U.S. patent application number 12/997113 was filed with the patent office on 2011-04-28 for method for embedding a biological sample in a transparent matrix for analysis using single plane illumination microscopy.
Invention is credited to Jurgen Haese, Wibke Hellmich, Helmut Lippert, Benno Radt, Olaf Selchow, Uwe Wolf.
Application Number | 20110094318 12/997113 |
Document ID | / |
Family ID | 40911087 |
Filed Date | 2011-04-28 |
United States Patent
Application |
20110094318 |
Kind Code |
A1 |
Hellmich; Wibke ; et
al. |
April 28, 2011 |
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.
Inventors: |
Hellmich; Wibke; (Jena,
DE) ; Radt; Benno; (Jena, DE) ; Lippert;
Helmut; (Jena, DE) ; Selchow; Olaf; (Jena,
DE) ; Haese; Jurgen; (Kahla, DE) ; Wolf;
Uwe; (Magdala, DE) |
Family ID: |
40911087 |
Appl. No.: |
12/997113 |
Filed: |
June 6, 2009 |
PCT Filed: |
June 6, 2009 |
PCT NO: |
PCT/EP09/04081 |
371 Date: |
December 9, 2010 |
Current U.S.
Class: |
73/863 |
Current CPC
Class: |
G02B 21/34 20130101;
G01N 1/28 20130101; G02B 21/32 20130101 |
Class at
Publication: |
73/863 |
International
Class: |
G01N 1/28 20060101
G01N001/28 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 11, 2008 |
DE |
10 2008 027 784.3 |
Claims
1. A method for positioning a sample in the detection area of an
objective in a microscope arrangement having the following method
steps: introducing a sample into an initially liquid, transparent
medium; changing the medium from the liquid state to the solid
state so that the sample is fixated inside the medium, but the
medium remains substantially transparent; and positioning the
solidified medium in the microscope arrangement in such a way that
the sample enclosed therein is situated in the detection area of
the objective.
2. The method according to claim 1, further comprising: introducing
one or more samples into the transparent medium which is initially
liquid; storing the medium with the samples in a sample reservoir;
removing a partial amount of the medium with a sample contained
therein from the sample reservoir; after the removing step,
changing the partial amount of medium from the liquid state to the
solid state, wherein the sample is fixated within the partial
amount of medium; and positioning the cured partial amount of
medium in the microscope arrangement in such a way that the sample
contained therein is situated in the detection area of the
objective.
3. The method according to claim 1; wherein the partial amount of
medium which is still in the liquid state with the sample contained
therein is sucked into the hollow space of a capillary, cannula or
disposable syringe; wherein the medium inside the hollow space is
then changed from the liquid state to the solid state; and wherein
the solid medium with the enclosed sample is ejected from the
hollow space.
4. The method according to claim 1, further comprising: storing the
transparent medium which is initially liquid on a medium reservoir;
removing a partial amount of the medium from the medium reservoir;
introducing a sample into the partial amount of medium which is
still liquid; after the removing step, changing the partial amount
of medium from the liquid state to the solid state, wherein the
sample (6) is fixated within the partial amount of medium; and
positioning the cured partial amount of medium in the microscope
arrangement in such a way that the sample contained therein is
situated in the detection area of the objective.
5. The method according to claim 4; wherein the partial amount of
medium which is still liquid is introduced into the hollow space of
a capillary, a cannula or a disposable syringe; wherein the partial
amount of medium which is still liquid is held inside the hollow
space; and wherein a sample is introduced into the partial amount
located in the hollow space.
6. The method according to claim 5; wherein one of the two end
openings of the hollow space containing the medium which is still
in the liquid state is hermetically closed; and wherein the method
further comprises: introducing the sample into the medium which is
still in the liquid state inside the hollow space through the
opposite second end opening; next, changing the medium inside the
hollow space from the liquid state to the solid state; and next,
ejecting the solid medium with the enclosed sample (6) is ejected
from the hollow space.
7. The method according to claim 1; wherein the change of the
partial amount from the liquid state to the solid state is carried
out under external influences on the medium.
8. The method according to claim 1; wherein a curable gel is used
as medium.
9. The method according to claim 1; wherein an automation of the
method steps in their entirety or an automation of some of the
individual method steps is provided.
10. The method according to claim 9; wherein a first sample is
introduced into the gel in the hollow space at predetermined time
intervals; wherein the gel is pushed forward; wherein a second
sample is introduced into the agarose gel; wherein the gel is
pushed forward again; and wherein a third sample is introduced into
the gel, and so on, until a given quantity n of samples has been
introduced into the gel, wherein distances a are adjusted between
the samples depending on the timing and forward feed speed.
11. A device for introducing a sample into the detection area of an
objective of a microscope arrangement, comprising: a reservoir for
a transparent medium which is initially still liquid; means
designed for: removing a partial amount of the liquid medium and
for introducing a sample into this partial amount; or introducing
samples into the liquid medium inside the reservoir and removing a
partial amount of the medium with a sample contained therein; means
for changing the removed partial amount of medium from the liquid
state to the solid state, wherein at least one sample is fixated
within the partial amount of the medium; and a device for
positioning and aligning the solidified partial amount of medium in
the microscope arrangement in such a way that the sample contained
therein is situated in the detection area of the objective.
12. The device according to claim 11, further comprising: a
manipulating unit which has a hollow needle in which a suction and
delivery piston is movably guided for sucking a partial amount of
the medium into the hollow needle or ejecting it from the hollow
needle.
13. The device according to claim 12; wherein the manipulating unit
is connected to the microscope arrangement by a holder, and the
holder is constructed with means for changing the position and
alignment of a capillary or cannula relative to the microscope
arrangement, wherein there is a change in position in coordinates
X, Y, Z and a rotational movement around the longitudinal direction
of the capillary or cannula by an angle .phi..
14. The device according to claim 11; wherein a capillary,
including the suction and delivery piston, is fastened to the
manipulating unit by means of connection elements which can be
disconnected manually.
15. A device for embedding a plurality of samples in a curable
transparent medium, comprising. an access for the medium, an access
for the samples; forward feed devices for the medium and the
samples; a device for introducing the samples individually into the
medium successively in a timed manner until a given quantity n of
samples has been inserted into the medium; and wherein the medium
is advanced in a timed manner, and distances a are adjusted between
the samples within the medium depending on the timing and forward
feed speed.
16. The device according to claim 12; wherein the hollow needle of
the manipulating unit is in the form of a capillary or a cannula.
Description
[0001] The present application claims priority from PCT Patent
Application No.
[0002] PCT/EP2009/004081 filed on Jun. 6, 2009, which claims
priority from German Patent Application No. DE 10 2008 027 784.3
filed on Jun. 11, 2008, the disclosure of which is incorporated
herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The invention is directed to a method and a device for
positioning and aligning a preferably biological sample in the
detection area of the objective in a microscope arrangement.
[0005] The method according to the invention can be applied
particularly in connection with single plane illumination
microscopy (SPIM). This microscopy method is a special method of
widefield microscopy by which image data for a three-dimensional
image of the sample are obtained on the basis of optical sections
through different planes of the sample.
[0006] 2. Description of Related Art
[0007] SPIM technology is described, for example, in Stelzer et
al., Optics Letter 31, 1477 (2006), Stelzer et al., Science 305,
1007 (2004), DE 102 57 423 A1, and WO 2004/0530558 A1.
[0008] The viewing direction on the sample is changed repeatedly so
that image data are not obtained from just one viewing direction on
the sample. Every time the viewing direction is readjusted, the
sample must be positioned and aligned relative to the detection
objective.
[0009] At present, the positioning and alignment of the sample to
be examined is carried out manually using commercially available
equipment and is therefore extremely time-consuming and is
accordingly only suitable for laboratory analyses of individual
samples.
[0010] However, particularly with regard to high volumes in
industrial applications, there is an increasing demand for
detection of a large number of samples successively in time with
the highest possible throughput per time unit.
SUMMARY OF THE INVENTION
[0011] Therefore, it is the object of the invention to propose a
method and at least one device by which it is possible to position
and align samples in the detection area of a microscope arrangement
with high efficiency. By the word positioning is meant within the
meaning of the invention the arrangement of the sample in the
detection area of the objective, while the word alignment refers to
the respective viewing direction of the objective on the
sample.
[0012] According to the invention, the following method steps are
carried out in a method of the type mentioned above: [0013] the
sample is introduced into a transparent medium which is initially
in liquid state, [0014] the medium is changed from the liquid state
to a solid state so that the sample is fixated inside the medium,
but the medium remains transparent or the transparency changes only
negligibly, [0015] 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.
[0016] In a first preferred embodiment of the invention, a
plurality of samples are introduced into the transparent medium
which is initially still in a liquid state. The medium with the
samples contained therein is stored in a sample reservoir.
[0017] When one of the samples is to be examined, a partial amount
of the transparent medium which is still in liquid state is removed
from the sample reservoir with the sample to be examined. After
being removed, the partial amount of medium is changed from liquid
to solid state, and the sample is fixated within the solidifying
partial amount of medium.
[0018] A partial amount can also be removed with a plurality of
selected samples. After removal, this partial amount of the medium
is changed from the liquid state to the solid state, and the
samples are fixated within the solidified partial amount of
medium.
[0019] The partial amount of medium is removed from the sample
reservoir, for example, in that it is sucked into the hollow space
of a capillary or cannula or, for example, into the hollow space of
a disposable medical syringe.
[0020] By capillary is meant within the meaning of the present
invention a hollow needle with a very small inner diameter and a
suction and delivery piston which is displaceably guided in the
interior; a suction effect is achieved when the suction and
delivery piston is displaced in one direction so that a partial
amount of the liquid medium is removed from the reservoir, while
the medium with the enclosed sample is pushed out of the capillary
when the suction and delivery piston is displaced in the opposite
direction.
[0021] By cannula is meant within the meaning of the invention a
hollow needle with an inner diameter greater than that of the
capillary and, consequently, without capillary action. A
displaceable suction and delivery piston can likewise be provided
in the interior of the cannula for the purpose described above.
[0022] A suction and delivery piston which can be used to suck in
or push out a partial amount of medium is likewise provided in the,
usually cylindrical, hollow space of the disposable syringe.
[0023] In a method step following the fixating of the sample, the
solidified medium is held in the microscope arrangement in such a
way that the sample fixated therein is situated in the detection
area. For this purpose, at least that portion of the solidified
medium in which a sample is located is pushed out of the hollow
space into the detection area. The portion of medium that is not
pushed out remains in the hollow space and is held therein, and the
alignment and position of the sample in the detection area is
influenced in a desired manner by means of specific changes in the
position of the capillary, cannula or disposable syringe.
[0024] If there are a plurality of enclosed samples, the medium is
held in the microscope arrangement in such a way that one of the
samples enclosed therein is initially situated in the detection
area, and after this sample has been examined the next sample
located in the partial amount is positioned and aligned and can be
examined.
[0025] In a second preferred embodiment of the invention, the
transparent medium which is still in the liquid state is stored in
a medium reservoir without samples.
[0026] In contrast to the first embodiment of the invention: [0027]
a partial amount of the medium which does not yet contain a sample
is initially introduced into the hollow space of a capillary, a
cannula or a disposable syringe in a first step, and [0028] one or
more samples are introduced into the medium already located in the
hollow space in a second step.
[0029] The inventive idea includes different ways of carrying out
the method: For example, it is conceivable to carry out both of the
above-mentioned steps directly one after the other in that the
medium in the liquid state is introduced first and, immediately
following this, the sample is introduced into the medium which is
still in the liquid state. This can be carried out with just a
sample by itself or also with a sample which is already embedded in
a smaller partial amount of the medium.
[0030] When a disposable syringe is used, for example, the liquid
medium can be introduced first and then one or more samples can be
introduced into the cylindrical hollow space of the disposable
syringe making use of gravitational force.
[0031] In contrast to this, it can also be provided that: [0032]
the medium introduced in the hollow space is first changed from the
liquid state to the solid state in an intermediate step before
introducing the samples, or [0033] a partial amount of the medium
which is in the solid state is introduced into the hollow space,
and [0034] the medium in the hollow space is not liquefied again
until a later time, [0035] the sample is introduced, and [0036] the
medium is solidified again in order to fixate the sample
therein.
[0037] This way of carrying out the method can be used
advantageously in connection with preparing a plurality of samples
which are already fixated prior to microscopic examination.
[0038] In each of the cases mentioned above, the solidified medium
is pushed out of the hollow space in order to position and align a
sample fixated therein in the detection area of the objective of a
microscope arrangement.
[0039] The change of the medium from the liquid state to the solid
state, or vice versa, is carried out under external influences,
particularly by heating or cooling. The solidification of the
medium can also be carried out under the influence of light.
[0040] Further, an automation of the method steps in their entirety
or an automation of some of the individual method steps lies within
the scope of the invention.
[0041] A curable gel, preferably agarose gel, is used as
medium.
[0042] The invention is further directed to a device for
positioning and aligning a preferably biological sample in the
detection area of an objective of a microscope arrangement
comprising: [0043] a reservoir for a transparent medium which is
initially still liquid, [0044] means designed for [0045] removing a
partial amount of the liquid medium and for introducing a sample in
this partial amount, or [0046] introducing samples into the liquid
medium inside the reservoir and removing a partial amount of the
medium with a sample contained therein, [0047] means for changing
the removed partial amount of medium from the liquid state to the
solid state, at least one sample being fixated within the partial
amount of the medium, and [0048] a device for positioning and
aligning the solidified partial amount of medium in the microscope
arrangement in such a way that a sample contained therein is
situated in the detection area of the objective.
[0049] The device is advantageously outfitted with a manipulating
unit which has a capillary, a cannula or a disposable medical
syringe in which a suction and delivery piston is movably guided.
Small volumes can be sucked in by displacing the piston and by the
vacuum pressure generated in this way. Accordingly, it is possible
to remove a partial amount on this order of magnitude from the
total reservoir of still liquid medium in a precise manner.
[0050] The manipulating unit will be explained more fully referring
to the example of capillaries, although the invention is not
limited to this.
[0051] For example, the manipulating unit can be designed to
receive a plurality of capillaries simultaneously. This proves
advantageous when a set of samples is prepared and distributed to a
plurality of capillaries, which samples are received by the
manipulating unit and exchanged with one another in a simple
manner, so that the samples received therein can be examined
efficiently one after the other in rapid sequence.
[0052] For the purpose of exchanging the capillaries in the sample
space, the manipulating unit can be outfitted with a turret
arrangement receiving the, capillaries. Accordingly, one of the
capillaries is moved into a position in which the suction and
delivery piston is grasped and the portion of the medium with the
enclosed sample is pushed into the detection area by means of the
suction and delivery piston. After being examined, the sample is
pulled back into the capillary by the suction and delivery piston
and is stored therein, the next capillary with the next sample is
moved into position, the suction and delivery piston is grasped,
and this sample is now pushed into the detection area.
[0053] In this connection, it is advantageous when the suction and
delivery piston comes into contact with the medium directly (i.e.,
without an air cushion) so as to facilitate metering of a highly
viscous medium such as a gel in particular. Further, this prevents
unwanted substances being sucked in via the air cushion (e.g., in
the form of aerosols) which is important above all when handling
living biological samples.
[0054] In a particularly advantageous embodiment of the arrangement
according to the invention, the capillary, including the suction
and delivery piston guided therein, is connected to the
manipulating unit by connection elements which can be disconnected
manually. In so doing, the capillary can also be located in a
sleeve which is then connected to the manipulating unit.
[0055] In this way, it is possible to disconnect the capillary,
including the suction and delivery piston guided therein, from the
manipulating unit, to immerse the suction opening of capillary in
the medium, to displace the suction and delivery piston in the
capillary, and to suck a partial amount of medium into the
capillary corresponding to a predetermined volume.
[0056] Depending on one of the modes of carrying out the method
which have already been described, the sucked in medium can already
contain a sample, or a sample is introduced subsequently in the
partial amount of medium located in the capillary.
[0057] When the medium and the sample are located in the capillary,
the medium is changed from the liquid state to the solid state with
the means according to the invention provided for this purpose in
order to fixate the sample within the partial amount of medium as
will be described in more detail below with reference to an
embodiment example.
[0058] The capillary which now contains the solid medium and the
sample enclosed therein is subsequently arranged at the
manipulating unit again by means of the connection elements.
[0059] The manipulating unit, per se, is fastened to the microscope
arrangement (e.g., by means of a straight-line guide) and it is
outfitted with means for changing the position and alignment of the
capillary relative to the microscope arrangement, wherein there is
a change in position of the capillary in coordinates X, Y, Z and a
rotational movement around the longitudinal direction of the
capillary by an angle .phi..
[0060] With the manipulating unit it is possible to move the end
portion of the capillary in which the sample is located into the
vicinity of the detection area initially by means of displacement
in coordinates X and Y and then, using the suction and delivery
piston, to push the solidified medium with the enclosed sample out
of the capillary in direction of coordinate Z until the sample is
positioned in the detection area. By rotating the sample around
angle .phi., the alignment of the sample (i.e., the viewing
direction of the objective on the sample positioned in the
detection area) is varied in the desired manner.
[0061] It lies within the scope of the invention to directly
manually initiate the movements of the capillary by means of
correspondingly constructed gear unit members and also to initiate
these movements by controlling motorized drives to which the
capillary is connected by gear unit members.
BRIEF DESCRIPTION OF THE DRAWINGS
[0062] 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;
[0063] 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;
[0064] 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;
[0065] 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;
[0066] FIG. 5 shows an alternative example for introducing samples
into a capillary shown in FIG. 1;
[0067] FIGS. 6 to 8 show another example for introducing individual
samples into a curable medium; and
[0068] FIG. 9 shows an example for a timed introduction of a
plurality of samples into a curable medium.
DETAILED DESCRIPTION OF EMBODIMENTS
[0069] 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.
[0070] The present invention will now be described in detail on the
basis of exemplary embodiments.
[0071] 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.
[0072] 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.
[0073] 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 .mu.L to 50 .mu.L 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.
[0074] 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.
[0075] 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.
[0076] 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
[0077] 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 .phi.. The directions R1 and R2 extend
parallel to coordinate Z in coordinate system X, Y, Z.
[0078] 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.
[0079] 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).
[0080] 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.
[0081] 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.
[0082] 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 .phi. within a range of 360
degrees, the detection direction relative to the sample 6 can be
changed as needed.
[0083] 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.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] After valve 21 is closed, the valve 20 is opened again, if
required, and liquid agarose gel 5 is again fed through access
17.
[0091] 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.
[0092] 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.
[0093] 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).
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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.
[0099] It is advantageous when the filling station and the
manipulating unit form a functional unit.
[0100] 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.
[0101] The following is carried out at given time intervals: [0102]
a first sample 6.1 is introduced into the agarose gel 5, [0103] the
agarose gel 5 is advanced with the piston 22, [0104] a second
sample 6.2 is introduced into the agarose gel 5, [0105] the agarose
gel 5 is advanced again, [0106] 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.
[0107] 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.
[0108] The samples 6 can subsequently be examined microscopically
as was described above.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] REFERENCE NUMBERS [0114] 1 capillary [0115] 2 suction and
delivery piston [0116] 3 end portion [0117] 4 sample reservoir
[0118] 5 agarose gel [0119] 6 sample [0120] 7 curing station [0121]
8 manipulating unit [0122] 9 receptacle [0123] 10 microscope stand
[0124] 11 actuating element [0125] 12 rotating and straight-line
guide [0126] 13 clamping device [0127] 14 drive element [0128] 15
illumination beam path [0129] 16 filling station [0130] 17, 18
access [0131] 19 straight-line guide [0132] 20, 21 valve [0133] 22
piston [0134] 23 closure [0135] 24 depression [0136] 25 curing
station [0137] 26 cutting device [0138] 27 knife [0139] 28 opening
[0140] R1, R2 directions [0141] .phi. angle [0142] a distance
[0143] A portion [0144] P position
* * * * *