Method for embedding a biological sample in a transparent matrix for analysis using single plane illumination microscopy

Abstract

The invention is directed to method for positioning and aligning a preferably biological sample in the detection area of the objective of a microscope arrangement. According to the invention, the method mentioned above has the following method steps: a sample is introduced into a transparent medium, preferably agarose gel, which is initially liquid; the medium is changed from the liquid state to the solid state, wherein the sample is fixated within the medium, but the transparency of the medium is retained; the solidified medium is positioned in the microscope arrangement in such a way that the sample enclosed therein is situated in the detection area of the objective. Further, a device is proposed for positioning and aligning a preferably biological sample in the detection area of the objective of a microscope arrangement.

Claims

1. A device for introducing a sample into a detection area of an objective of a microscope arrangement, comprising: a reservoir for a transparent medium which is initially still liquid; a needle-piston unit comprising: a hollow needle; and a suction and delivery piston which is movably guided in the needle for sucking a partial amount of the medium into the hollow needle or ejecting it from the hollow needle; and a manipulating unit comprising: a receptacle configured to receive the needle-piston unit; and a connection portion configured to connect to a holder of the microscope arrangement; wherein the manipulating unit is configured to manipulate the needle-piston unit so as to: position and align the partial amount of medium, after the partial amount of medium is solidified within the needle, in the microscope arrangement in such a way that a sample contained in the solidified partial amount of medium is situated in the detection area of the objective; and change a position of the sample along a coordinate X, a coordinate Y, and a coordinate Z; and rotate the sample around a longitudinal direction of the hollow needle by an angle φ; wherein the needle-piston unit is fastened to the manipulating unit by means of connection elements configured to be manually disconnected from the hollow needle; wherein the hollow needle of the needle-piston unit is in the form of a capillary or a cannula.

2. The device according to claim 1, further comprising: a means for introducing the sample into the partial amount medium after the partial amount of medium is arranged in the needle.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a capillary with a suction and delivery piston guided so as to be movable in its interior and a sample reservoir in which the end portion of the capillary is immersed;

(2) FIG. 2 shows the capillary according to FIG. 1 in a curing station, shown schematically, in which heat energy is extracted from a transparent medium which is initially still in liquid state in order to solidify it;

(3) FIG. 3 shows a schematic illustration of the mode of operation of a manipulating unit designed for positioning and aligning a sample in the detection area of a microscope objective;

(4) FIG. 4 shows the manipulating unit according to FIG. 3, wherein the sample is positioned in the illumination beam path of a microscope and is aligned on the sample in a first viewing direction of the microscope objective on the sample;

(5) FIG. 5 shows an alternative example for introducing samples into a capillary shown in FIG. 1;

(6) FIGS. 6 to 8 show another example for introducing individual samples into a curable medium; and

(7) FIG. 9 shows an example for a timed introduction of a plurality of samples into a curable medium.

DETAILED DESCRIPTION OF EMBODIMENTS

(8) It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for purposes of clarity, many other elements which are conventional in this art. Those of ordinary skill in the art will recognize that other elements are desirable for implementing the present invention. However, because such elements are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements is not provided herein.

(9) The present invention will now be described in detail on the basis of exemplary embodiments.

(10) FIG. 1 shows a capillary 1 with a suction and delivery piston 2 which is guided in the interior so as to be displaceable in directions R1 and R2. Combinations of capillaries 1 and suction and delivery pistons 2 of this kind are known, per se, as pipettes and are used for dispensing liquids.

(11) The capillary 1 can be made of glass or plastic and can be provided with a volume scale (not shown in the drawing) arranged laterally in longitudinal direction. The suction and delivery piston 2 is generally made of a flexible plastic, but can also be formed of a stainless steel rod linkage with a plunger arranged thereon.

(12) When the end portion 3 of the capillary 1 is dipped into a sample reservoir 4 in which a transparent, initially liquid medium in the form of agarose gel 5 and a plurality of samples 6 are located and the suction and delivery piston 2 is displaced inside the capillary 1 in direction R1, a partial amount of the agarose gel 5 of for example, about 30 μL to 50 μL is sucked into the capillary 1 and this partial amount of agarose gel 5, including one of the samples 6, is removed from the total reservoir of samples 6 in a precise manner.

(13) The sample 6, surrounded by the agarose gel 5 which is likewise removed, is transported by the capillary 1 to a curing station 7, shown schematically in FIG. 2, where the agarose gel 5 is cooled. The agarose gel 5 is increasingly solidified as heat energy is extracted while remaining transparent, and the sample 6 is fixated in the agarose gel 5.

(14) The capillary 1 with the sample 6 fixated in the agarose gel 5 is now transported to a manipulating unit 8, shown schematically in FIG. 3, and fixed therein by means of a receptacle 9. The manipulating unit 8 is in turn fastened to a microscope stand 10, only a partial area of which is shown for the sake of clarity.

(15) It is advantageous when a connection of the manipulating unit 8 to the microscope stand 10 is provided by means of straight-line guides which ensure a displacement of the manipulating unit 8 relative to the microscope stand 10 in coordinates X and Y.

(16) The manipulating unit 8 is outfitted with an actuating element 11 which is supported in a rotating and straight-line guide 12 and is accordingly displaceable in directions R1 and R2 and rotatable around an angle φ. The directions R1 and R2 extend parallel to coordinate Z in coordinate system X, Y, Z.

(17) The actuating element 11 has a clamping device 13 which encloses the end of the suction and delivery piston 2 remote of the sample 6. The clamping device 13 causes displacements of the actuating element 11 in directions R1 and R2 and also the rotation of the actuating element 11 to be transmitted to the suction and delivery piston 2. A drive element 14 serves to initiate the displacements in directions R1 and R2 and the rotational movement.

(18) After the capillary 1 is locked in the manipulating unit 8 and the clamping connection between the suction and delivery piston 2 and the actuating element 11 is produced, the sample 6 is located in the vicinity of the detection area which is represented here by the illumination beam path 15 in the form of a light sheet which is formed and provided for subsequent examination of the sample 6 by the method of single plane illumination microscopy (SPIM).

(19) Before starting the examination, it must be ensured that the sample 6 is situated in the illumination beam path 15. To achieve the configuration shown in FIG. 4, the actuating element 11 is displaced in direction R2 by the rotational movement of the drive element 14 based on the diagram shown in FIG. 3, and the displacing movement is transmitted by means of the clamping device 13 to the suction and delivery piston 2 and then to the agarose gel 5 with the sample 6 enclosed therein.

(20) Since the capillary 1 is not included in this displacing movement because it is locked in the manipulating unit 8, the agarose gel 5 with the sample 6 is pushed out of the capillary 1 until the configuration illustrated in FIG. 4 is achieved and the sample 6 is situated in the illumination beam path 15.

(21) The detection direction of the microscope objective, not shown in the drawing, is perpendicular to the drawing plane. By rotating the capillary 1 by an angle φ within a range of 360 degrees, the detection direction relative to the sample 6 can be changed as needed.

(22) In this way, the positioning and alignment of samples 6 in the illumination beam path 15 and detection area of the microscope, respectively, can always be reproduced.

(23) A first variant for filling the capillary 1 was described with reference to FIG. 1. An alternative variant which satisfies the demand for increased throughput per time unit in the examination of samples 6 is shown by way of example in FIG. 5.

(24) In this case, a filling station 16 is provided in which an empty capillary 1 is initially inserted. The filling station has an access 17 for agarose gel 5 and an access 18 for samples 6. Further, a guide 19 is provided for a piston 22 in order to displace the latter in a straight line in directions R1 and R2. The filling station is preferably combined with a curing station possessing possibilities for temperature control and for supplying and removing heat.

(25) Valves 20 and 21 which are preferably electronically controllable and are alternately opened and closed depending on the control are arranged in accesses 17 and 18.

(26) The filling station 16 is operated in such a way, for example, that the valve 20 is initially open and liquid agarose gel 5 is displaced through the access 17 until it is below the piston 22 and is displaced farther into the capillary in direction R2.

(27) The agarose gel 5 is pressed in direction R2 into the capillary 1 or sinks (for example, when the piston 22 is removed) into the capillary 1 under the influence of gravitational force or capillary force. To prevent the agarose gel 5 from flowing out through the lower end of the capillary 1, a closure 23 is placed on this end as soon as agarose gel 5 is located in the capillary 1. The valve 20 is then closed.

(28) Valve 21 is now opened and a sample 6 is displaced through access 18 until it is below the piston 22 and is displaced farther into the capillary in direction R2.

(29) After valve 21 is closed, the valve 20 is opened again, if required, and liquid agarose gel 5 is again fed through access 17.

(30) Alternatively, instead of supplying a sample 6 by itself, a sample 6 which is already embedded in a partial amount of agarose gel 5 can be fed through access 18. This partial amount is then pressed into the capillary 1 along with the embedded sample 6 by means of the piston 22 or sinks into the capillary 1 under the influence of gravitational force and combines with the agarose gel 5 already located therein.

(31) Subsequently, the valves are closed and the piston 2 is displaced in direction R2 so that it contacts the agarose. After the agarose cures, the sample can be moved up and down by rotating the drive element 14.

(32) It is advantageous when the upper opening of the capillary 1 (i.e., the opening of the capillary 1 opposite the direction of gravitational force) is conically expanded so that the agarose gel 5 can flow into the capillary 1 more reliably (not shown in the drawing).

(33) It also lies within the scope of the invention to construct the filling station in the manner shown in FIG. 6. Filling with agarose gel 5 is accordingly initially carried out as described above. However, the rounded or otherwise shaped end of a tool guided through access 18 is then pressed into the agarose gel 5 which is still in liquid state, whereupon the agarose gel 5 is cured, and the tool is removed again after curing so that a depression 24 (e.g., in the shape of a hollow cone or a trough) remains in the cured agarose gel 5. The valves are not shown in FIG. 6 for the sake of clarity, especially since their function has already been described referring to FIG. 5.

(34) After the tool has been removed, a sample 6 is advanced into the depression 24 through access 18 as is indicated in FIG. 7. Just the sample by itself or the sample located in a partial amount of agarose can be supplied. If required, an additional partial amount of agarose is introduced.

(35) When the sample 6 has been advanced into the depression 24, the agarose gel 5, including the sample 6 located in the depression 24, is advanced by the piston 2 until the configuration shown in FIG. 8 is achieved. If required, the agarose gel 5 is now liquefied again by temporarily supplying heat in order to embed the sample 6 completely in the agarose gel 5.

(36) It is advantageous when the filling station is designed so as to be compatible with a microscope so that the sample can be inserted in the depression 24 and oriented while being observed.

(37) Subsequently, the same process as that described referring to FIG. 5 may be carried out, wherein the capillary 1 is removed from the filling station 16 and is prepared for microscopic examination as was described above with reference to FIG. 3 and FIG. 4.

(38) It is advantageous when the filling station and the manipulating unit form a functional unit.

(39) However, the inventive idea also includes a mode of operation for filling capillaries or cannulas which expands on the mode of operation described with reference to FIGS. 6 to 8. The following description refers to FIG. 9.

(40) The following is carried out at given time intervals: a first sample 6.1 is introduced into the agarose gel 5, the agarose gel 5 is advanced with the piston 22, a second sample 6.2 is introduced into the agarose gel 5, the agarose gel 5 is advanced again, a third sample 6.3 is introduced, and so on, until a given quantity n of samples 6.n have been inserted in the agarose gel 5, wherein distances a are adjusted between the samples 6.1, 6.2, . . . , 6.n depending on the timing and forward feed speed.

(41) In this case, the agarose gel 5 is solidified by extracting heat at position P with the same timing with which the samples 6.1, 6.2, . . . , 6.n are embedded in the agarose gel 5, and the sample 6.2 located at position P is accordingly fixated in the agarose gel 5.

(42) The samples 6 can subsequently be examined microscopically as was described above.

(43) A portion A of the agarose gel 5 in which a sample (e.g., sample 6.1 in this instance) is embedded is severed by means of a cutting device 26 which is guided through the housing wall of the curing station 25 and provided with a knife 27 which is displaceable in directions S1 and S2.

(44) The severed portion falls through a funnel-shaped opening 28 out of the curing station 25 under the influence of gravitational force and can be supplied for further analytic methods.

(45) It is noted that the suction and delivery piston 2 and the piston 22 described in the embodiment examples have different functions inasmuch as the piston 22 has no suction function, which is achieved, for example, in that it is guided with a sufficiently large play in the hollow cylinder. Nevertheless, the piston 22 can be exchanged for a suction and delivery piston 2 insofar as the corresponding function is desired for handling the agarose gel 5 and sample 6.

(46) While this invention has been described in conjunction with the specific embodiments outlined above, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the preferred embodiments of the invention as set forth above are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the inventions as defined in the following claims.

REFERENCE NUMBERS

(47) 1 capillary 2 suction and delivery piston 3 end portion 4 sample reservoir 5 agarose gel 6 sample 7 curing station 8 manipulating unit 9 receptacle 10 microscope stand 11 actuating element 12 rotating and straight-line guide 13 clamping device 14 drive element 15 illumination beam path 16 filling station 17, 18 access 19 straight-line guide 20, 21 valve 22 piston 23 closure 24 depression 25 curing station 26 cutting device 27 knife 28 opening R1, R2 directions φ angle a distance A portion P position