• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    In a device (1) for rapid freezing under pressure of a water-containing sample (3), in particular a biological preparation / is in a high pressure chamber (11), in which a sample holder (30) with a sample held therein (3) introduced and sealed pressure-tight a high pressure coolant is supplied to the location of the sample holder (30) therein. At the same time, the high-pressure chamber (11) has a viewing window construction (2) with a pressure-tight window (20) through which light from outside can be directed onto the sample (3) located in the sample holder (30). The window (20) may include a transparent window member made of a high pressure resistant material, wherein the window member (20) is held by a pressure and temperature resistant window storage provided in the high pressure chamber.
    公开号:AT512287A4
    申请号:T50051/2012
    申请日:2012-03-02
    公开日:2013-07-15
    发明作者:Reinhard Lihl；Rainer Wogritsch；Heinz Plank
    申请人:Leica Microsysteme Gmbh；
    IPC主号:
    专利说明:

    P12478
    DEVICE FOR THE LIGHT TEMULATlON AND CRYOPRONIZATION OF BIOLOGICAL SAMPLES
    The invention relates to a device for rapid freezing under pressure of a water-containing sample, in particular a biological preparation, namely with a high-pressure chamber into which a sample holder can be introduced with the sample held therein and sealed pressure-tight, wherein in the high-pressure chamber, a coolant to place Furthermore, the invention relates to a method corresponding to the device for high pressure freezing.
    A Hockdruckgefriereinrichtung of the type mentioned is described in DE 100 25 512 Al, wherein in addition by a prestressed pneumatic cylinder, a particularly rapid increase in pressure is achieved.
    A device for high pressure freezing of biological and industrial samples produced by the Applicant is known as "Leica EM HPM100". successfully distributed. With the "Leica EM HPM100 " it is possible, a sample with liquid nitrogen under a pressure of up to 2100 bar within a few milliseconds to a low temperature, in particular below -100 ° C, to cool (cryopreservation). The sample has a thickness of up to 200 μτη and is vitrified in the cryopreservation by the rapid cooling process, i. the formation of crystalline ice is largely inhibited because crystallization could distort or even destroy the microstructure of the sample.
    In the "Leica EM HPM100 " A sample cartridge is used to hold the sample under high pressure conditions during the freezing process. The sample cartridge is made of high-strength plastic and comprises three components, namely two half cylinders with a channel through which cooling liquid can flow, and a carrier plate with an opening for receiving the sample. The pressure at the location of the sample is generated by the cooling liquid, which is brought to a pressure of 2100 bar for this purpose.
    Fig. 8 shows the basic structure of the high pressure freezing system 9 which is the "Leica EM HPM100 " underlying and also usable for freezing systems according to the invention. The sample cartridge 89 is inserted into the high-pressure chamber 90 via a charger 95 £ 014.1 302012/50051. For the freezing process, the high-pressure chamber 90 is closed by locking the loading device 95 by means of locking pins 94. The freezing process is achieved by the supply of liquid nitrogen (LN2) under high pressure, which is introduced into the space of the cartridge 89 abruptly. For this purpose, the system 9 is equipped with a coolant supply 8 In the coolant supply 8, LN2 is passed from a coolant reservoir 81 via a coolant pump 82 with a downstream check valve 83 to a compressed air operated pressure booster 84. Here, the used coolant LN2 is heated to the target pressure of e.g. Brought 2100 bar. The output of the pressure booster 84 is connected to the high pressure chamber 90 through a supply line via a high pressure valve 85. The high pressure valve 85 is configured to abruptly supply the high pressure LN2 to the interior of the high pressure chamber 90 upon opening, so that the sample held in the cartridge 89 is high pressure frozen in a very short time. A pressure sensor 91 and a temperature sensor 92 monitor the freezing process. Via an outlet opening 93 (outlet with silencer), the chamber 90 can be expanded to allow the removal of the cryopreserved sample.
    In addition, the high pressure chamber may provide a supply of another liquid ("fill liquid"), e.g. an alcohol (in particular ethanol), wherein this filling liquid is supplied from a container 86 via a pump / dosing system 87 with a downstream check valve 88 of the high-pressure chamber 90. For example, in certain applications, the alcohol may be used to fill the chamber prior to freezing; In many cases, the use of the alcohol offers the advantage of faster pressure build-up when exposed to LN2, but the use of the filling liquid is not mandatory, especially since different types of samples should not come into contact with alcohol. Good Abkühhmgsergebnisse can be achieved with the apparatus shown here without the use of a filling liquid such as alcohol in particular. For specific research and development areas, the behavior of a sample due to stimulation with light, in particular the visible region, is examined, in particular by examining the state achieved by photostimulation. Such studies are carried out, for example, on biological objects such as plant cells or cyanobacteria. However, the photostimulated state is under normal conditions (i.e., under biosource conditions, in the 2 / 1® (02-03-2012! Print €> d; Q5 # 3-2012 £ 014.1 102012/50051
    Usually at ambient temperature and pressure), so additional measures will be required to fix the transient stimulated state of the sample. For this purpose, the cryopreservation described above is useful; A rapid freezing of the sample under high pressure is desirable here, because in biological samples vitrification without ice crystal formation can be achieved. In the known Hochdruckgefriereinrich-lines, however, irradiation of the sample only outside of the high-pressure chamber is possible because the interior of the high-pressure chamber is not or completely insufficiently accessible to light. However, since the charging of a sample into the high-pressure chamber takes a few seconds and can hardly be shortened due to equipment limitations, the charging time of a sample is thus well beyond the lifetime of the examined light-stimulated state, which is why it is difficult to impossible in known devices, the cryopreservation sufficient quickly after the optical stimulation of the sample.
    Under "high pressure" Here, a pressure range is understood that leads to a significant reduction (ie, by several Q of the melting point of the water.) According to its state diagram at 2045 bar, pure water has the highest possible lowering of the melting point, which is lowered to -22 ° C. at this pressure value However, pressures below this value of 2045 bar are usually sufficient, especially since in biological samples, of course, never pure water.With the experiments performed by the inventors good results of cryopreservation were already achieved at pressures below 2000 bar, namely at 1600 bar, so that the pressure ranges considered here are several 100 bar, preferably between 1000 and 2200 bar.
    It is therefore an object of the invention to provide a high-pressure freezing device which enables a light stimulation of the sample by an intense light pulse and immediately following a cryopreservation of the stimulated sample in a simple manner. Here, the time interval between stimulation and freezing process and the conditions of the stimulation should be as largely and reproducibly adjustable.
    This object is achieved by a device of the type mentioned, in which according to the invention, the high-pressure chamber has a viewing window construction with a pressure-tight window through which light can be supplied from the outside and directed to the sample located in the sample holder or einstrahlbar. : 3/16 02-03-2012 iPrinted: 05-03-2012 IE014.1 102012/50051
    The invention is based on the insight that the desired short period of time between photostimulation and freezing under high-pressure conditions can be achieved by performing the light stimulation on the sample already charged in the high-pressure chamber. Thereby, the Uchtstimuherte state can be preserved sufficiently quickly without unwanted delays must be taken into account. Thus, the invention makes an examination of the photostimulated sample accessible, for example in a cryo-electron microscope, wherein prior to the actual examination optionally further preparation steps, such. Thin section in a cryo-ultra-microtome, can be turned on.
    In a particularly advantageous embodiment of the invention, the window is executed by means of a window member made of a high-pressure resistant material which is transparent at least in part (especially of course in the areas through which the light is incident) for the supplied from the outside of the high-pressure chamber light, wherein the window element is held by a pressure and temperature-resistant window storage provided in the high pressure chamber.
    Furthermore, it is favorable if in the high-pressure chamber a sample holder is provided, which is made of a material which is transparent to the light supplied from the outside of the high-pressure chamber. In this case, the sample holder (sample cartridge) may have a sample carrier and at least one transparent tray, wherein the sample carrier has an opening for receiving the sample. In addition, the at least one shell part has a channel for a coolant, wherein in the mounted sample holder the sample carrier is surrounded by the at least one shell part and the channel of the at least one shell part leads from an external mouth to the region of the opening of the sample carrier. This allows photostimulation of the sample and subsequent cryopreservation in the same positioning in the sample holder and thus facilitates reliable preservation of the photostimulated state. By accommodating in the sample cartridge, the sample remains stably positioned throughout the stimulation-preservation process; In addition, the sample cartridge forms a "chamber in the chamber", which directs the coolant specifically to the location of the sample and thus ensures a delay-free cooling process. 02-03-2012 -5-iRrinted: P12478 05-03-2012 rto 201: 2/50051
    In particular, light can be radiated directly from a suitable light source with the required light intensity through the pressure-tight window. Alternatively, a light guide in the form of a fiber optic cable can be supplied to the viewing window from the outside, with its end can be pressed against the viewing window. In this way, light with selectable wavelength and / or intensity and / or pulse duration can be conducted to the sample via the light guide. For this purpose, the viewing window construction may favorably have an opening serving as a light passage, the axis of which is aligned with the location of the sample and into which a light guide connectable to a light source can be brought to the window.
    In order to ensure a timed sequence of the stimulation-preservation process, a device associated with the control device, which is adapted for driving a light source of the light to be supplied to the sample, expedient, wherein the output of a sensor or switching means of Kühlmittelkrei-ses connected to the control device is and the light source is controllable at an adjustable time interval to the trigger signal of the sensor.
    A further aspect of the invention relates to a method for quickly freezing a water-containing sample, in particular a biological preparation, in a high-pressure chamber, the method comprising the steps of: irradiating the sample with light through a pressure-tight window of the high-pressure chamber, applying a sample to the sample cryogenic liquid - in particular liquid nitrogen - under pressure (in particular high pressure conditions), wherein the irradiation of light according to a predeterminable temporal correlation takes place on the basis of a trigger signal for applying the sample with the cryogenic liquid.
    In this case, it is favorable if the sample is received in a sample holder which is transparent to the light used for the irradiation (at least in those regions through which light is to pass), and the sample holder containing the sample is inserted and locked in the high-pressure chamber. In addition, the triggering signal from a switching means for applying the sample with the cryogenic liquid or a switch means associated switching signal can be derived, in which case the signal thus derived can be used as a trigger signal for irradiating the sample optionally taking into account an adjustable delay time. 02-03-2012 print € d: Ö5H03-2Ö12 E014.H Π02012 / 50051 PI2478 -6-
    The invention together with further advantages and further developments will be explained in more detail below with reference to an exemplary, non-limiting exemplary embodiment, which is illustrated in the accompanying drawings. The drawings show:
    Fig. 1 is a perspective view of the Hochdruckgefrierarüage according to the embodiment;
    Fig. 2 is a sectional view of the system of Fig. 1 and in particular the viewing window therein;
    Fig. 3 is an enlarged detail of the sectional view of Fig. 2;
    4 shows the sample cartridge in a perspective view;
    FIG. 5 shows the sample cartridge of FIG. 4 in an exploded view; FIG.
    FIG. 6 shows the carrier plate of the sample in a longitudinal section in a perspective view; FIG.
    Fig. 7 is a block diagram of the light stimulation control; and
    Fig. 8 is a schematic representation of the high-pressure freezing system according to the prior art.
    The exemplary embodiment relates to a high-pressure freezer which is based on the structure of the "Leica EM HPM100". is based and continues to develop. The basic structure of the high-pressure freezing system described with reference to FIG. 8 is also used in the apparatus of the embodiment. However, it should be understood that the invention is not limited to this embodiment or to such high-pressure freezers based on said Leica device but also encompasses other implementations of the stimulation-preservation process according to the inventive concept described in the claims.
    Fig. 1 shows the entire Hochdruckgefrieranlage 1 according to the embodiment in a perspective view. A high-pressure chamber 11 has a high-pressure line 12 for a cryogen, in this case liquid nitrogen (LN2), as well as a holder 13 for a cartridge (FIGS. 2 and 4). As already stated by the "Leica EM HPM100 " Her known, the holder 13 is inserted and fixed with the sample cartridge in the chamber 11, wherein by means of (not shown) seals on the holder, the interior of the chamber is sealed. The longitudinal axis of the chamber along which the holder is inserted extends horizontally, and in FIG. 1, a seal is also shown on the chamber 11, 2, 2, 2, 2, 2, 2, 2, 2, p12 [E014 .1 P12478 -7- mounted sensor 14 for measuring the temperature inside the chamber recognizable; opposite to the sensor 14, not visible in Fig. 1, a sensor for measuring the pressure value in the chamber interior. In addition, with the reference numeral 15, the alcohol supply with check valve recognizable.
    In Fig. 1, the housing 27 of the means for locking on both sides of the chamber 11 can be seen. Housed in the housings 27 are the locking bolts 94 (FIG. 8) described at the outset as well as these actuating pneumatic cylinders whose compressed-air connections 28 can be seen in FIGS. 1 and 2. The locking pins or pneumatic cylinders are monitored by a sensor (not shown in Fig. 2, element 71b in Fig. 7) whose signal is used to confirm that locking has taken place. This ensures that a freezing process can only take place when the chamber is locked. For this example, the piston of one of the pneumatic cylinders may be provided with a magnet which triggers a reed contact of the sensor 71b upon actuation of the piston. Such a reed-contact sensor can be easily mounted and positioned on the outside of the housing 27 of the pneumatic cylinder (eg, in an external groove) at a suitable location. In the embodiment shown, a fixed timing is configured in which the interlock acknowledgment signal is configured the start of the freezing process is carried out by the sensor 71b, optionally after a device-related and / or an additional adjustable delay time.
    According to the invention (and in contrast to the "Leica EM HPM100"), the high-pressure chamber 11 is provided with a viewing window structure 2 for irradiation of the sample inserted in the chamber 11 in situ with externally irradiated light, e.g. white light or blue light (maximum at a wavelength of 460 nm).
    The sectional views of FIGS. 2 and 3 illustrate the viewing window structure 2. The sectional plane extends vertically, through the central axis of the viewing window and transverse to the longitudinal axis of the chamber. The viewing window structure 2 is housed in a window opening 19 of the chamber 11 and fixed by a screw connection. A viewing window 20 is held by an insert 22 having a tubular base shape with a central opening 21 coincident with its axis with the central axis of the window 20. The insert 22 together with the window opening 19 em window bearing of the window 20th
    [02-03-2012 iPanted: 05-03-2012 E014.1 1102012/50051 PI2478 -8-
    In the along the axis of the insert extending opening 21, which serves as a light path, one end of a light guide 23 is received, preferably anschiagend to the window 20. The axis of the light path, which thus defines the Einstrahlxichtung the light through the light guide 23, is directed to the held in the cartridge 30 sample 3 and can, as shown in this embodiment, perpendicular to the longitudinal axis of the chamber 11th
    To the insert 22 includes a window holder 24 which has a shoulder 25 on the outside of rohrfömugen basic shape, and cooperating with this cap screw 26. The latter is screwed into an internal thread of the opening of the chamber and presses on the shoulder 25, which is for the flameproof Fixing the window 20 provides.
    As a viewing window 20, e.g. a so-called visual cell may be used, e.g. the view cell Sitec 740.01 (Sitec-Sieber Engineering AG, Aschbach, Switzerland) with a colorless sapphire as a window element. These vision cells meet the requirements of the invention in terms of tightness, pressure and temperature resistance of the viewing window, with an optical window width of 6 to 28 mm.
    Alternatively, a block of industrial transparent diamond can be used as a viewing window. The diamond block may for example be soldered to a pressure-tight screw; Brazing the diamond material is possible under vacuum and results in a reliable connection. This unit of screw and diamond block can be used as a viewing window 20 according to the invention. Instead of a transparent diamond, any high-strength material satisfying the requirements of compressive strength and transparency in the light region used, such as, in particular, diamond, sapphire, etc., may be used.
    It should be noted that, in contrast to the viewing window 20 and the light path 21, the remaining construction of the chamber 11 is opaque because it is made of components of e.g. Steel or aluminum is made and any plastic parts used (such as seals) are also made of opaque plastic or rubber. This prevents unwanted influence of the sample by disturbing light away from the photostimulation. (8/16 102-03-2012 printed: 05-03-: 2012 E014.1: 102012/50051
    P1247R -9-
    Fig. 4 shows the sample cartridge 30 which is held in the end piece 16 of the holder 13.
    Fig. 5 shows the cartridge 30 in an exploded view. The end piece 16 consists e.g. made of stainless steel; it is designed cup-shaped (the cup is oriented parallel to the longitudinal axis, thus horizontally in the embodiment) and has a recess 17 on the side corresponding to the location of the viewing window 20. To the end piece 16 includes two opposite inserts 18 (also made of steel) with plano-concave shape. The inserts 18 serve as spring tongues and perform a spring action to hold the parts of the sample cartridge together. The inserts 18 are laterally offset for reasons of space requirements for the viewing window; in other embodiments, these elements may be mounted in the vertical direction.
    The cartridge 30 consists of a cover part 31 and a bottom part 32. Between the parts 31,32 of the cartridge, a support plate 33 is held. The cover part 31 is shown in Fig. 4 only indicated, which improves the view of the support plate 33. The cover part 31 and the bottom part 32 have a substantially semi-cylindrical basic shape, wherein on the inner flat surface of the bottom part 32 and optionally also the cover part 31 along the longitudinal direction, a channel 34 is provided, can be passed through the coolant to the sample. In this way, LN2 can flow from both sides to sample 3, which promotes rapid cooling. In the embodiment shown, in order to additionally enhance the supply of LN2, one channel 34 is provided from both end faces.
    This allows the LN2 to flow through the cartridge 30, substantially along the longitudinal axis of the cartridge, corresponding to the direction of the dotted arrow L in Fig. 5. In the holder 13 (inside the cup, near or in the "bottom surface"). an opening is provided which leads to an outlet opening 29 located outside the chamber 11 and thus serves as an outlet 93 for gases or liquids displaced by the LN2 (including a possible filling liquid such as ethanol). This exit path to the opening 29 is dimensioned so that the passage of the cartridge with a certain amount LN2 (0.25 1) is possible, without this leading to a pressure reduction within the time required for cryopreservation (about 300 to 500 ms); Thereafter, the escape of the chamber filling through the outlet opening 29 results in a relaxation of the pressure in the chamber 11. Without such opening for the escape of displaced fluids, the desired pressure could be achieved in the rapid LN2 supply, but not a rapid cooling. > 9/16 02-03-2012 jPrinted: 05-03-2012 E014.1 1102012 ^ 0051 PI2478 -10-
    In the parts 31, 32 of the cartridge, lateral notches 35, e.g. In pairs opposite lying formed, be designed for blocking the cartridge against movement in the longitudinal direction and fixing the components to each other.
    The components 31, 33 of the cartridge 30 are made of a transparent plastic, e.g. Polycarbonate or polystyrene produced. At least the lid part 31 is made of transparent material; For reasons of easier handling (and also the likelihood of confusion), it makes sense that the bottom part 33 is made of transparent material.
    The support plate 33 is e.g. Made of PEEK (polyetheretherketone), which facilitates the processing. On the one hand, the abovementioned materials have high stability with regard to pressure resistance and low-temperature behavior over the pressure and temperature range used and, on the other hand, are sufficiently transparent in the light range used for the optical stimulation, in this case for wavelengths> 400 nm. In addition, the shape as a half cylinder is also advantageous in terms of optics, since a bundling of the light can take place to the sample out.
    6 shows the carrier plate 33 in a longitudinal section along a vertical plane, so that the space for receiving a sample 3 is shown cut away. The carrier plate 33 has laterally a respective notch 36 which corresponds to the notches 35 of the lid and bottom parts 31, 32 and has in its center a circular opening 37, wherein the sample 3 between two discs 38 of eg Sapphire is held. The distance between the discs can be adjusted by spacer rings 39. The sapphire discs 38 also serve to protect the sample 3, since the rapid flow of the LN2 needed for cooling would otherwise entrain the sample. The thickness of the sapphire discs 38 is e.g. 110 μιη with a diameter of the opening 36 of 6 mm. In a variant with an opening of 3 mm diameter, a smaller thickness of the slices of 50 μm may be sufficient. The material sapphire is preferred because it is characterized by high stability and is also a good substrate for cell cultures and bacteria. In addition, sapphire has the additional advantage of high thermal conductivity, which ensures rapid cooling of the sample.
    The entire cartridge 30 is dimensioned such that within a period of preferably 500 ms the required pressure (for example 2000 bar-200 MPa) can be established and maintained, whereby rapid freezing of the sample 3 is achieved in the same time interval. 02-03-2012 -11- PI2478
    Fig. 7 shows a block diagram of the trigger control 7 of the light stimulation, which allows a temporal control of the light pulse for stimulation and the freezing process, if desired controlled by computer. By means of a sensor 71, e.g. in the form of a reed contact, the inlet valve for the LN2 is monitored. The sensor is e.g. When the inlet valve is opened to start pressurization and cooling, the light stimulation activates and triggers the sample. The actual pressure / cooling begins a certain time later than the opening of the inlet valve; this time period can be determined in advance (e.g., by measurements over the temperature sensitivity by oscilloscope monitoring · for this, the temperature sensor must have a corresponding time resolution) and in the case shown here is e.g. about 30 ms. The beginning of the light stimulation is triggered directly by the sensor signal, thus resulting in an irradiation time, which corresponds to the said period of time, in this case thus 30 ms.
    By another sensor 71a attached to another switching component of the cooling system 8, another time interval of irradiation before freezing can be achieved. In the embodiment shown also the operation of the locking bolt 94 (more precisely: the confirmation signal of the lock) can be selected as the time, which is possible because, as described above, the lock also triggers the start of a freezing process. With the sensor 71b, e.g. is attached to one of the pneumatic cylinders 28 of the operation of the locking bolt, results e.g. a delay time of approx. 500 ms. In the block diagram, the sensors 71,71a, 71b are shown as an example of any number of closeable sensors Instead of a sensor signal can of course also the switching signal of an electrically controlled valve as an input signal for driving the trigger control 7 are used.
    The sensor 71 is connected to a trigger input of the programmable LED light source 73 together with optionally further sensors via a connection distributor 72 which serves to select one of the connected sensors. The light source 73 radiates, as already described via a fiber optic light guide, a light pulse as soon as the trigger signal is activated. In this case, via a control interface, in this case e.g. a serial RS232 port of known type, by means of a computer 74 in the programmable light source 73, a delay time and the duration of the light pulse can be set arbitrarily. The delay time is used to set the time between activation of the -12- P12478
    Sensor and the beginning of the pressure / Abkühlvorgangs to shorten, i. to any desired value less than 30 or 500 ms.
    The light source 73 may preferably be in the form of a high-intensity LED or semiconductor laser. For example, the light source may be an LED light source of the LZ1 type (e.g., LZ100B200 for blue light, 460 nm wavelength) of LED Engin, Inc. (San Jose, CA, USA). A suitable programmable light source is, for example, the "Schott LLS". Schott AG (Mainz, Germany). A light source of this type offers pulses with adjustable pulse rate and pulse width, high light intensity (up to 275 lm) and different wavelengths, is compatible with all common fiber optic types and has interfaces that allow programming and control via computer. The light sources mentioned can be selected and used for different types of light - blue in the exemplary embodiment, as well as red, dark red (i.e., long-wave red), orange (short-wave red), yellow, green, short-wave blue, white, IR, UV, etc .; Depending on the light used, if appropriate, the transparent material of the cartridge 30 is suitably adapted.
    The entire procedure of preparing a sample using freezing according to the invention under high pressure conditions is thus in a typical example of the embodiment: positioning the sample in the carrier plate and assembling the cartridge; - loading the cartridge into the holder; - Inserting the holder together with the cartridge in the high-pressure chamber and locking the holder; - performing cryopreservation after light stimulation; - Relieving the pressure (via an outlet opening as described above or by opening an exhaust valve); - Removal of the cartridge and further preparation of the sample (in the cartridge or taken from this).
    The individual steps may vary in other embodiments depending on the chosen application.
    The actual cryopreservation after light stimulation involves the steps of light-stimulating the sample through the viewing window and then the high-pressure IRRinted. P12478 1) 5-03-: 2012 102012/50051 preserving the sample by inducing the flow of refrigerant (LN2) into the chamber (e.g., by opening the inlet valve). These two processes are triggered according to a predeterminable temporal correlation
    In the temporal synchronization of these two events including a predeterminable time delay, the specific details of the apparatus used Hock-druckgefheranlage can be exploited. For example, in the embodiment described, it can be utilized that - as already mentioned - between the actuation of the coolant system 8, e.g. the opening of the inlet valve 85, and the onset of the freezing process, there is a delay time which has a fixed duration and generally provides sufficient time for light stimulation. Therefore, the light stimulation is triggered by said operation of the coolant supply 8. This allows a sequence of the two events within a very short time interval of less than 500 ms.
    Of course, a variety of modifications and developments of the invention shown is possible without departing from the scope of the invention, which is not limited to the embodiment shown, but is determined by the claims. For example, any other known cryogen suitable for the particular application can also be used as the cryogenic liquid instead of LN2. The light stimulation can be carried out by irradiation with any type of desired light, monochrome or with desired light spectra, whereby not only the visible range, but depending on the need also IR and / or UV light can come into question. Also, the irradiation of the stimulation light from the light source may be direct (i.e., without the use of a light guide) by positioning the light source directly at the opening of the window insert.
    Vienna, 02. March 2012 13/16 02-03-2012
    权利要求:
    Claims (9)
    [1]
    iBrinted: .05-032012 E014.1 102012 / 5ΘΘ51 P12478 -14- REQUIREMENTS 1. Device (1) for quick freezing under pressure of a water-containing sample (3), in particular a biological preparation, with a high-pressure chamber (ll) into which a Sample holder (30) with a sample (3) held therein and can be closed pressure-tight, wherein in the high-pressure chamber (11) a coolant 2mm location of therein sample holder (30) can be fed, characterized in that the high-pressure chamber (11) a viewing window construction (2) having a pressure-tight window (20) through which externally supplied light can be directed to the sample holder (30) located in the sample (3).
    [2]
    2. The device according to claim 1, characterized in that the window (20) by means of a window member of a high pressure resistant material, which is transparent to the supplied from the outside of the high-pressure chamber light, is executed, wherein the window member (20) of one in the high-pressure chamber provided pressure and temperature-resistant window bearing (22) is held.
    [3]
    3. Apparatus according to claim 1 or 2, characterized in that in the high pressure chamber, a sample holder (30) is provided, which is made at least in part of a material which is transparent to the supplied from the outside of the high pressure chamber light.
    [4]
    4. The device according to claim 3, characterized in that the sample holder (30) has a sample carrier (33) and at least one transparent shell part (31, 32), wherein the sample carrier (33) has an opening (37) for receiving the sample (3 ) and the at least one shell part (31, 32) has a channel for a coolant, wherein in the mounted sample holder (30) of the sample carrier (33) of the at least one shell part (31, 32) is surrounded, wherein the channel of the at least one Shell part leads from an outer mouth to the region of the opening of the sample carrier
    [5]
    5. Device according to one of the preceding claims, characterized in that the Sichtfensterkonstru ktion (2) has a light path whose axis to the place of 14/16 02-03-2012 iPnnted; Sample (3) is aligned and into which a light guide (23) connectable to a light source (73) can be brought to the window (20).
    [6]
    6. Device according to one of the preceding claims, characterized by a control device associated therewith, which is adapted to drive a light source (73) of the light to be supplied to the sample, wherein an output of a sensor or switching means (71, 71a) of a coolant circuit, the coolant is fed into the chamber can be connected and the light source is ansieuerbar in an adjustable time interval to the trigger signal of the sensor.
    [7]
    7. A method for rapid freezing of a water-containing sample (3), in particular a biological preparation, in a high pressure chamber (11) comprising the steps of: - irradiating the sample (3) with light through a pressure-tight window of the high pressure chamber (11), - Sample (3) with a cryogenic liquid under pressure, wherein the irradiation of light according to a predeterminable temporal correlation takes place on the basis of a trigger signal for the application of the cryogenic liquid to the sample.
    [8]
    8. The method of claim 7, wherein previously the sample (3) in a sample holder (30), which is at least partially transparent to the light used in the irradiation, is recorded, and containing the sample sample holder (30) in the High pressure chamber is used and locked.
    [9]
    9. The method of claim 7 or 8, wherein the trigger signal from a switching means (85,17,17a, 17b) for the application of the sample with the cryogenic liquid or a switch means associated switching signal is derived, and the trigger signal as a trigger signal for the Irradiation of the sample is optionally used, taking into account an adjustable delay time. Vienna, 2 March 2012 15/16 [02-Q3-2Q12
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    同族专利:
    公开号 | 公开日
    JP2013181987A|2013-09-12|
    AT512287B1|2013-07-15|
    DE102013003164A1|2013-09-05|
    US9097632B2|2015-08-04|
    JP5298253B2|2013-09-25|
    DE102013003164B4|2019-06-27|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    ATA50051/2012A|AT512287B1|2012-03-02|2012-03-02|Apparatus for light stimulation and cryopreservation of biological samples|ATA50051/2012A| AT512287B1|2012-03-02|2012-03-02|Apparatus for light stimulation and cryopreservation of biological samples|
    DE102013003164.8A| DE102013003164B4|2012-03-02|2013-02-26|Apparatus for light stimulation and cryopreservation of biological samples|
    US13/778,720| US9097632B2|2012-03-02|2013-02-27|Device for the light stimulation and cryopreservation of biological samples|
    JP2013040538A| JP5298253B2|2012-03-02|2013-03-01|Photostimulation and cryopreservation equipment for biological samples|
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