WO2019025205A1

WO2019025205A1 - Capillary cell and use of a capillary cell

Info

Publication number: WO2019025205A1
Application number: PCT/EP2018/069696
Authority: WO
Inventors: Thomas Fritzsche; Ulf-Dietrich Braumann
Original assignee: Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
Priority date: 2017-07-31
Filing date: 2018-07-19
Publication date: 2019-02-07
Also published as: DE102017213138B3; DE102017213138B9

Abstract

The invention relates to a capillary cell (1, 70, 110), in particular for use in an optical microscope, more particularly a light sheet microscope (40), said capillary cell having an outer casing (3, 72, 112) consisting of a material that is light-permeable in the visible spectrum. The casing also has a side wall and a lower cover. A support device (9, 82, 122) is provided inside the casing, said support device comprising inter alia a permeable structure.

Description

Capillary cell and use of a capillary cell

The present application is a capillary cell according to the preamble of claim 1 and a use of a capillary cell in a microscopy method, such as a Lichtblattmikroskopieverfahren or a selective piain Illumination microscopy (SPIM) method.

Modern microscopy methods, especially in the visual field, offer numerous possibilities in biological research. Among other things, long-term observations of biological, especially living, samples are possible in many microscopy methods. In particular, the SPI M methods and light sheet microscopy are methods that enjoy great popularity in the imaging of biological cell processes.

In a light sheet system, the illumination and detection axes are orthogonal to each other. To move the sample to be examined in the intersection of the illumination and detection axis is often resorted to a capillary cell, in which the sample to be examined is introduced. Subsequently, the capillary cell or the sample contained therein can be moved plane-wise along the detection axis so as to see different levels illuminated by the light sheet. An example of such a capillary cell is shown in DE 10 2012 108 158 AI. The capillary cell described therein comprises, in addition to an envelope with a limiting side wall and an upper and a lower lid, a closure and an inflow, by means of which the capillary cell can be flushed through with a solution. The drain is in the first one

Cover and the inflow arranged in the opposite lid. As a result, the sample to be examined, for example, in the solution to be investigated floating. In the capillary cell shown in DE 10 2012 108 158 AI, however, it is disadvantageous that long-term studies are difficult to carry out, since the capillary cell is flowed through permanently.

Document CA 2 684 221 A1 shows a device or a method in which a fluid, in which particles to be examined are located, flows through a capillary. The particles moving with the fluid are measured as they flow through a measuring range. Via porous structures, substances can be added to the fluid inside the capillary, such as, for example, growth-inhibiting substances, poisons or cell-damaging chemicals. The documents DE 10 2014 004 851 AI and US 2016/0 001 285 AI show a vertical reaction vessel in the form of an insert. The insert is adapted to form a receptacle, with at least one top opening located at the top, and a second opening located at the top for pressure equalization and an overflow. The reaction vessel consists of a dimensionally stable base body, wherein the dimensionally stable base body forms at least one non-capillary reaction space as the inner volume with at least one semipermeable membrane as the side wall.

The document EP 0 515 883 A2 shows a device for the safe removal of blood from a storage vessel with an aperture holder, in which a

Aperture exhibiting part is held and to which a capillary is attached. The storage vessel can be closed by a cover part. The lid part has an area through which the capillary can be inserted into the storage vessel when the lid part is closed. The document US 5 126 238 A shows a hollow fiber bioreactor system and a method for cell proliferation. In the context of the system and method, the use of a hollow fiber membrane or a bioreactor comprising a hollow fiber membrane is suggested as well as a circuit connected to a first inflow and a first outflow and having means for introducing fluids into the circulation, the reagents and Contain nutrient-containing basal medium.

It is the object of the present invention to provide a capillary cell which can be used inter alia for the long-term observation of samples.

The object is achieved by a capillary cell with the features of claim 1. Further embodiments can be found in the subordinate claims.

The capillary cell comprises an outer shell of a light-permeable material in the visual spectrum (380 nm-800 nm) or near infrared spectrum (800 nm-1500 nm), preferably in the visual spectrum-transmissive region. Examples of suitable materials are glass, in particular borosilicate glass, quartz glass, optical glass or special optical glass, and plastics such as fluorinated ethylene propylene (FEP), polymethyl methacrylate (PMMA), polystyrene (PS), cellulose acetate butyrate (CBS). The shell comprises at least a side wall and a lower lid. The envelope also has an inflow and an outflow by means of which a nutrient solution can be flushed through the inflow into the envelope and drained off through the outflow.

Furthermore, the capillary cell has a support device for a sample to be examined, which is connected to the shell. The support device divides the sheath into a lower chamber extending between the lower lid and the receptacle and an upper part extending between an upper boundary of the sheath and the support device. The support device is designed such that it comprises a permeable structure. The support device is preferably attached to the side wall and allows no further gap between the side wall and the support device. In this way, for example, in the lower one Chamber filled nutrient solution, which is circulated for example by the outflow and the inflow, only pass through the permeable structure of the support device in the upper part of the capillary cell.

Furthermore, the support device is suitable for carrying biological samples on top, i. the upper part of the Kapillarzelle facing side of the support device to be arranged. In this way, the sample is in a fixed location and can thus be made available for long-term observations. In order to allow sufficient supply of the biological sample with nutrient solution, nutrient solution flushed into the lower chamber can be absorbed by the permeable structure of the sample to be examined.

Depending on the nature of the support device, the sample to be examined can pull the nutrient solution upwards solely due to the acting capillary forces through the permeable structure. An example of such a support device with a permeable structure, for example, a filter, for example, a ceramic filter with a pore size of 2 μιτι. A nutriprimed lower chamber of the capillary cell wets the lower side of the support and the permeable structure, which is so moistened with the nutrient solution and the nutrient solution is sucked through the filter to the top of the support due to osmotic forces. Since the sample to be examined is placed on the upper side of the support device, it absorbs the nutrient solution and is thus either kept alive for long-term observation or ensures the biological functionality of the sample to be examined.

The lower cap of the capillary cell may for example be integrally connected to the side wall. In one embodiment, the capillary cell has an upper lid, which thus closes the upper part of the capillary cell to an upper chamber. Also, this cover may be integrally connected to the side wall. However, in other embodiments, the top lid is detachably connected to the sample. The

Reclosability of the capillary cell through an upper lid has the

Advantage that the sample to be examined, in a simple manner can be stored on the support device. The support device is mechanically stable enough to carry the weight of the sample to be examined. Further embodiments can be found in the subordinate claims.

In one embodiment, the inflow and outflow are directed into the lower chamber so that the lower part of the sheath is a flow chamber. In this embodiment, the entire lower chamber may be flushed with a nutrient solution, such as agarose. As an inflow, for example, openings in the lower lid or openings in the side wall, which limits the lower chamber, are introduced. In order to connect the inflow and outflow with a micropump system, for example, the outflow and the inflow can be equipped with hoses, which enable a simpler connection of the micropump to the inflow and outflow. Such hoses may for example consist of plastics such as polyvinyl chloride (PVC), polyurethanes (PUR), fluorinated ethylene propylene (FEP) or silicone or glass. In another embodiment, the inflow is passed through the lower lid and connected to another capillary. That capillary is inside the capillary cell shell or inside the sidewall and carries the nutrient solution. The capillary preferably protrudes through the support device and extends to the upper limit of the shell or up to an upper lid on which the drain is arranged. In the region of the flow through the support device or through the permeable structure, the further capillary can be interrupted so that a nutrient solution passing through the further capillary can moisten the support device or the permeable structure or the permeable structure can absorb the nutrient solution.

In a further embodiment, the further capillary is formed with a semipermeable membrane, wherein the semipermeable membrane adjoins the lower chamber, so that a fluid exchange between the lower chamber and the further capillary can take place. Particularly preferably, the further capillary also projects into the upper part and also includes here a semipermeable membrane. In other embodiments, however, the semipermeable membrane of the further capillary is located exclusively in the lower chamber or in the lower chamber and in the region of the support device.

In the embodiments described so far, it is possible, on the one hand, to supply the sample to be examined with the lower chamber through which nutrient solution flows and, on the other hand, to prevent the sample to be examined from being suspended on the support device or the upper part of the capillary cell. Some of the samples to be examined, such as hippocampal slices or hippocampal structures of animals, must be substantially unflushed for long-term study. In a further embodiment, the envelope comprises an upper lid which forms the upper boundary (even if this lid were, for example, still surmounted by the side wall). This upper lid may have a gas inlet and a gas outlet system so that the upper chamber bounded by the upper lid and the support means is flushable with a gas. For many studies it is necessary not only to provide the sample with nutrients from a nutrient solution, but also to ensure that the sample to be tested is kept in a particular gas environment. For example, there are many samples to be tested, which can preferably survive in a nitrogen atmosphere. In order to ensure simple survival of the samples under laboratory conditions, it is necessary to flush the upper part of the chamber with a gas. In this case, the gas inlet system may have only a common inlet and outlet or have two separate inlet and outlet openings, which are respectively in the upper lid or in the side wall which defines the upper chamber, are introduced.

In a further embodiment, the permeable structure is a filter, in particular a ceramic filter or a microfilter. It is also possible to design the permeable structure in the form of a semipermeable membrane, for example a proton exchange membrane. A typical pore size can be from Ο, ΐμιτι to ΙΟΟμιτι. The capillary cell presented here can be used in various sizes. For example, the height of the capillary cell can be between 3 and 12 cm. The diameter of a round capillary with a substantially circular side wall may be in the range between 500 μιη and 4 cm, preferably between 2 mm and 15 mm. For other geometries, an interval of the possible cross-sectional area of the capillary cell can be calculated from the above diameters. Although so far only a round configuration of the side wall has been explained, it is also possible to choose, for example, a rectangular, in particular square, or elliptical basic structure for the capillary cell. The choice depends primarily on the size of the samples to be examined and the materials available for the capillary cell.

The volume of the capillary cell may be, for example, 7 mm ³ to 20 cm ³ in the lower chamber. The upper part can hold a similar volume. The volume of the capillary cell, the lower chamber and the upper part depends inter alia on the diameter of the capillary cell. The support device may have a volume of typically 0.1 mm ³ to 5 cm ³ , and / or the support device may have an area of typically 2 mm ² to 5 cm ² .

As already mentioned, the capillary cell presented here is particularly suitable for use in a light sheet microscopy method.

The invention will be explained in more detail below with reference to a few concrete exemplary embodiments. Show it:

Fig. 1A schematic representation of a capillary cell;

Fig. 1B Use of such a capillary cell in a light-sheet microscope;

FIG. 2 shows further embodiments of a capillary cell, which is shown in longitudinal section; FIG. and Fig. 3 further embodiment of a capillary cell, which is also shown in longitudinal section.

FIG. 1A shows a capillary cell 1 with an outer shell 3, which is defined by a side wall 5 and a lower cover 7. Furthermore, a support device 9 is shown within the shell 3, which is designed as a permeable structure. Between the lower cover 7 and the supporting device 9 there is a lower chamber 11, wherein in the side wall 5, which delimits the lower chamber 11, an inflow 13 and an outflow 15 are present to supply the lower chamber 11 with a nutrient solution, For example, from agarose, to flow through and so to create a constant flow. The side wall 5 is formed of a glass or plastic and is substantially round. The diameter of the circular side wall is substantially D = 2.5 cm. The support device 9 is a ceramic filter, which analogous to the diameter D = 2.5 cm also a circular

Has a disc with a diameter of 2.5 cm. The ceramic filter is glued to the side wall.

If a nutrient solution is now flushed into the inflow 13, it initially fills the lower chamber 11 and, in particular, moistens a lower side

17 of the support device 9. Due to the moistening and the prevailing capillary forces, the nutrient solution is sucked through the support device 9 from the lower side 17 to the upper side 19, and thus can be a sample to be examined located on the upper side 19 of the support device 9 provide with the nutrient solution.

At the upper boundary 21 of the side wall 5 is an opening through which the sample to be examined can be placed on the upper side 19 of the support device 9. The capillary cell 1 shown here is suitable, for example, to deposit a rat hippocampus on the surface 19 and to supply it with nutrients by means of a nutrient solution flushed into the lower chamber 11. The hippocampus prepared in this way can be introduced together with the capillary cell 1 into a light-sheet microscope assembly and examined over a period of days or weeks in order to investigate the activity of the hippocampus in a long-term experiment. It will be explained with reference to FIG. 1B how an examination of the sample in the capillary cell can be carried out by means of light-sheet microscopy. FIG. 1B shows the capillary cell 1 with a sample 30 arranged therein, which is arranged on the support device 9. For the sake of simplicity, the inflows and outflows of the lower chamber 11, as shown in Fig. 1A, not shown. The light-sheet microscope 40 has a plurality of parts, only a few of which are explicitly shown. Among other things, a detection objective 42 can be seen and a matrix screen 44, on which the light waves (shown here schematically as reference symbol 46) impinge and, for example, are assembled into images by a computer unit. The sample 30 is transilluminated out of the plane of the drawing with a light sheet 48. This light sheet or the capillary with sample can be pushed back and forth along the direction z, so as to illuminate different levels of the sample 30 to be examined. In this way, different levels can be displayed on the matrix screen 44 and assembled into an overall image or a three-dimensional model of the sample to be examined.

The capillary cell 1 is located within a sample chamber 50, which is intended to prevent the objective 42 or the objective for the irradiation of the

Light sheets 48 are touched by the capillary cell 1 and thus could be damaged. In order to discharge the capillary cell 1 into the sample chamber 50, it can be connected, for example, at its upper end to a holder 60, so that a discharge into the sample chamber 50 is simply possible. If the sample to be examined or the lower chamber of the capillary cell is now flushed with a nutrient medium, the sample to be examined 30 can remain in the experimental setup for several days or even weeks, and images of different levels can be taken continuously.

Based on the Fign. 2 and 3 further embodiments of capillary cells will be explained. 2, a capillary cell 70 is shown in longitudinal section. Similar to the capillary cell 1 shown in FIG. 1A, the capillary cell of FIG. 2 may have a round, oval, quadrangular or rectangular cross-section. The capillary cell has a sheath 72 having a sidewall 74 and a bottom lid 76 integral with the sidewall connected, and an upper lid 78 which is designed to be removable, so that the sample 80 can be placed on the support device 82. The lower chamber 86 disposed between the lid 76 and the lower surface 84 of the support 82 has an inlet 88 and a drain 90. The inlet 88 projects into the lower chamber 86 as a small capillary 92 that does not protrude over the entire length of the lower chamber 86 and supplies it with a nutrient medium needed for the sample 80. This nutrient medium may be carried out through the drain 90, which also includes a small capillary 94. In this way, a flow chamber is created in the lower chamber 86. The support device 82 is at the edge

96 impermeable formed, for example, a PMMA, and provided in its center 98 with a permeable material, such as a semipermeable membrane. Due to the semipermeable membrane 98, the sample 80 to be examined can absorb the nutrient solution in the lower chamber due to suction or capillary action. The upper chamber 100 is connected to a gas purging system 104 via an inlet 102. The gas of the gas purging system 104 is, for example, nitrogen or carbon dioxide or another gas or gas mixture, which requires numerous samples to be examined in order to be permanently functional or suitable for the experiments. Although only a single inlet 102 is shown in FIG. 2, a separate outlet may be present in addition to the inlet. The inlets and outlets shown in FIG. 2 are held in a small capillary shape. This may affect or improve the flow characteristics within the lower and upper chambers, respectively. In order to access the capillary cell 70 for the light sheet microscopy or SPIM process, it is made of a borosilicate glass. The support device with the solid region 96 and the semipermeable membrane 98 is manufactured in advance and subsequently introduced into the capillary cell 70. In this case, it is glued to the side wall 74. Thus, it is only possible via the semi-permeable membrane 98 to exchange media between the upper and lower chambers.

2 shows a variant of the capillary cell 70 shown in FIG. 2. The capillary cell 110 has a shell 112 with a side wall 114 and a bottom cover 116 and an upper cover 118. The upper cover 118 is designed to be detachable again, see FIG that a sample to be examined 120 can be stored on the support device 122. The support device 122 shown here is for example a filter. The jig 122 and the lower lid 116 define a lower chamber 124. The upper lid 118 and the support surface 122 define an upper cell 126. The capillary cell 110 also has a rinsing system for supplying the sample 120 to be examined with a nutrient solution. Unlike in the examples shown above, however, the nutrient solution is passed through a capillary 128, whose inflow 130 is arranged on the lower lid 116 and whose outflow 132 is on the upper lid 118. The further capillary 128 thus passes over the entire height of the capillary cell. In this case, the further capillary 128 is also guided by the bearing surface 122. If a nutrient solution is now flushed into the further capillary 128 by the inflow 130, the nutrient solution can enter the lower chamber 124, since a delimiting wall 134 of the further capillary 128 is a semi-permeable membrane through which the nutrient solution passes, and thus gradually can fill lower chamber. Alternatively, the semi-permeable membrane is located exclusively in the area of the support 122, ie, only along the small portion 136. The further capillary 128 has solid, ie impermeable, walls in the upper chamber 126 which do not allow any nutrient fluid to escape into the upper chamber 126. As already shown in FIG. 2, the top cover 118 also contains a gas inlet 102, which can be connected to a gas flow system 104. In this way, the sample 120 can also be rinsed with the gas.

Claims

claims

A capillary cell (1; 70; 110), in particular for use in an optical microscope, in particular in a light-sheet microscope (40), wherein the capillary cell (1; 70; 110) has an outer sheath (3; 72; visual and / or near infrared spectrum translucent material; the shell (3; 72; 112) has a side wall (5; 74; 114) and a bottom cover (7; 76; 116); and the shell (3; 72; 112) has an inlet (13; 88; 130) and an outlet (15; 90; 132),

characterized,

in that a support device (9; 82,122) for a sample (30; 80) to be examined is connected to the cover (3; 72; 112), wherein the support device (9; 82,122) supports the cover (3; 72; 112) into a lower chamber (11; 86; 124) extending between the lower lid and the support means (9; 82,122) and an upper one between an upper limit (21) of the sheath (3; 72; 112 ) and the support device (9; 82, 122) extending part (23, 126) compartmentalized; and the support means (9; 82, 122) comprises a permeable structure (9; 98; 122).

The capillary cell (1; 70) of claim 1, wherein the inflow (13; 88) and the outflow (15; 90) are directed into the lower chamber (11; 86) such that the lower chamber (11; 86) the sheath (3; 72) is a flow chamber.

The capillary cell (110) of claim 1, wherein the inflow (130) is passed through the lower lid (116) and connected to a further capillary (128) leading through the support (122) and the drain (132) is connected to a , the inflow (130) opposite end of the further capillary (128) is connected.

4. The capillary cell (110) according to claim 3, wherein the further capillary (128) has a semipermeab- at least adjacent to the lower chamber (124). le membrane (134), wherein the semi-permeable membrane (134) is preferably also arranged in the upper part (126).

A capillary cell (1; 70; 110) as claimed in any one of the preceding claims, wherein the upper boundary (21) of the sheath (3; 72; 112) comprises an upper lid (78; 118) covering the upper portion (23,126 ) as the upper chamber (23, 126) and a gas inlet system (102) in the upper chamber (23, 126) is guided, so that the sample to be examined (30; 80) is flushable with gas.

A capillary cell (1; 70; 110) according to any one of the preceding claims, wherein the transmissive structure (9, 98, 122) is or comprises a filter or semi-permeable membrane (98).

The capillary cell (1; 70; 110) of claim 6, wherein the filter is such that a liquid can rise due to capillary forces.

A capillary cell (1; 70; 110) according to any one of the preceding claims, wherein the translucent material is a glass or a plastic.

9. The capillary cell (1; 70; 110) according to any one of the preceding claims, wherein the lower chamber (11; 86; 124) has a volume of 7 mm ³ to 20 cm ³ , and / or the support device (9; 82, 122 ) has a volume of 0.1 mm ³ to 5 cm ³ , and / or the support device (9; 82, 122) has an area of 2 mm ² to 5 cm ² .

10. Use of a capillary cell (1; 70; 110) according to one of the preceding claims in a light-beam microscope.