toilet. After all needs were met, Remus felt human enough to brave looking in the mirror. He promptly wished he hadn't. Blood shot eyes stared back from a face that was heavily lined and deathly pale from late nights and liquor. Since he'd walked out of Tonks' flat, his hair had become shaggier and unkempt. Even his mother would've found him unlovable in this state.
"Drag your arse into the shower, mate, or shove over. I've got to piss."
The corners of Remus' mouth turned up as viewed the man who embodied the adage that there was always somebody who had it worse. Blearily, his mate warned, "If you use up all the hot water, though, I will finally commit the murder I was imprisoned for."
"Not to worry, I need cold to clear my head."
"Good, fix some food once you're out, its gone elevenses." Sirius shut himself in the water closet, while Remus stepped into the shower, fully clothed.
After frigid water brought his lethargic brain to life, he hastily changed the temperature to warm. While stripping off sodden clothes, Remus mumbled, "You could've stripped down Tonks' defences along with her clothing." Reaching for the soap, a memory came to mind.
Tonks sat beside him on her bed. She was recovering from their disastrous Halloween mission. Taking his hand, she pressed it against her cheek. She tried to smile, and his heart swelled at the gallant effort. He was more than happy to oblige when Tonks said, "Memories don't wash away. I want you to replace them."
Washing his hair, he wondered if a more intimate embrace would've helped Nymphadora let go of the past and realise the depths of her feelings. Would making love on Bonfire Night have made them stronger, or just made her run scared again? If he'd taken what she'd offered, would they be waking up in tangled sheets right now, skin against skin? Allowing second guesses to wash away with the soap, he disengaged the water charm and shook his head to remove both water and tormenting doubts.
Towelling off, Remus went into the bedroom, chuckling to hear a thud against the lavatory door. Sirius had found the clothes he forgot to remove from the shower. Pulling on an old, comfortable shirt and trousers, he returned to the lounge and opened a window to allow in much needed fresh air. The November day was cold but dry.
"Mr. Lupin! Good Morning!"
Leaning out, he looked down and saw his landlord standing below on the pavement. It was his customary place to read the Quibbler away from his wife's disapproving gaze. Gesturing at the corner shop located beneath Remus' flat, the man grinned. "Ambika has brought Masala Dosas for Tiffin. My children demand you come share them with us."
Before he could regretfully decline, two five year olds rushed out and called, "Bring Snuffles too! Bring Snuffles too!"
Patil laughed heartily. "Yes, yes, Mr. Snuffles, the magic dog, must join us, or my life will not be worth living."
Unable to hold out, Remus smilingly agreed to come down in a few minutes. Retracing his steps, he knocked on the lavatory door and said, "Hurry up, the Patils have offered to feed us."
Sirius bounded down the steps in Animagus form shortly afterwards. Tiffin, or a light meal, was never passed up. His coat was damp, but Aashi and Samy didn't care, hugging and petting the dog joyfully when the visitors joined the family at the table in the back room of the shop.
"He smells clean, like people shampoo," the little boy said, rubbing the top of the dog's head.
His sister, Aashi, pleaded, "May I bring Snuffles to show my play group?"
"They saw three dogs last week. The children don't need to see another," Mrs. Patil remonstrated gently.
"But Snuffles is a magic dog!" Samy and Aashi said together.
The boy appealed to his father, "Snuffles has the soul of a man. He is ever so smart, and can answer our questions with a shake of his head, or…"
Patil interrupted, "When I named you Samyak, I did not know the fate I was bestowing upon myself." Remus saw the smile the man tried to hide when he completed the rebuke. "I am always having to say…"
"Enough!" his children shrieked, falling into giggles, amused by the running joke about the boy's name.
Made from rice and lentil flour, the crepe like cuisine was crispy and stuffed with mashed potatoes lightly cooked and fried with onions and spices. The Masala Dosas were served with coconut chutney and very delicious. Snuffles made happy growl-y sounds as he inhaled a dozen dosas.
"I have never seen or heard an animal appreciate food as much as this one," Ambika declared with a pleased expression.
Remus smiled to himself. He'd thanked his hostess and praised her cooking, but Mrs. Patil, like most women, was won over by Sirius'…or Snuffles'…unabashed appreciation. While the children took turns feeding the magic dog a glass of tea with milk and sugar, their father said in an undertone, "This Tournament at Hogwarts, it will not be dangerous for the other students to watch, will it?"
The man's wife stood and began noisily clearing the table. Patil smiled sheepishly. "Ambika is not overly fond of our niece Parvati."
"That's 'cause she said Mum was a lesser incarnation of her namesake!" Aashi informed while leaning against the black dog's side.
"Mum said Parvati should have been named Minakshi, after the fish-eyed goddess!" Samy added, grinning.
Snuffles gave several woofs. The children begged to be allowed to play fetch in the garden. Shooing them off, their mother said apologetically, "I hope they have not given you a mistaken notion of our family." Her smile was strained as she continued, "We are very large and very close…spending every holiday together…and birthdays…amongst many other parties…yes, very, very close."
Remus didn't quite know how to respond, so he just nodded. When Ambika left the room, he said, "Mr. Patil…"
"Neil?" he repeated, before saying, "Pardon my surprise, and please, call me Remus."
"I enjoy the incredulity, actually." Against his dark skin, Neil's smile flashed white. "Neil means blue, which was my father's favourite colour." He chuckled. "You may have noticed the importance we place on the meaning of names." Changing the subject, Patil brought the topic back to Hogwarts. "So, Remus, the Tournament will be safe for Padma and Parvati?"
"Yes, of course, Dumbledore has things well in hand." The former professor wished he could believe that about Harry as well, but as Sirius had said repeatedly during the last few nights, too many odd happenings and disappearances were being ignored by the Ministry.
The men talked of recent events. Remus gently joked, "No articles from the Quibbler you'd like to share?" He thought the man would laugh, remembering the 'Triwizard Tournament or Twister Championship' headline they'd chatted about. Instead, the jovial face became sombre.
"They printed a most distressing article on werewolves. It was written by someone with no knowledge whatsoever and has shaken my trust in the paper." Brightening, Neil confided, "The Intruder is even more entertaining."
The children came back with Snuffles. Thanking the hospitable family who had never treated him as something dark and frightening, Remus returned upstairs with his 'pet'.
"You've got to get back with Tonks, old man. We haven't gone on a run in days and I'm getting out of shape. Those kids about did me in." Sirius dropped into a chair and moaned, "Beer…must…have…beer."
Opening the coolant spelled cabinet, Remus tossed a bottle and withdrew a pitcher of water to pour himself a glass.
"Not wanting a bit of the hair of the dog that bit you, mate?"
His friend's bark of laughter made Remus admit wryly, "My drinking nights are over, Sirius. I've felt sorry for myself long enough. I'm going to have faith that Tonks will come to me soon."
"Moony, you're a good man. So good, in fact, that while you're picking up my order of ferrets from that bloke in Knockturn Alley and delivering them to Buckbeak, I'll be closing my eyes in order to better focus on all the ways you're such a fine example of wizardhood."
"Wizardhood? Is that a word?"
"Is now."
Unable to argue what the Marauders had called 'Black logic'; Remus left the flat to complete a few errands. After seeing to Buckbeak, he stopped by Charing Cross Road. He had no work to require his presence, but his father's old friend had owled and asked him to come in.
The rare bookshop was doing a brisk business. The proprietor, Andrew Hopkins, looked up from the sale he was making at the counter and smiled. "Good afternoon, Mr. Lupin. I will be with you in my office in a few moments."
Puzzled by the formal tone, Remus inclined his head in acquiescence and strolled to the back of the shop. A few regular customers met his gaze and turned away. With a sinking feeling, he continued on until reaching the office.
"I'm afraid I'm a bearer of bad news, Remus." The older man's smile was pained as he entered and took his seat behind the desk. Blue eyes lacked their customary twinkle as the silver haired wizard explained, "Someone is using that scurrilous Quibbler article to spread the word you are not to be trusted, as werewolves are known to be deeply involved in Dark arts. Yesterday afternoon, the client you'd hoped would request another translation stopped by to ask that I pass on his decision to use the services of another."
It was a blow. Remus had anticipated spending the funds for translating Ovid's Tristia, sad poems, on a lavish Christmas present for Tonks. A bitter thought arose. I won't need a present if I don't have a partner. Refusing to give into negativity, he said to the man waiting, "I'm sorry to hear that."
"As was I, my friend."
"I suppose this means I'll be looking for a new line of work, once again."
Andrew's nod conveyed his sympathy that prejudice would ensure he received no more commissions. Trying not to sound harsh, Remus spoke the name of the man who would take satisfaction in bringing Tonks' new partner low, "Rosier."
"I can't think of anyone else who gains by discrediting you."
Anger welled and was immediately tamped down. From the time Remus was bitten, his mother had stressed again and again that he couldn't get angry like other boys. If he lost his temper with another child, their parents could report him to the authorities. He had learnt never to show extreme anger or aggression, much less act on any of those feelings. Another man could demonstrate what he thought of the other wizard's underhanded tactics, but Remus would be labelled a danger to society and locked away in Azkaban. Still grappling to control dangerous emotions, he looked up when a man in his mid twenties appeared in the doorway.
"I'm sorry to intrude, but the assistant directed me back." Something about the wizard struck a familiar chord. Holding out a card, the younger man said, "I'm David Dunne. I used to run a bookshop…"
"Off Diagon Alley," Remus finished, looking from his card in Andrew's hand to the man's tense face. "Where did you get my card?"
Shifting uncomfortably, the wizard admitted, "I met an Auror, Tonks. She told me this shop might need another assistant. Said a guy named Lupin would put in a word for me with the owner, that you understood…my condition..."
Andrew leaned forward. "If your condition remains private knowledge, I am indeed looking to fill a position, Mr. Dunne. Would you care to discuss terms?"
Remus smiled at Andrew. "I'll leave you to it. I believe I'll return home and take my dog for a walk in the park, since my schedule has been cleared. Pleasure meeting you, Mr. Dunne."
"You too," David replied. "When you see her, tell Tonks I hope she's doing better."
"What do you mean?"
He'd meant to sound calm, but Dunne raised his hands and said, "If I was mistaken, then I apologize for causing alarm. It's just that, when Auror Tonks dropped by my flat to give me the card, she looked pale, and her hair seemed so dull that I thought…" He shrugged awkwardly.
Thanking the other werewolf for his concern, Remus left the shop and walked aimlessly down the pavement. He hoped Tonks wasn't physically ill, but he also hoped his stubborn love was miserable without him- to the point where she couldn't let another day go by without telling him how she really felt.
Returning to his flat with a feeling that happiness was almost in his grasp, Remus shook Sirius awake. "Let's go for a walk in the park."
"No, mate, my paws…feet…whatever…are tired."
"Cami will be there."
The rejuvenating effect of those words was almost magical. Immediately bright eyed and bushy tailed, Snuffles was eager to leave and pulling against the leash, straining to walk faster as they headed to the park.
"Snuffles! Come to me, you big, beautiful, boy!" Tonks' friend Camillia greeted the dog with a deep affection that the canine enjoyed receiving as much as she enjoyed giving.
Sitting on a bench, the wizard watched the pair play and wondered how Cami would react if she knew the 'Newfoundland mix of her dreams' was an Animagus. Would Sirius be the man of her dreams? Guiltily relieved that his friend had decided not to reveal his feelings until Harry was in the clear, Remus tried to read the book he'd brought along. It was an exercise in futility. Glancing up sometime later, he saw a young woman walking toward him and tossed the book down.
Tonks looked every bit as washed out as Dunne had said, and yet she'd never been more beautiful. The corner of his mouth turned up in tenderness. She wiped her tears with the backs of her fingers like a child, but standing before him to admit her feelings, Nymphadora proved that she was an exceptional woman.
"The reason why I lied, and kept things from you, why I couldn't bear the thought of you being hurt, and why I was willing to sleep with you to prove that I care…"
"Yes?" It was hard not to jump in, to assure her that he understood how the past could shadow the present and make love a risk almost too high to take. His gentle prompt seemed to be what Tonks needed to encourage her to say,
"I love you, with all my heart, and without you my world, like my hair, is colourless crap."
He smiled at the declaration that was as unique as the woman who made it, cupping her face with hands that trembled. "I love you, with a heart that's yearned to hear you say those words for so long, a part of me wants to howl for joy." Remus kissed her instead, trying to convey a heart overflowing with love that was indescribably precious and beautiful.
"Tonks…you're pink!"
Snuffles began barking excitedly upon hearing Cami's laughing observation. Remus opened his eyes and grinned. "Your hair…it's pink." The colour seemed to epitomize happiness. His fingertips brushed her face. "Like your cheeks." His gaze and head lowered. "And your lips." Although he didn't usually display unreserved passion in public, Remus returned her ardour with a kiss that made up for the time he'd lost with the love of his life.
I hope you've enjoyed the companion piece to the final chapter of 'Once in a Blue Moon'. I certainly enjoyed writing the 'Tonks confesses her love' scene from Remus' point of view, using the dialogue from 'Moon' with his reactions and emotions colouring the scene, so to speak. If you did too, please tell me in a Review, and don't forget that even if I have to knock on the neighbours of relatives' doors, asking to borrow a cup of internet, lol, chapter one of A Wolf in the Moonlight will post next Friday!


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It's official: the language that can save your life is English. Don't take my word for it; read the response of Traian Basescu, the Romanian president, when invited to address a conference in France, in French: "I regret that I am not a French speaker, because I was a captain in the Merchant Navy and English is the language that can save you."
Two groups of British people will, regrettably, take comfort from the president's honesty: zealous patriots may celebrate the apparent supremacy of English above the language of our nearest neighbour; and prospective travellers could interpret the president's comments as confirmation that there is no point learning a foreign language. Wherever you travel, they will conclude, speaking English is a life-saver.
How sad. As you will, no doubt, have discovered, even a smattering of a local language is an asset. Not only are you better placed to argue with taxi drivers at the airport and order something vaguely palatable and non-lethal for dinner, but being able to converse with the local people helps you unlock all sorts of doors and adds an extra dimension to a journey. The better your linguistic abilities, the more you gain from a trip - and the more you can give back to the host community.
That's the theory, at any rate. But how do the leaders of the travel industry measure up? I talked to the people responsible for sending tens of millions of Brits abroad each year, and found a very mixed ability class. In which languages, I asked, (a) are you fluent; (b) can you get by; and (c) can you avoid arrest and decline unsavoury propositions?
This last category prompted some interesting responses. Michael Cawley, the deputy chief executive of Ryanair, looked at me oddly when I posed the question, then said: "I'm fluent in Irish and English, but why on earth would I want to decline any unsavoury proposition?" And Mark Ellingham, the founder of Rough Guides, maintained: "It is easier to avoid arrest, or indeed unsavoury propositions, by appearing entirely incapable of any intelligent communication. Arresting someone who can't understand the basics ('You're nicked', etc) is clearly taking away part of the fun, and looking a bit of a headache in the hours ahead."
He confessed that, "I'm a pitifully bad linguist", but went on to demonstrate an admirable tenacity at overcoming the handicap of having English as a mother tongue: "I've done Spanish, Italian, and a teach-yourself-Greek course. I can communicate half-decently (and a little better than that in a bar, late at night) in Spanish and Italian. And I am almost fluent in French in Morocco, though much less so in France."
TOP OF the class is Neil Taylor, founder of Regent Holidays and the man who organised the first tourist trips from the UK to destinations as diverse as the People's Republic of China, Albania and Cuba. He is fluent in French and German; gets by in Mandarin and Estonian; and has got out of trouble in Spanish, Italian and Albanian (though he regrets only in the Gheg dialect from the north of the country).
The bosses of Britain's mainstream tour operators are more adept at language than many of their package-holiday customers. Manny Fontenla-Novoa, who runs Thomas Cook, is fluent in Spanish, the language of our favourite package holiday destination. He can also get by in Portuguese and Italian, and knows the basics in French and German. The latter must be particularly useful since Thomas Cook is now German-owned. Thomson, too, is part of a German company, so it is good news that Peter Rothwell, who runs Thomson, is fluent in both French and German.
Britain's leading long-haul holiday company, Kuoni, is Swiss-German owned - and the managing director, Sue Biggs, claims to be able to decline unsavoury invitations in Swiss-German as well as Thai and Swahili. She gets by in French and Italian, and claims fluency in "English and Yorkshire".
Paul Goldstein, of the adventure operator Exodus, says he is fluent in American, Canadian and Australian as well as English; he can get by in French; and has avoided arrest in Spanish. "Of course," he adds (with foreign tongue firmly in cheek), "I know the basic tenets of the Central African Chewa dialect, a sadly deteriorating vernacular, which was invaluable when I found myself there for three months in 1992."Reuse content


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- Horoscopes
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North Stonington — Phil Perry of Black Hall Club remains in the running for the 112th Connecticut Amateur golf championship, advancing to the quarterfinal round of match play.
Perry, who's seeded 29th, upset No. 4 Rick Hayes of Lake of Isles in 22 holes and knocked off No. 20 Ross Kronberg of Great River Golf Course, 2 and 1, in match play on Wednesday at Lake of Isles.
In today's quarterfinal, Perry faces No. 5 John Flaherty of TPC River Highlands. Tee time is 8:10 a.m. The semifinal round will be held in the afternoon and a 36-hole final set for Friday.
Perry will attempt to reach the semifinals for the first time since 2008.
It was a day of upsets as the top two seeds - Sean Gaudette and Dave Szewczul - were eliminated. No. 3 Patrick Albanesi, an 18-year-old playing out of Hartford Golf Club, is the highest remaining seed.
No. 12 Nick Torrance of Lake of Isles won his first match, 5 and 4, over No. 21 Evan DeLuca of The Course at Yale before losing to Flaherty in 20 holes. No. 11 Kyle Buschmann of Fox Hopyard defeated No. 11 Richard Malafronte of Hunter Golf Club, 6 and 5, and then lost to No. 6 Branden Chicorka of Eclub of River Valley, 6 and 5.
• Zach Weigel (Dartmouth) had two hits, including two-run double, with two runs as the Mystic Schooners beat the Valley Blue Sox, 9-3, for their fifth straight win. Stephen Laurino (Marist) had two hits, including a two-run homer, for Mystic (6-1) and Jared Hendren (Heartland CC) had two hits, a run and an RBI. Also, Toby Handley (Stony Brook) doubled and scored twice.
New York-Penn League
• Mauricio Dubron's RBI single with one out in the 12th inning scored Raymel Flores as the Lowell Spinners spoiled the Connecticut Tigers home-opener with a 2-1 win before 4,010 at Dodd Stadium. Oscar Perez allowed just two hits over the final four innings and struck out two to earn the win for Lowell (3-3). Deiner Lopez also tripled and scored. Kasey Coffman scored on Ismael Salgado's bases loaded walk with two outs in the bottom of the seventh for the Tigers (2-4). Jacob Kapstein added two hits.
H.S. baseball
• Montville juniors Buddy Dewaine and Jordan Zeppieri were among eight area players named to the Connecticut High School State Coaches' Association All-State team. Ledyard senior Jordan Kowalski and Waterford senior Tom Pezzolesi joined Dewaine and Zeppieri on the Class M team. New London senior Eddie Santiago and East Lyme senior Austin Toback and were selected to the Class L team. Seniors Bill Buscetto (Old Lyme) and Willie Rios (St. Bernard) were both chosen to the Class S team.
H.S. football
• New London's Jose Garcia and Nico Ramos were both drafted to play for Team Nutmeg at this month's CHSCA Super 100 Classic game. The game features the state's top seniors, who were drafted to their respective teams. Ledyard's Khary Childs-Carrasquillo and Calvin Greene, Tuzar Skipper and Ramel Williams of NFA will play for Team Constitution. The game will be played at Rentschler Field on June 28 at 4:30 p.m.
Senior Legion
• Kavin Jakuba scored the game's only run on a wild pitch in the bottom of the eighth inning to give Ledyard-Pawcatuck at 1-0 victory over New London in Zone VI. John Moody pitched two perfect innings in relief of Mike Dellaporta to earn the win while Lucas Bolduc pitched well in defeat for New London, going the distance and allowing only four hits. Connor Gilbert had two hits for L-P while Brian Cespedes had a double for New London.
Junior Legion
• J.T. Mullins pitched a complete game and also added two hits and two RBI, powering Niantic past Norwich, 8-1, in Junior Legion. Lucas Sousa-Ross had two hits and scored two runs and Corey Levesque added a single, double and three RBI for Niantic (4-2).
Salem rallies behind woman seriously injured in January crash
State looking into New London police's reporting of traffic stops
Tractor-trailer rollover closes I-95 southbound lanes in Old Lyme
Norwich budget cuts would mean layoffs, school impacts, no swimming at Spaulding Pond
Police: Off-duty officer punched elderly Foxwoods employee in the face
Proposal could lead to resignation of Norwich utilities commission leaders


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Come and share your story of love, loss, single parenting and hope with others experiencing a similar journey.
CancerCare will be facilitating an eight-week Bereavement Support Group for those raising children who have recently experienced the loss of a loved one to cancer. This support group takes place on Tuesday evenings.
To register, or for more information, please contact Claire Grainger, LCSW at 201-301-6811 or email@example.com.


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- Project Details
Title Donnons une chance à la Compétence. Transparence et activité professionnelle indépendante pour travailleurs migrants, grâce aux services d'un Centre d'Evaluation
Project Number 2009-1-IT1-LEO05-00195
Year 2009
Project Type Projets de transfert d'innovation
Status Clôturé
Country IT-Italie
Marketing Text Donnons une Chance à la Compétence s'occupe des problèmes liés à l'intégration socio-économique des groupes de migrants, ainsi que de l'amélioration de la condition de la femme sur le marché du travail. DCC adapte et expérimente la méthode, déjà utilisée pour les femmes migrantes, unissant orientation, analyse et développement des compétences actives et aide pratique dans l'insertion sur le marché du travail. La nouveauté réside dans l'intégration de l'analyse dynamique des compétences au sein d'un processus d'orientation se déroulant aussi bien en présence qu'à distance.
The issue of socio-economic integration of migrant groups, and improvement of women conditions in the Labour Market is a key challenges both at national and at EU level: these groups, even in a variety of forms, face problems as discrimination, lack of recognition of competences or even qualification, when presenting themselves in an foreign working context. Language problems, lack of knowledge of local needs, culture of work and social marginalisation are often additional obstacles to a satisfactory and successful integration process.
GC AC pursues the following objectives:
- Improve the quality of VET systems and employment services, by transferring an added value set of tools already experimented in similar contexts;
- Support the development of local education, training and labour market active policies, by adopting an adapted and experiment set of tools, practices and instruments in the context of competence assessment and transparency, migration, weak target groups' empowerment;
- Experiment and transfer a successful and integrated model, successfully experimented in Germany with migrant women, resulted able to support them within the insertion in the Labour market;
- Improve the employability, or the labour condition, of final beneficiaries in the relevant territories-countries, through integrating the AC instruments and tools in the organisations concerned;
- Have a detailed picture of Labour market needs, similarities and differences, and synergies in the concerned contexts, in order to plan tailored services and policies.
The transfer is made of three components: (1) train of trainers seminars, (2) Assessment Center tools and methods; (3) E-platform for competence documentation and self assessment process, that will be experimented and tested within three different contexts (Center and Southern Italy, Poland) and transferred in four contexts (Center and Southern Italy, Poland, Romania), supporting the creation of stable Networks of professionals and stakeholders which represent a key factor for sustainability and impact strategies. Consortium is involving key public authorities in transfer contexts, experienced VET organisations, social partners. This composition will ease the transfer process and the expected impact on local, national and EU level, allowing a large scale involvement of relevant stakeholders, durable effects on labour markets and beneficiaries and the concrete possibility of stable adoption.
The implementation of the project shall proceed through the successive development of the WP's, meaning the various thematic and transversal aspects will be clearly linked so as to generate synergies and ensure a maximum impact:
1. Labour market analysis. The methodological approach of the needs assessment analysis will be those of the social empirical research – case studies, interviews, etc. and based upon qualitative and quantitative official data available. The results observed will serve as a basis for the implementation of WP2.
2. Adaptation of method. The first part of work will be constituted by the adaptation of Assessment Center exercises and tools, and train of trainers toolkit, the second part will be the review of e-learning platform for documentation of competences and self assessment.
3. Transfer of methods and experimentation. This is the heart of the project, made of 3 main components: a) train of trainers seminars to improve targets' competences and use the toolkits with beneficiaries, b) toolkit of tools of AC (description of competences, instructions, etc), c) E-platform for competence documentation and self assessment process, for final users having attended AC sessions. The final activity in each component will be the review of tools after the test with target groups and beneficiaries. These components are divided into the WP 3 and WP 4, in order to manage the transfer in a rational way, being able to operate a more effective management, counselling, monitoring and evaluation.
4. Dissemination strategy will use a variety of tools, along the whole lifetime of the project, to ensure targeted communication (institutional – public bodies, professional – VET and counseling actors and Centers, end-users oriented) and the multipliers effect pursued by GC-AC. The combination of IT resources (website, newsletters, CD-Rom, etc.), printed tools (leaflets, booklets, etc.), and in presence activities (press conferences and releases, seminars) ensures a wide possibility to reach the different target groups, easy the transferability and sustainability process.;Location: all project locations.
5. Monitoring and evaluation activities allow for permanent review and quality control, and will be oriented towards the "logical framework method".
Marché du travail
Formation tout au long de la vie
Égalité des chances
Validation, transparence, certification
Étude interculturelle
Autres Activités de Services
Hébergement et Restauration
Product Types
Matériel d'apprentissage
Enseignement à distance
Matériel pour l'enseignement
Méthodes de distribution
Site Internet
Product information During the project implementation, partners produced the following products: National Reports Market Needs Assessment, Integrated labour market needs Assessment,three brochures concerning the Assessmentcenter methodology and testing; project web-site; GCAC on-line platform, Final publication, Cd-rom, two networks in Assessementcenter, outputs of the Project Management and Evaluation Reports.
Projecthomepage http://www.gcac.eu


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30 April 2016
May at Transgression
We have a couple of holiday dates this month, The park will be open from 10am on Monday 23rd and Monday 30th May.
Foam Dart Thunder returns on Saturday 14th May @ 7.30pm, The skatepark will be closed for normal sessions at 7pm on this day.
Tickets for FDT May are already sold out so we highly recommend following them on Facebook to avoid missing out on Junes event!!
We are busy putting together our Summer Camp schedule so be sure to check our website regulary for all the latest news…
Our reception is undergoing a few changes and our new shop will be announcing an opening date very soon!!!