• 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:

    公开号:NL2005116A
    申请号:NL2005116
    申请日:2010-07-20
    公开日:2011-01-25
    发明作者:Reinhard Lihl；Guenter Resch
    申请人:Leica Mikrosysteme Gmbh；
    IPC主号:
    专利说明:

    Title: Transfer of a sample carrier in the correlative electron microscopy
    The invention relates to a holding device for holding a sample carrier for electron microscopy and for transferring the sample carrier from a light microscope to a cryopreparator for the cryopreparation of samples for electron microscopy.
    The invention further relates to a method for transferring a sample carrier for electron microscopy from a light microscope to a cryopreparator for cryopreparation of samples for electron microscopy.
    Cryo-electron microscopy has shown to be particularly suitable for structural biological investigations. With this technology a water-containing sample is cryofixed, that is, it is cooled very quickly and avoiding the formation of ice crystals. The objects to be examined, for example cells, enzymes, viruses or lipid layers, are thereby embedded in a thin glazed ice layer. The major advantage of cryofixation is that the biological structures can be preserved in their natural state and can be examined in their physiological environment. Among other things, a biological process can be stopped at any desired moment by cryofixation and examined in this glazed state in an electron microscope.
    To precisely determine the right moment of cryofixation, the correlative method between light microscope and electron microscope, also referred to as CLEM ("correlative light-electron microscopy"), is of great advantage. The correlative method between light microscope and electron microscope allows the biological sample to be first observed in a light microscope until the desired state is reached. The sample is then transferred to a cryopreparator and cryofixed for observation with an electron microscope.
    Cell cultures are often used as samples. These often grow directly on the sample carrier. CLEM makes it possible to first examine the cells in the living state in a light microscope, to select certain cells or to await a state of a cell and to retain this state by rapid freezing.
    In another correlative method, the light microscope examines the sample that has already been cryofixed. However, compared to the CLEM method described above, this method has the disadvantage that the biological sample or cells cannot be examined in the living state.
    Regardless of the nature of the sample preparation, a high-resolution image obtained with a transmission electron microscope requires that the sample be thin enough. Samples for transmission electron microscopy are usually 30-100 nm thick and preferably 50-80 nm thick. In other methods with a transmission electron microscope (for example, Intermediate Voltage Transmission Electron Microscopy (IVEM)), the samples can also be considerably thicker. Samples with a certain thickness can be obtained by cutting with the aid of an ultra-microtome, whereby a cryofixed sample (cryo-cut) is cut into very thin disks. Another preparation method relates to the application of thin liquid films to an electron microscopy carrier. A thin liquid film is herein frozen very quickly, whereby ice crystal formation is avoided. For this purpose, a carrier for electron microscopy ("network", "grid") is immersed in a liquid containing the sample, or the sample liquid is applied to the dawner by means of a pipette, the excess liquid is removed, for example, with the aid of a filter paper, and liquid fluid remaining on the support is cryofixed by immersion in a bath of, for example, liquid ethane. Cryofixed samples can be examined directly in a frozen state in a cryo-electron microscope because they are resistant to the high vacuum prevailing in the electron microscope. In the freeze substitution, which is described, for example, in patent EP 1 267 164 B1, the water in a cryofixed sample is exchanged with a polymer and after curing the polymer, the sample is sliced with the ultra-microtome at room temperature; the examination with an electron microscope also takes place at room temperature.
    Automatic and semi-automatic cryopreparators for cryofixing are known in the art. Patent WO 02/077612 A1 (see also EP 1 370 846 B1 and US 020040157284) describes such a device with which the cryopreparation can be carried out almost automatically. This device is on the market under the trade name Vitrobot ™. With this device, the sample carrier is fixed in a holding device. Excess sample liquid on the carrier is, if necessary, derived with the help of filter paper ("blotting" process). The sample is then vitrified by rapidly immersing the support in a coolant (ethane). Another device from the Gatan company (www.gatan.at) with the trade name Cryoplunge ™ is more simply constructed and not fully automatic.
    Because biological processes often end very quickly, it is desirable in the above-described correlative method between light microscope and electron microscope (CLEM) to achieve rapid transfer of the sample carrier from the light microscope to an electron microscope.
    However, the transfer of a sample carrier from a light microscope to an above-mentioned cryopreparator is associated with a considerable investment of time. For these devices, there is also no special transfer from the sample carrier from the light microscope to the cryopreparator. Therefore, the transfer time caused by manually picking up the sample carrier of a light microscope, for example by means of tweezers, is too long for many examinations.
    It is therefore an object of the invention to obviate the drawbacks of the prior art and to enable rapid transfer of the sample carrier from the light microscope to the cryopreparator including the freezing of the sample.
    This object is achieved with a retaining device mentioned above, which according to the invention comprises an enclosing element, the enclosing element being detachably enclosable in an enclosing device arranged on the light microscope and on the cryopreparating device.
    Furthermore, this object is achieved with an above-mentioned method, which according to the invention comprises the following steps: - attaching the sample carrier to a holding device, - releasably enclosing the holding device with the sample carrier held therein by means of a sample device arranged on the holding device. containment element in a first containment device of the light microscope and the examination with a light microscope of a sample present on the sample carrier, device, and - detachably enclosing the holding device with the sample carrier held therein by means of the containment element arranged on the holding device in a second containment device of the cryopreparator and cryopreparation of the sample.
    Because the sample carrier is already mounted in the holding device during the examination with the light microscope and the picking up of the sample carrier between the two observation methods is thus eliminated, the transfer time can be considerably shortened thanks to the invention. After examination with the light microscope, the sample carrier attached to the holding device with the sample can be quickly removed by detaching the inclusion connection between the inclusion element and the inclusion device of the light microscope, quickly transferred, fixed in the inclusion device of the cryopreparation device by embedding the retaining device, and finally being cryofixed. The sample carrier with the sample thus remains attached to the retaining device all the time, starting with the examination with the light microscope, during the transfer from the light microscope to the cryopreparator, until freezing in cryogen. Thanks to the invention, it is possible to position reproducibly in the light microscope and in the cryopreparator.
    The transfer time itself can be kept very short and, depending on the distance from the light microscope to the cryopreparator, can only be a few seconds.
    The term "sample carrier" refers to all carriers suitable for electron microscopy and for sample preparation for electron microscopy. In particular, the term "sample carrier" refers to the grids already mentioned above ("grid carrier", "network", "net"), whereby the grids have differently shaped holes (holes, slits, etc.) or a number determined by a grid meshes and / or are film-coated (e.g., Quantifoil-coated grids) and / or are vapor-deposited with carbon. Other carriers which, however, can be used in the cryopreparation of samples for electron microscopy are, for example, sapphire discs as described in patent EP 1 267 164 B1.
    The holding device is designed in such a way that the usually very refined and small sample carrier, in particular a grid (diameter 2-3 mm), is certainly fixed. Usually a grid is fixed in its peripheral area by a pair of tweezers. Grids with lips are also very beneficial.
    This special type of sample carriers (also referred to as "grid with tab", "tabbed grid" or "handle grid") has, in addition to the outer edge next to the standard grid radius, a lip, which can be gripped, for example, with tweezers.
    For a light handling of the retaining device, in particular during the transfer, which usually takes place manually, it is advantageous if the retaining device is substantially elongated. The retaining device is preferably made of tweezers.
    In a preferred embodiment the containment element has a first area for fixing the retaining device in the respective containment device of the light microscope or the cryop repair device, respectively, and a second area for clamping, for example, a tweezers with a very fine tip. The sample carrier is held by means of the tweezers. The tweezers can be easily exchanged if necessary, for example when the tweezers tip is worn. Although the aforementioned embodiment is preferred, the retaining device can also be made in one piece.
    The invention is particularly suitable for the transfer and for the cryop repair of samples with cells, in particular a cell culture.
    For samples, which include cell cultures, inverse microscopes are usually used. The sample carrier is usually located in a transparent culture container, for example a cell culture dish or a petri dish, with a cover glass as bottom. The objective is arranged under the cover glass or the culture holder. The edge of the culture container means that the retaining device does not extend horizontally, but extends into the culture container at an angle. This causes problems with a conventional sample carrier, in that the surface of the sample carrier must be arranged in parallel and in contact with the cover glass. It is therefore particularly advantageous if the surface of the sample carrier is orientable at an angle with the substantially elongated holding device. The use of a sample carrier with lip, in particular a grid with lip as described above, has proved to be particularly effective for this embodiment. This lip can be bent either elastically or plastically, so that a parallel position can be achieved from virtually the entire grid surface to the cover glass. After the examination with the light microscope, the holding device can be quickly removed from the light microscope and transferred to the cryopreparator. After freezing, the lip can, for example, be removed from the grid by means of a scalpel, razor or micro-scissors and the grid can be mounted in a frozen state in a sample carrier for electron microscopy.
    In order for the sample carrier in the light microscope to approach the cover glass in a focused and controlled manner, it is advantageous if the retaining device for positioning the sample carrier is moved into the desired position by means of an adjusting device arranged on the containment device of the light microscope.
    When the retaining device is fixed in the cryopreparator, it is advantageous if the retaining device is rotatably mounted about its longitudinal axis to allow "blotting" of the desired side of the net.
    In order for the retaining device to be fast and repeatable with precision by means of the containment element in the respective containment device and to be removed therefrom, the following coupling mechanisms have proved themselves to be advantageous in practice: In a first advantageous embodiment the containment element is of a ball containment coupling enclosed in the containment device. In a sub-embodiment, the enclosing element comprises an enclosing groove into which a spring-mounted coupling part (ball enclosure) arranged in the relevant enclosing device of the light microscope or the cryopreparating device can be enclosed. There are many options for carrying out the recording, such as a dovetail or T-shape. An L-shape has proven to be particularly advantageous, because it can thereby prevent installation in two positions (180 ° symmetry) and enable a clear positioning of the sample carrier. The L-shape allows installation in only one position. This has the advantage that the correct position of the sample carrier can always be taken into account, because the sample carrier must take a certain position both during the examination with the light microscope and when "blotting" and will not be rotated through 180 °. Errors can be avoided in this way.
    In another advantageous embodiment, the containment element can be fixed in the containment device by means of a magnetic coupling. However, the embodiment with the ball lock coupling is preferred on the basis of the even higher precision.
    The invention, including other advantages, will be explained in more detail below with reference to a non-limiting exemplary embodiment, which is shown in the accompanying drawing. In the drawing: FIG. 1 is a perspective view of a holding device for a sample carrier for electron microscopy, which is fixed on a table of a light microscope,
    FIG. 2 shows a cryopreparator in perspective view with the retaining device of FIG. 1 fixed in the cryopreparator. 1
    FIG. 3A is a perspective view of a holding device according to the invention,
    FIG. 3B is a perspective view of the containment device, wherein the retaining device of FIG. 3A can be embedded in accordance with the bullet containment principle, and
    FIG. 4 is a sectional view of the holding device of FIG. 3A and the containment device of FIG. 3B in an enclosed state.
    FIG. 1 shows a perspective view of a holding device 100 for holding and examining a sample intended for cryoelectron microscopy in a light microscope. In the example shown, it is a cell culture sample. For this, the cells are cultivated under sterile conditions in tissue culture containers on sample carriers for electron microscopes. The cells grow as a monolayer on the sample carriers. For the examination with a light microscope, a sample carrier, in the example shown, a grid 101, is removed and fixed in a holding device 103 that can be attached to the light microscope. With the aid of the retaining device 103 a faster transfer of the grid 101 from the light microscope to a cryopreparator (see Fig. 2) is made possible. The example shown is an inverse light microscope.
    For a lighter handling, the retaining device 103 is substantially elongated. The retaining device 103 in this exemplary embodiment is of two parts and comprises both tweezers 103a for fixing the grid 101 and an enclosing element 103b, which on the one hand receives the tweezers 103a and on the other hand fixes the retaining device 103 to an enclosing device 104, which is mounted on a table 108 of the light microscope is arranged. A coupling mechanism between the containment element 103 and the containment device 104 according to the bullet containment principle is further shown below in FIG. 3A, 3B and 4.
    As in FIG. 1, the grid 101 comprises a lip 107 on its outer edge next to the standard grid radius, to which the tweezers 103a can engage. The retaining device 103 is now mounted in the light microscope such that the tweezers 103a with the grid 101 extend inwards into a cell culture dish 102 located on the table 108. The edge 106 of the cell culture dish 102 implies that the retaining device 103 does not extend horizontally, but extends at an angle α (Greek letter alpha) into the culture dish 102. The grid 101 lies on a cover glass 102a for examination. The cover glass 102a forms the bottom of the cell culture dish 102. Cell culture dishes of this kind are commercially available (e.g. Fluorodish Cell Culture Dish 35 mm, glass 23 mm diameter, 0.17 mm thickness from the company World Precision Instruments Ine.). The cells on the grid 101 are thereby facing the cover glass. The objective (not shown) is arranged below the cover glass 102a or the cell culture dish 102. In order to achieve the parallel position required for examination with a light microscope virtually from the entire grid surface to the cover glass 102a, the lip 107 held by the tweezers 103a is preferably bent elastically with respect to the grid surface. For examination with the light microscope, the grid surface is consequently oriented at an angle with respect to the longitudinal axis (L) of the holding device 103. A plastic bending of the lip 107 is also conceivable, however, an elastic bending is preferred because the surface of the grid 101 for the subsequent cryopreparation must again be oriented substantially parallel to the longitudinal axis (L) of the retaining device 103.
    In order for the grid 101 in the light microscope to be directed and controlled to approach and position the cover glass 102a, the holding device 103 and the grid 101 mounted therein can be moved by means of an adjusting device 105 arranged on the containment device 104 of the light microscope and into the desired position.
    After examination with the light microscope, for example when a desired biological condition of the sample has been reached, the retaining device can be quickly removed from the light microscope by detaching the containment element 103 from the containment device 104 and transferred to the cryopreparation device and being cryofixed (for a detailed description, see Fig. 2). After freezing, the lip 107 is removed from the grid 101 by means of a scalpel, razor or micro scissors and the grid 101 including the sample thereon is mounted in a frozen state in a sample holder for the electron microscopy.
    Because the user can easily grasp the grid 101 outside the preparation room with the tweezers 103, the holding device 103 can be manually and quickly and with repeatable precision attached to and removed from the light microscope table 108 as well as in the cryopreparator. A suitable coupling mechanism according to the ball enclosure principle is shown in more detail below in FIG. 3A, 3B and 4. The tweezers 103a can also be easily changed if necessary.
    FIG. 2 shows a perspective view of an automated cryopreparator 200 for specimen preparation for an electron microscope. The device 200 comprises as essential components a climate-controlled preparation chamber 201 and a cooling device 202, in which a container with a cryogen is located. Various stepper motors as well as a control are accommodated in the housing back part 203 of the device 200, which are not further shown here. In FIG. 2, the preparation chamber 201 is in its upper position, in which the holding device 103 with the grid 101 secured therein can be fixed by means of the containment element 103b in an containment device 204 in the cryopreparator 200. The mechanism for enclosing the enclosure element 103 in the enclosure device 204 is the same as that for the light microscope (see Fig. 1). After fixing of the holding device 103, the preparation chamber 201 is moved downwards in the direction of the cooling device 202 by means of a stepper motor. The holding device 103 with the sample is now located in the preparation chamber 201. Abundant sample liquid can - as already described above - be removed from the grid surface by means of "blotting" with filter paper. The retaining device 103 is rotatable 180 ° in both directions about its longitudinal axis L ", to allow" blotting "of both sides. After the blotting process, the grid 101 is moved very rapidly downwards by means of the vertical movement of the retaining device 103 in the cryogen container (not shown) of the cooling device 202 located below the preparation chamber 201, so that it is located on the grid 10 biological sample (cells) is vitrified.
    After freezing, the grid with the frozen sample is removed from the holding device 103 for microscopy in an electron microscope. The cryogenic container is removable from the device 200 in order to be able to place the glazed sample in a sample container for a cryo-electron microscope. After freezing, the holding device is hoisted up to just above the cryogen container. The gas temperature in this area is approximately -160 ° C. The retaining device 103 is manually removed from the containment mechanism and the grid 101 is deposited in a transfer container which is cooled by means of liquid nitrogen and is preferably located next to the cryogen container.
    This transfer holder is again transported to a charging station for cryo-electron microscope holders (Gatan company: www.gatan.com).
    FIG. 3A shows a perspective view of the retaining device 103. The retaining device 103 is, as already explained above, constructed in two parts and comprises a pair of tweezers 103a for fixing the grid 101 (with lip 107) and also an enclosing element 103b, which on the one hand the tweezers 103a and, on the other hand, can be enclosed in an enclosing device 104 of the light microscope and also in an enclosing device 204 of the cryopreparating device 200. The containment device 104 is shown in FIG. 3B; the enclosure device 204 of the cryopreparator device is similarly shaped.
    The mounting of the retaining device 103 in the containment device takes place by horizontally sliding the containment element 103b of the holding device 103 into the containment device 104 (mounting direction represented by the level 109). In FIG. 3A, on the underside of the containment element 103b, an inclined surface 110 (for example slanted at 45 °) can be seen, which comprises a V-shaped containment groove 111. The containment groove 111 serves as an enclosure for a spring-mounted coupling part 112 (ball enclosure 112) arranged in the enclosure device 104. This is illustrated in FIG. 4, which shows the holding device 103 of Fig. 3A and the containment device 104 in an enclosed state. The ball enclosure 112 is located in the enclosure groove 111.
    There are many options for carrying out the recording, such as a dovetail or T-shape. In the example shown, an L-shape 114 has been chosen in order to avoid mounting in two positions (180 ° symmetry) and to allow for unambiguous positioning of the grid 101. The L-shape 114 allows the assembly to take place in only one position.
    The L-shape 114 has the advantage that the correct position of the grid 101 can always be taken into account, because the grid 101 must assume a certain position both during the examination with the light microscope and when "blotting" and not over Will be rotated 180 °. Errors can be avoided in this way.
    Instead of the spring-loaded ball enclosure, a magnetic coupling can also be used as coupling piece 112.
    The embodiment described above is only one of many examples and, therefore, not to be regarded as limiting.
    权利要求:
    Claims (11)
    [1]
    A holding device (103) for holding a sample carrier for electron microscopy (101) and for transferring the sample carrier (101) from a light microscope to a cryopreparator (200) for cryopreparating samples for an electron microscope, characterized in that the holding device (103) comprises an enclosing element (103b), the enclosing element (103b) being detachably enclosable in an enclosing device (104, 204) arranged on the light microscope and on the cryopreparator (200).
    [2]
    Holding device according to claim 1, characterized in that the holding device (103) is made substantially elongated.
    [3]
    Holding device according to claim 2, characterized in that the surface of the sample carrier (101) is orientable at an angle with respect to the substantially elongated holding device (103).
    [4]
    Holding device according to one of claims 1 to 3, characterized in that the holding device (103) is adapted to hold a sample carrier with a lip (107).
    [5]
    Holding device according to one of claims 1 to 4, characterized in that the holding device (103) is rotatably mounted in the cryopreparator (200) about a longitudinal axis (L ').
    [6]
    Holding device according to one of Claims 1 to 5, characterized in that the containment element (103b) can be enclosed in the containment device (104, 204) by means of a ball containment coupling (111, 112).
    [7]
    Holding device according to one of Claims 1 to 5, characterized in that the containment element can be enclosed in the containment device by means of a magnetic coupling.
    [8]
    Method for transferring from a sample microscope for electron microscopy (101) a light microscope to a cryopreparator (200) for cryopreparating samples for an electron microscope, characterized by the steps of: - mounting the sample carrier (101) in a retaining device (103); - releasably enclosing the holding device (103) with the sample carrier (101) held therein by means of an enclosing element (103b) arranged on the holding device (103) in a first enclosing device (104) of the light microscope and examining with a light microscope a sample located on the sample carrier; - detaching the holding device (103) with the sample carrier (101) retained therein from the light microscope containment device (104) and transferring the holding device (103) to the cryopreparator (200); and - releasably enclosing the holding device (103) with the sample carrier (101) held therein by means of the containment element (103b) arranged on the holding device (103) in a second containment device (204) of the cryopreparator (200) and cryopreparation of the sample.
    [9]
    Method according to claim 8, characterized in that a sample carrier with connecting pieces (107) is used as the sample carrier (101).
    [10]
    Method according to claim 8 or 9, characterized in that the holding device (103) for positioning the sample carrier (101) is moved by means of an adjustment device (105) arranged on the containment device (104) of the light microscope.
    [11]
    Use of a retaining device according to any of claims 1 to 7 for transferring and cryopreparating samples with cells, in particular a cell culture.
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    同族专利:
    公开号 | 公开日
    AT508017A4|2010-10-15|
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    AT508017B1|2010-10-15|
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    法律状态:
    2015-02-25| V1| Lapsed because of non-payment of the annual fee|Effective date: 20150201 |
    优先权:
    申请号 | 申请日 | 专利标题
    AT11442009|2009-07-22|
    AT0114409A|AT508017B1|2009-07-22|2009-07-22|TRANSFER OF A SAMPLE CARRIER IN CORRELATIVE ELECTRONIC MICROSCOPY|
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