DE102005061811A1 - Sample chamber for admission of liquid or liquid-containing biological samples, comprises two chamber walls spaced from each other, a sample area, an observation area, media connection connected to the sample area, and tempering equipment - Google Patents

Abstract

The sample chamber for admission of liquid or liquid-containing biological samples, comprises two chamber walls spaced from each other, a sample area limited by the chamber walls, an observation area (30), a media connection (13, 14) connected to the sample area and arranged for the introduction of a liquid medium in the area, and a tempering equipment. In the sample area, a sample is accessible in the observation area for analysis. One of the chamber walls is formed in the observation area from optically transparent material. The sample chamber for admission of liquid or liquid-containing biological samples, comprises two chamber walls spaced from each other, a sample area limited by the chamber walls, an observation area (30), a media connection (13, 14) connected to the sample area and arranged for the introduction of a liquid medium in the area, and a tempering equipment. In the sample area, a sample is accessible in the observation area for analysis. One of the chamber walls is formed in the observation area from optically transparent material. Capillary force gradients are formed between the chamber walls and the media connection. One of the capillary force gradients is formed between the media connection and a liquid tube and is applied to the observation area of a capillary force vector. A maximum capillary force is pressed in the observation area between the chamber walls of the fluid medium. A capillary force gradient is formed along the liquid tube by a surface that increases hydrophilic characteristics and/or decreases hydrophobic characteristics. The capillary force gradient is formed along the liquid tube through spacer gradients, which distinguish a reduction of the light width of the sample area between the media connection and the observation area. One of the chamber walls is formed along the spacer gradients by high liquid-attracting surface and/or low liquid-repulsing surface. The media connection is formed adjacently to the spacer gradient and to the observation area. A capillary force gradient is adjusted spatially and/or temporally changeable. One of the chamber walls is formed in the observation area from glass or plastic and/or carries a scaling. The chamber wall possess a thickness of less than 200 mu m. One the chamber walls possesses a maximum wettability at the observation area. The media connection is arranged relative to the observation area on different sides of the sample chamber. The liquid tube is formed between the observation area and each medium connection. The sample chamber exhibits a gas connection (15) for gas supply or gas removal and/or for pressure balance. Spacers are arranged between the chamber walls. The liquid tube is limited on single-piece of an inner wall and extends between the chamber walls. The spacers are formed through the inner wall, which is porous or partially permeable. The inner wall exhibits a liquid attractive and/or liquid repulsive surface. The width and/or form of the liquid tube changes between the media connections and the observation area. A gel surface is intended for admission of gel in the sample area. The liquid tube exhibits the maximum capillary force in the gel surface and a part of the fluid tube flows through the gel surface, which is limited on single-piece of the inner wall. The inner wall size is equal to the size of a standard-cover glass for the microscopy. The chamber wall possesses different large surfaces, where the larger chamber wall completely covers the smaller chamber wall and projects on an edge with contact area. The media connection is arranged at one of the chamber walls and flows to the contact area. An elastic material covering device (20) is intended and is laterally locked with the sample area. The covering device exhibits a stair profile (21), which is laterally laid on the chamber wall. A plastic transparent chamber body exhibits a holder for the chamber wall and is formed as single-piece. The chamber body exhibits a continuous opening in which the stair profile is formed at the internal edge of the opening. The opening of the chamber body exhibits a one-sided conical enlargement for the approximation of an optical measuring instrument at the sample area. The chamber body contains a liquid- and gas media line, which flows into the sample area at the media connection. The media connection is arranged in a wall of the chamber body and exhibits a funnel-shaped recess for forming spacer gradients. The media lines are arranged coaxially in lockable manner and exhibit an elastic material locking device. The sample chamber is equipped with an injection device, which is importable in the sample area and is equipped with an electrode mechanism. The injection device exhibits a hollow injection channel, which exhibits a point at a free end and a lateral outlet opening. The electrode mechanism comprises electrodes, which are arranged on one of the chamber walls and projects through openings in one of the chamber walls in the sample area. The chamber body is formed as multi-way chamber body and is made of plastic, titanium, glass or stainless steel through autoclaving or UV-irradiation. Independent claims are included for: (1) an injection channel; and (2) a method for admission of liquid medium in a sample chamber.

Description

The The invention relates to a chamber for receiving liquid or liquid-containing Samples, for example of samples containing biological materials in dissolved or suspended form. The invention particularly relates a sample chamber in which biological samples are kept alive or cultivated and be subjected to an investigation. The invention relates also methods for handling samples, in particular methods for Cultivation of biological samples in a sample chamber.

The investigation of biological materials, such. B. biological cells or microorganisms is often under the requirement to leave the study conditions during the study or during a longer observation period unchanged, so that, for example, cells remain alive. For this purpose, a sample must be placed in a chamber in which on the one hand the desired examination, such. As a microscopic image or a fluorescence measurement can be made and on the other hand ensures the adjustability of the desired living conditions. These include, for example, a constant inflow of a supply medium into the chamber, a removal of cellular metabolites from the chamber and a mechanical protection of the cells. In addition to liquid media cells must also be supplied with substances that are only gaseous or vaporous under normal conditions, such. B. CO ₂ or O ₂ . A common problem with conventional sample chambers is that the adjustment of the living conditions can be disruptive to the requirements of the particular examination desired (and vice versa).

For example it is at high-resolution, far-field microscopic Investigations of importance that a sample chamber along the optical axis has a well-defined, small overall thickness, to make sure the entire aperture angle of the microscope (Lenses and / or condenser) can be exploited. In The rule is a total thickness of less than 2 mm or high-resolution techniques even less than 350 microns needed. For typical cover glass thicknesses of approx. 150 μm remains for the sample space A chamber only has a free space of approx. 50 μm. In the Practice has shown that it is extremely difficult in such a narrow sample space biological cells for longer examination periods z. B. to get alive in the range of hours and with nutrients and gases. If no media exchange occurs, contact on the cells already within 30 to 60 minutes morphological changes on, which can falsify the examination result.

at the conventional one Techniques, in particular, have the following problems. If biological Cells with a surrounding medium simply between a carrier glass and a cover glass, which has a laterally open chamber will form between the glasses a narrow, slit-shaped Sample space formed with a good optical quality (see, for example, A. R. Hibbs in "Confocal Microscopy for Biologist "Edn. 3, BIOCON, Ringwood East, Australia, 1999). However, they are disadvantageous the low mechanical stability the chamber, the risk of cells being damaged by the coverslip, and the only limited possibility for the exchange of liquid media. The media exchange is a stress factor for the cells, it is because of the mechanical instability the chamber complicated and associated with a risk of contamination. A gas exchange can only rich by diffusion into the outer Randbe done the sample space. The use of a concave carrier glass (so-called Harrison-Maximov chamber) is additionally disadvantageous because high-resolution optical Investigations can only be carried out on one side.

From R.G. Harrison ("Anat. Reg. ", Vol. 1, 1907, Page 116) and A. A. Maximov ("Contribs. Embriol. Carnegie Inst. Publ. ", Bd. 16, 1925, page 47) are chambers with a nutrient supply by a mechanically driven flow described. The flow of the medium is disadvantageous because the driven flow mechanically influence the cells and if necessary remove substances, with which the cells condition and shape their environment. The Microscopic imaging may be due to a movement of the cells in the flow and be disturbed by vibrations of the chamber. Another disadvantage is the complex structure of conventional flow chambers and the limited Supply of cells with substances from the gas phase.

As a general rule there is an interest in a chamber, in particular biological Samples as carriers for the high-resolution Far-field microscopy is useful and in a cell culture without a mechanically driven media flow is possible as well as a sterile Gas and / or media exchange z. B. to maintain the cultivation conditions or to carry out ensures a drug treatment are.

The These problems occur not only in the investigation of biological Samples on, but also in the handling of other liquid or liquid-containing Samples in which a gentle intake in a closed Chamber in the presence of a gaseous Medium desired such as in the study of interfacial reactions.

The The object of the invention is to provide an improved chamber for receiving of samples to overcome the disadvantages of the conventional techniques and in particular an extended scope and has a simplified structure. The object of the invention In particular, it is to provide a chamber in which defined Conditions gentle liquid and optionally gaseous Media supplied and / or dissipated can be. The chamber should also for the investigation of small sample volumes be suitable. The task It is also an object of the invention to provide improved methods for receiving and To provide analysis of samples in sample chambers.

These Task is performed by a sample chamber and a procedure with the characteristics the claims 1 or 50 solved. Advantageous embodiments and applications of the invention will be apparent from the dependent claims.

Based device the invention is based on the general technical teaching, in a sample chamber for receiving a sample in a sample chamber to provide at least one capillary force gradient. The sample chamber has two spaced apart, at least in Partial areas parallel chamber walls on, between which the sample space is formed as a column-shaped free space. The formation of the capillary force gradient means that an inner Surface of the Sample space such as a surface of a chamber wall at least in a subarea z. B. materially or geometrically modified is, so that according to the gradient of the gradient on a surface touching liquid Medium a changing Attraction exercised becomes. In general, the capillary force gradient can be characterized by a sudden or increase stepwise increasing capillary force. Prefers however, is a steady and continuous course of change the capillary force, possibly with locally different gradients.

Of the Capillary force gradient is between at least one media port, which flows into the sample chamber, and at least one observation area formed in which a sample an optical examination accessible is. According to the extent of the observation area is between the chamber walls on a liquid Medium exerted a maximum capillary force. For a liquid medium is the wetting highest in the observation area.

The Formation of the at least one capillary force gradient means that a potential of capillary force is created. By the orientation the capillary force gradient has this capillary force potential outside the observation area, in particular at the lateral edge of the sample chamber Maximum and in the observation area a minimum. The geometric Area in the sample chamber, where a liquid medium along the Kapillarkraftgradienten arranges, is here also as liquid line or fluid channel designated. Advantageously, the liquid in the sample chamber along the at least one liquid line localized by the capillary force so as to allow the liquid line only through the chamber walls is limited. A liquid medium introduced into the sample chamber moves along the liquid line automatically to the observation area.

The inventive sample chamber has the following additional advantages. First, by the to the observation area increasing capillary force on at the media port provided liquid Medium drawn into the observation area, without passing through the sample chamber externally driven flow must be built. The loading of the sample chamber is self-organizing. Second, touched the liquid medium at least in the observation area both chamber walls simultaneously. Thereby can for optical Investigations along the optical axis within the chamber Refractive index differences can be avoided or minimized. thirdly It is particularly beneficial that it is for the investigation for example with a microscope, if the sample is only in a small Chamber area is present, but there with a high density. In the cultivation of biological cells on coverslips must a culture not on the entire free surface of the coverslip, but only be formed in the observation area. This allows itself available at low numbers Cells, which grow up adherently with a sufficiently high density in the observation area to allow for mutual conditioning of adjacent cells to reach.

Of the Capillary force gradient also gives benefits on the output side. If over the Media connection is sucked with a vacuum force liquid comes it leads to a liquid tear, as soon as the capillary force is greater than the vacuum force is. This will cause unintentional dehydration the sample space in the observation area avoided.

Finally, there the liquid through the interaction with the inner surface of the sample space at this is held, restrictions conventional Overcome sample chambers to a horizontal operating position. According to the invention, the Chamber with vertically or inclined relative to the horizontal inclined chamber walls perpendicular to a horizontal optical axis of a Microscopes are arranged and used.

The inventive sample chamber advantageously forms a flow chamber, especially for microscopic Investigations of multiphase systems containing at least two gaseous, liquid, gesförmigen and solid phases. Applications arise in particular in physics in the study of diffusion, evaporation, Condensation and absorption processes, in chemistry during investigations of surfaces and interfacial processes, z. B. to artificial Membranes, and in biology in the study of z. B. living (cultured) cells, cell groups or cell components.

The Sample chamber forms a two-dimensional capillary, partially along the at least one liquid line with a liquid Medium filled can be on both sides of the chamber walls and otherwise at least is bounded on one side by a gas or another liquid medium, which preferably makes direct contact with the (first) liquid medium exhibit.

At least one of the chamber walls the sample chamber according to the invention is at least equal to the extent of the observation area made of an optically transparent material. advantageously, may be a sample in the observation area of at least one of the chamber walls ago an optical examination, in particular with light having wavelengths in particular from the UV over the VIS to the IR area be subjected. Preferably, both chamber walls have the Transparency, so that bilaterally investigations are feasible. Particularly preferred is the formation of the chamber walls at least in the observation area made of glass or plastic, since these materials are mostly biological Samples are inert and available with optical quality. It will be glass or Plastic with optical quality verwen det, which the utilization of the complete aperture angle of at least one lens and / or the condenser of the microscope and minimizing or avoiding aberrations in the optical Make sure picture. If the transparent chamber wall is a scaling, such as B. bears a coordinate system, advantages for the evaluation of a microscope image. If the scaling is in the observation area, so can Dimensions derived directly from samples or sample parts become. The scaling can in particular be linear (for example with a series of parallel scale lines) or areal (for Example with a square grid structure) be formed.

Especially preferred are chamber walls with a thickness smaller than 200 μm (For example, 140 microns up to 180 μm) used, since with it an extremely small total thickness of the sample chamber along the optical axis of an optical inspection device can be achieved. The total thickness of the chamber along the optical Axis can be less than 5 mm, in particular less than 2 mm (thickness a conventional one Slide) be. When using coverslips for microscopy, the Total thickness even less than 400 microns, especially less than 300 microns.

Of the Capillary force gradient will vary depending on the desired Application of the sample chamber and in particular depending from the liquid used Medium formed. The person skilled in the art can use the material of the inner surface of the sample space based on his knowledge of the wetting properties of the used medium.

If according to a first embodiment of the Invention of capillary force gradient by chemical composition at least one inner surface the sample space, in particular at least one of the chamber walls formed is, both can chamber walls inside level, parallel surfaces have. The provision of at least on the inside levels surfaces the chamber walls gives advantages for the optical examination of a sample in the observation area and for the Setting a defined chamber volume. One or both chamber walls may be along the liquid line a distance from the media connection to the observation area a increasingly fluid-attracting or decreasing liquid repellent surface (or Surface coating) exhibit. For this purpose, for example, a liquid-attracting, on the chamber wall, the adhesion effect reinforcing the wall material Substance with a surface density increasing towards the observation area arranged. According to the second alternative, a liquid repellent, the adhesion effect decreasing substance may be arranged with decreasing density. The least a chamber wall can also be used both to form the capillary gradient with liquid attracting as well as liquid repellent substances with opposing ones Flächendichtegradienten be equipped.

If, according to a preferred variant of the invention, the surface increasingly attracting or decreasing liquid to the observation area is respectively formed correspondingly by an increasingly hydrophilic or decreasing hydrophobic surface, particular advantages arise for the absorption of water-based media, such as e.g. As aqueous dissolved or suspended samples. Another advantage of this variant is that substances for hydrophilic or hydrophobic equipment of solid surfaces known per se and in the design the sample chamber according to the invention can be used without further ado.

According to one alternative embodiment of the Invention, the Kapillarkraftgradient by a distance gradient formed between in the sample space. Along at least one the fluid line forming part of the area between the media connection and the observation area decreases the vertical distance between the inner surfaces of the Sample space, especially between the chamber walls. At least one of the chamber walls has an increasing thickness towards the observation area, so that corresponding to the observation area, the clear width of the Reduced sample space and increases the capillary action. The distance gradient advantageously causes a provided at the media connection liquid is pulled under the action of capillary force in the sample space. Of the Part of the sample space, in which the mutual distance of the inner surfaces is minimal and therefore maximum capillary forces on the liquid medium act, forms the observation area. Advantageously, too in the formation of the capillary force gradient by a distance gradient the inner surfaces the chamber walls in the observation area plane and parallel, so that optical Investigations are simplified.

The both embodiments of a material and geometric capillary force gradient can according to a further modification of the invention can be combined. First Is it possible, that along the distance gradient one to the observation area increasingly adhesive acting, z. B. hydrophilic and / or decreasing adhesion-reducing acting, z. B. hydrophobic surface is formed. advantageously, Thus, the increasing capillary action is reinforced. Secondly For example, the capillary force gradient may be along a first leg between the media port and the observation area by the distance gradient and along another leg through the increasingly liquid attracting and / or decreasing liquid-repellent surface become. Preferably, the first leg is the distance gradient provided adjacent to the media port while the second leg is formed adjacent to the observation area.

According to the invention can be provided be that the capillary force potential in the sample chamber spatially variable is what benefits for one flexible adaptation of the sample chamber to a specific application. The Kapillarkraftgradient can be adjusted with a predetermined course by, for example, said distance gradient and / or the different liquid repellent or liquid-attracting surfaces are changed. The change can For example, mechanically by an external force, electrical, magnetically, piezoelectrically, thermally and / or chemically effected become. Alternatively or in addition For example, the capillary force potential in the sample chamber may vary with time.

According to one particularly preferred embodiment In the invention, the capillary force gradient is not just along one Distance between the at least one media connection and the observation area formed, but also in the rest Surroundings of the observation area. In this design takes the on a liquid Medium between the chamber walls acting capillary force in the entire sample space respectively to the observation area towards. The observation area forms an at least local maximum wettability. This embodiment The invention has the advantage that in the sample chamber liquid-free Form parts of the sample space that are filled with a gas or vapor.

advantageously, the liquid present in the sample chamber is moved along the liquid line in contact with a surface area with the heightened Capillary force arranged. When the liquid volume is less than is the volume of the sample space, there is a collection of the liquid in the observation area, characterized by the maximum capillary force distinguished. The fluid line serves for contacting through the liquid medium, for example for its Collection or guide through the sample chamber. For Investigations on biological samples can be made of the liquid line the temporary fixation of the samples, z. B. serve as a cell culture surface. In adjacent surface areas (in the following also "open spaces" called) with the lower capillary force, on the other hand, become sample liquids or liquid Sample components less strongly attracted or even repelled. On the areas There is no or almost no liquid medium. The rehearsal room is about the areas appropriately filled with a gas or vapor (gas phase).

In the chamber, liquid and gaseous media can be arranged in a predetermined geometry corresponding to the distribution of the at least one liquid line and the open spaces, so that z. B. biological cells in a liquid sample easily and effectively under constant conditions with gases, eg. B. CO ₂ or O ₂ can be supplied. Within the closed sample chamber, an interface is formed at least between the liquid phase and the gas phase, through which substances can diffuse from the gas phase into the liquid sample. The defined Ge The condition of the liquid line ensures that the diffusion into all parts of the sample can take place under desired conditions in accordance with a prescribable protocol.

By the setting of predetermined geometric conditions, in particular the size of the liquid line can the relationship the interface relative to the volume of the fluid line Sample for optimal supply of the sample, especially the biological Cells in the sample are optimized. The fluid line can in particular in strips be formed with such a small width that substances from the gas phase from both sides of adjacent open spaces through Diffusion can be transported in the entire sample.

Generally the media exchange in the sample chamber according to the invention via a individual media connection. Preferably, however, at least two media connections provided, of which the first media connection for media supply and the second media port can be used for media output. The capillary force potential is at every media outlet and in its immediate environment lower than in the vicinity of the liquid line (and especially at the lateral edge of the sample chamber), but higher than in the observation area. Particularly preferred are the media connections on arranged relative to each other opposite sides of the sample chamber, wherein from the first media port, a first gradient with increasing Capillary force towards the observation area and a second gradient with decreasing capillary force formed towards the second media port are. Advantageously, the sample chamber in this design be used as a flow chamber.

A another embodiment The invention is characterized in that in the sample chamber a gas connection is provided, through which a gaseous or vaporous medium in the sample room or removed can be and is possibly provided for pressure equalization. Of the Gas connection can be one of the mentioned media connections or an additional one Be connection. The provision of the gas connection advantageously allows the targeted composition of the substances in the multiphase system in the sample chamber.

If between the chamber walls the sample chamber spacers are arranged, is advantageously the adjustment of the vertical distance of the chamber walls simplified. The distance can depend in particular on the concrete Conditions, such as B. the type of liquid sample to be examined chosen become. If the spacers by known spacers or be formed strips, there are advantages for a simplified construction of the sample chamber.

According to one Another preferred feature of the invention, the chamber walls are different size surfaces on, wherein they are arranged in the chamber so that the larger chamber wall the smaller chamber wall on at least one, preferably two, opposite each other Overhanging edges. This makes possible, that the chamber walls in the assembled chamber on different support elements rest. Advantageously, can those of conventional Techniques known spacer (spacer) can be avoided. advantageously, are thus in the adjustment of the distance between the chamber walls, z. B. between the coverslips Overcome limitations, so far due to the availability and stability passed by spacers. Furthermore, the available surface of the chamber walls optimally utilized. The chamber can have an extremely low overall thickness own and in particular thinner as a standard carrier glass for the Be microscopy. Particularly preferred is a design in which the larger chamber wall the complete chamber wall completely Covered along the entire edge. For example, as chamber walls, two rectangular or round, for example, circular coverslips used with different sizes.

A subarea the larger chamber wall, the in the composite chamber, the smaller chamber wall protrudes, is called a contact area. The contact area is in direct connection with the capillary force gradient. advantageously, can thereby a provided in the contact area liquid pulled by the increasing capillary force to the observation area become. According to the extent of the contact area of the Sample chamber of the sample chamber according to the invention a bigger light Possess width as between the chamber walls. In the contact area can thus a larger amount of media be kept as a reservoir from which the medium in the observation area is pulled. Furthermore, limitations become more conventional Chambers overcome, whose clear width is greater than the diameter of an injection cannula must be. Even with a gap width the sample space of the sample chamber according to the invention of less as 100 μm can injection needles or Liquids for hydration a larger diameter exhibit.

If the at least one media connection is in direct connection with the contact area and z. B. adjacent to the contact area or in the contact area opposite upper surface is arranged, advantages may result from the transfer of supplied liquid medium directly from the media connection on the force gradient to the observation area.

As a general rule is between the chamber walls of the Sample space as a layered Capillary space given in the unladen state of the sample chamber is empty. According to one modified embodiment However, the invention may have at least one inner wall in the sample space be provided, which is substantially perpendicular to the chamber walls between this extends. The inner wall forms a predetermined limit along the fluid line, in the case of this embodiment a liquid Medium not only by the capillary force, but also by the Interior wall is located. Preferably, two inner walls are provided, which extend on the two sides of the liquid line. The interior walls can Advantageously, in addition to the Kompartimentierungsfunktion spacers form, with which the vertical distance between the chamber walls adjusted becomes. For example Interface tests can it according to a another embodiment The invention may be advantageous if the inner walls are porous or semi-permeable. This means that from the liquid line fro the liquid Medium not through the inner walls can pass through, but these are permeable to substances that in the liquid Medium solved or in the gas phase of the rest Sample chamber are included.

Special Benefits for the effectiveness of fluid management in the sample chamber can become result, if the inner wall on its pointing to the liquid line Page a liquid-attracting or liquid repellent surface. Furthermore, the surface can be the inner wall a Kapillarkraftgradienten analogous to the above Have capillary force gradient, so that the effect of Kapillarkraftpotenzials the liquid line reinforced becomes.

One important advantage of the sample chamber according to the invention consists in the high variability the geometric properties of the capillary force potential. advantageously, can the fluid line through the sample chamber is changing along its longitudinal extent Width and / or shape on wise. The liquid line can, for example straight or curved, in particular circular arc or S-shaped or meandering curved.

Of Furthermore, the field of application of the sample chamber according to the invention be extended when in the sample room, especially in the observation area a gel area is provided, which serves to receive a gel-like substance. For many Applications, it is advantageous if in the gel area a three-dimensional Gel based on water, such as. As an agar or collagen gel is arranged. For the investigation of the interaction of a sample in a liquid medium, such as z. B. of biological cells with the gel, it is advantageous if the capillary force potential in the gel area is at least a minimum or if at least a part of the liquid line is the gel area crosses.

According to others Variants of the invention may be the gel area of the remaining fluid line separated by an inner wall, as described above. The inner wall consists in particular of chemical or biochemical Applications of a porous or partially permeable wall material or a partially permeable one Membrane. Furthermore, the sample chamber with a gel connection be equipped, through which the gel material is filled in the gel area.

According to one advantageous variant of the invention, the chamber is equipped with a covering device, with the sample space between the chamber walls laterally closed is. With the cover, the gap-shaped sample space is on all sides closed in the directions parallel to the planes of the chamber walls. Advantages of the lateral covering device are the provision a closed compartment, in which, in addition to the liquid filled area gaseous or vaporous Additives can be introduced are.

The Covering device is preferably formed as a step profile, on the the chamber walls in different heights with at least two opposite edges, preferably however, along the whole edges rest and are fixable. The staircase-shaped steps of the Step profiles form carrier elements for the Arrangement of the chamber walls with the desired Distance. The step heights can unlike traditional ones Spacers with high precision and stability are formed, even if minimum distances of the chamber walls in the sub-mm range should be set. If the step profile with a variety of steps, if necessary with different step heights, can become Benefits for the setting of sample rooms with different clear widths result.

Particular advantages for a precise and simple alignment of the chamber walls arise when the chamber according to the invention is equipped with a chamber body on which the chamber walls are positioned relative to each other in parallel with the desired mutual distance. Advantageously, the chamber body forms a stable and accurate support for the chamber walls. If the chamber body is integrally formed, the structure of the sample chamber according to the invention is advantageously simplified. Furthermore, the compactness (flat design) and stability of the chamber can be improved. If the chamber body is made of a translucent material, there are particular advantages in the supply or removal of liquid media in the sample chamber. In particular, it can be observed whether a liquid medium is bubble-free.

advantageously, There are no limitations with respect to the shape of the invention in the invention Chamber body, the z. B. a flat cylinder (with a circular or elliptical base) or form a flat cuboid (with a rectangular base) can. The external dimensions of the chamber body (vertical to the optical axis of a microscope for observation of the observation area), which is essentially the outer dimensions of the Floor space of the chamber body are preferably equal to or nearly equal to the size used by default coverslips for the Microscopy chosen. Typical dimensions are z. B. in a circular Floor space 12 mm or 25 mm or, in the case of a rectangular base, 22 mm x 22 mm, 24 mm · 24 mm or 24 mm x 50 mm. Particularly preferably, the dimensioning is the same or almost the same the size of one Standard carrier glass for the Microscopy (76 mm x 26 mm x 1.5 mm).

The Inventive chamber can advantageously be made from a few parts easily as an easy to handle Composition compatible with conventional examination techniques is and, for example, fits in standard microscope tables. The Chamber can be used as a closed chamber, as a flow chamber or as open chamber can be used.

According to one preferred embodiment of Invention has the chamber body a continuous opening, at the inner edge of the chamber walls are fixed. At this Design forms the chamber body a frame with the function of said cover device, wherein the step profile is at the inner edge of, for example, rectangular or round opening of the chamber body located. The step profile forms a first edition for the smaller one Chamber wall with a circumferential step according to the thickness of Chamber wall and the desired clear height the sample space, wherein above the umlau fenden stage at least one additional edition for the second chamber wall is formed. Advantageously, the chamber walls aligned by placing in the step profile in the chamber body and on the chamber body glued to the step profile. Another squeezing the coverslips as with conventional techniques can be avoided. Alternatively, the step profile in a chamber body without the through opening be formed.

According to one Further modification of the invention can at least on one side the inner opening of the chamber body a conical widening provided to the surface of the chamber body be. With this expansion, there is advantageously room for the approximation of a optical measuring device, z. B. a lens of a microscope and its movement relative to the sample surface created in the chamber.

advantageously, can in the chamber body at least one media line be integrated, through which a connection between the sample room and the outside environment, z. B. an outer media reservoir is formed. The at least one media line leads from an opening in the surface of the chamber body, the one outer connection makes, inside, to the rehearsal room. The at least one media line allows advantageously an increased Operational safety and an extended scope of the chamber according to the invention. About the Media line can in particular cells introduced into the chamber for cultivation or liquid or gaseous Media are directed into or derived from the sample space. The chamber can in particular with at least two media lines be used as a flow chamber.

For a media feed into the sample space according to the invention at least one liquid media line, the for the supply of liquid Media is designed and at least one gas media line for the exchange of substances provided in the gas phase. The liquid media line opens between the chamber walls preferably in an inner, in the operating position of the chamber for Example horizontally oriented wall of the chamber body out to the rehearsal room. It is preferably a funnel-shaped depression in the wall of the chamber body provided, from the liquid Medium on the sample surface between the chamber walls flow can.

According to the invention, a the media lines run in another media line, wherein in particular a coaxial construction is preferred. In this design can benefits for give the compactness of the sample chamber according to the invention.

In general, the chamber according to the invention can be permanently connected via the media line with external reservoirs for liquid or gaseous media during the investigation. Advantages for setting the conditions in the sample room or for the realization of certain treatment protocols may result if the compound is temporarily disconnected. For this purpose, the at least one media line or the chamber body is equipped with a locking device with which the media line can be opened or closed.

When Locking device can generally known per se valves or shut-off valves may be provided. For one compact design of the chamber and easy connection of the media line to external reservoirs However, it is preferred if the locking device by an elastic Material is formed, through which the media line in a subarea is interrupted. The elastic material allows as with a Septum on the one hand a safe and permanent closure of the Samples room to the outside and on the other hand a penetration with an injection device, such as B. an injection cannula. When the injection device is removed from the locking device again is because of the elasticity the material used, for. B. of elastic silicone, the closure the sample space given again. Alternatively, the media line consist in a partial area of the elastic material. In this Fall will be a closure or a release of the locking device by a deformation of the elastic material z. B. with a leading to the surface of the chamber body Stamp realized.

The Injection device of the chamber according to the invention preferably contains at least one injection cannula, in the media line in the chamber body through the locking device is insertable. When the injection cannula at a free end a sharpened Tip and at a distance from this has a lateral outlet opening, can Advantageously, injuries to the elastic material of the locking device be avoided.

The Hypodermic needle, which at one end for connection to a liquid conveyor, such. B. a syringe plunger is set up and spaced from the opposite, free end has a radially laterally opening outlet opening, provides an independent one Subject of the invention. With this injection cannula can advantageously elastomeric closures, such as B. a septum or the barrier device of the sample chamber according to the invention repeatedly punctured without injury. The injection cannula can z. B. from an inert metal, for. As steel or a plastic. Preferably, the free end of the injection cannula has a sharpened Cut or point on the injury z. B. a septum to be reduced. To further reduce an injury during By piercing a septum, the lateral exit opening can make a in the wall of the cannula have lowered, preferably rounded edge.

The has injection device according to the invention according to a preferred variant of several injection cannulas on, with the same lengths a common base part and arranged together or separately with at least one liquid reservoir can be connected. The combination on a common base part has the advantage that all injection needles simultaneously connected to the sample chamber or separated from it can be.

Further advantageous variants of the injection device are characterized from that the injection needles relative to a flat surface of the Base part form a predetermined angle. This angle is especially depending from the geometry of the sample chamber, with which the injection device combined, and the rest geometric conditions of the concrete application selectable and is preferably 90 ° or if necessary, an angle other than 90 °. Furthermore, the shape of the base part with the injection cannulas is preferably to the outer shape of the chamber body of inventive sample chamber customized.

If the sample chamber according to a another embodiment the invention is equipped with an electrode device, can advantageously result in further applications of the sample chamber. The electrode device can be used, for example, for measurements, eg. B. impedance measurements on a sample, or for electrical treatments, eg. B. Electrofusion or electroporation treatments be provided the sample. In a first variant, the electrode device Elekt roden, on at least one of the chamber walls firmly are attached. Advantageously, in this case, the geometric Alignment of the electrodes relative to the sample, in particular relative to Observation area are fixed. The electrodes can be used for Example may be arranged with a specific pattern on the chamber wall. In a second variant, electrodes of the electrode device protrude through at least one of the chamber walls in the sample chamber, in particular in the observation area. In this case, there are special advantages for the flexible adjustment of the electrodes relative to the sample, for example relative to suspended biological cells in the sample chamber.

According to a further embodiment of the invention, the sample chamber can be equipped with a tempering device. Advantageously, with the tempering in the sample chamber, the temperature, for. B. for human samples physiologically adjusted 37 ° C, can be adjusted. The tempering device can be used for heating and / or cooling be designed the sample chamber. The tempering device is preferably arranged on at least one of the chamber walls. For example, an ITO (indium tin oxide) coating may be provided on at least one of the chamber walls for resistance heating. For cooling z. B. be provided a Peltier element. Optionally, the tempering for temperature measurement, z. B. be designed with a thermocouple. In this case, advantageously extends the functionality of the temperature control (measuring / heating / cooling). The temperature in the sample chamber can be regulated in particular with the tempering device.

One Method for receiving a liquid sample in the chamber according to the invention represents another independent The present invention provides the movement of a liquid medium. such as B. a liquid Sample, a medium containing the sample or a medium for supply a sample already arranged in the chamber to the observation area generally in two steps. First the medium is provided at the media port for delivery to the sample space. This takes place, for example, with a liquid conveying device, such as As a pump, syringe, pipette or o. G. Injection device. Subsequently the movement of the medium to the observation area takes place below the effect of capillary forces along the capillary force gradient. This movement is preferably done exclusively by the capillary force without exercising of flow forces by means of an externally driven flow. Alternatively, the sample chamber may also be used as a flow chamber become. For this purpose, the capillary force is combined with flow forces.

With the term sample is here used to designate any flowable composition, the one liquid contains or from a liquid consists. The liquid may be the substance to be examined in the chamber or merely a medium surrounding the substance to be examined. The sample for example, a solution or suspension. The substance to be examined can with the liquid transported to the chamber or another way, eg. B. before a composition of the chamber deposited on at least one of the chamber walls become. Particularly preferred is the application of the chamber according to the invention in the study of samples containing biological materials, such as Of cells, cell components (cell organelles), microorganisms, Viruses or cell groups. Non-biological materials can also with the chamber according to the invention be examined with advantage, such. B. substances in certain Microenvironments or interfacial reactions to be examined.

According to one preferred application of the invention is in the sample chamber, in particular in Observation area arranged a biological sample. She is in Adhesive sample space on at least one of the chamber walls adhesively fixed or in liquid Medium between the chamber walls freely suspended.

According to one preferred variant of the method according to the invention is the biological Sample on one of the chamber walls arranged before the sample chamber is assembled. For example Cells are on the chamber wall, in particular according to the Extension of the observation area in a separate cultivation facility, z. B. a culture dish until a desired cell density is reached. Subsequently the chamber wall is inserted into the chamber body and with the second chamber wall combined. According to a modified embodiment of the The biological sample can be mixed with the liquid medium already in the process composite sample chamber out and in particular in the observation area be cultivated.

The Cultivation of biological samples in the observation area of the composite and closed sample chamber represents an important advantage of Invention over usual Sample chambers. Biological samples can in the sample chamber with the substantially two-dimensional, columnar sample space along the predetermined sample area be arranged while over adjacent open spaces a gaseous Medium is located, leaving a phase boundary within the chamber is substantially perpendicular to the columnar extension. By the supply of liquid and gaseous or vaporous Substances will transfer the sample over Enough nutrients for a substantially unlimited period of time.

If according to one Another preferred feature of the method, the liquid medium or in particular the biological sample in the observation area Being subjected to a microscopic examination may have properties of the examination subject via longer periods under substantially unchanged Conditions are determined.

Further Details and advantages of the invention will become apparent below Reference to the attached Drawings described. Show it:

1 a schematic plan view of a first embodiment of a sample chamber according to the invention,

2 a schematic sectional view through the sample chamber according to 1 along the line II-II,

3 a sectional view of another embodiment of a sample chamber according to the invention,

4 and 5 Illustrations of capillary force potentials in various embodiments of a sample chamber according to the invention,

6 and 7 a sectional view and a plan view of a further embodiment of a sample chamber according to the invention,

8th and 9 Views of an injection device for in the 4 and 5 shown sample chamber,

10 schematic perspective views of different variants of inventive sample chambers with capillary sample spaces,

11 schematic illustrations of the distribution of liquid and gaseous phases in the sample chamber inventive sample chambers, and

12 Illustrations of the connection of sample chambers according to the invention to gas and liquid reservoirs.

The 1 and 2 schematically illustrate a first embodiment of a sample chamber according to the invention 100 for receiving a liquid sample 1 , The sample chamber 100 includes an upper chamber wall 11 and a lower chamber wall 12 , between which the sample space 10 is formed. Laterally in a direction parallel to the extension of the chamber walls is the sample space 10 through the cover 20 closed in this embodiment by a along the edge of the chamber walls 11 . 12 extending spacers 21 is formed.

The chamber walls 11 . 12 consist of flat glass plates with a thickness of z. B. 150 microns and an area of z. 1 cm x 1 cm. The spacer consists z. B. plastic, z. As polyethylene, metal or glass, with a height of 1 mm, so that the chamber walls 11 . 12 correspondingly have a distance of less than 1 mm, in particular less than 100 microns.

The inner surface of the lower chamber wall 12 carries a strip-shaped, hydrophilic coating of surfactants, eg. As polylysine, agarose or collagen, the liquid line 31 forms. The fluid line 31 has a continuous form extending over the at least one chamber wall. It extends between the lateral edges of the chamber wall or the mouths of the media lines in the sample space. With the areal density of the hydrophilic coating increases toward the center of the chamber wall 12 the capillary force acting on an aqueous sample steadily increases. The field of maximum capillary force is the observation area 30 (white dashed), in which an optical examination of the sample 1 should be done. In the neighboring areas, which are the open spaces 32 form, no coating is given. Between the open spaces 32 and the liquid line 31 a coating limit is given (wetting level). The hydrophilic coating collects the liquid sample 1 along the liquid line 31 , wherein under the effect of the surface tension between the chamber walls 11 and 12 a rounded fluid structure is spanned (see 2 ).

Along the wetting line between the liquid line 31 and the adjacent open spaces may be different from the lower chamber wall 12 extend to the upper chamber wall an inner wall which is formed by a partially permeable membrane and forms a barrier for a liquid medium.

The geometric shape of the liquid line 31 is dependent on the function of the sample chamber 100 selected. For example, they are a central observation area 30 and guide areas with the capillary force gradient 33 intended. In the observation area 30 the observation of the sample takes place 1 while the management areas of the supply or removal of liquid media, eg. B. serve with nutrients. The shape of the liquid line 31 can be modified depending on the particular application. For example, annular fluid lines may be provided. In the observation area 30 the gradient may continue, alternatively wetting may be constant there.

In the sample chamber 100 lead six media connections 13 - 15 , The opposite sides of the sample chamber 100 arranged media connections 13 . 14 are in the upper chamber wall 11 the end of the fluid line 31 arranged opposite each other and each serve to supply or discharge of liquids in the sample chamber 100 , The remaining media connections 15 are connected to the open spaces and serve for the supply or removal of gases. Because of the geometric shape of the liquid line 31 (please refer 1 ) the open spaces 32 are separated from each other, must suitably for each of the open space 32 at least one independent gas media connection 15 be provided.

The media connections 13 - 15 are connected to liquid and gas media lines (in the 1 and 2 Not shown). As media lines, glass capillaries are used, for example, through the spacer 21 into the respective parts of the sample space 10 be guided and to the associated chamber wall 11 or 12 open adjoining. The liquid media conduit for sample delivery opens at the capillary force gradient 33 ,

3 illustrates in an exaggerated way the formation of a distance gradient 34 that goes along the chamber wall 11 through the sample chamber 100 extends. By moving along the distance gradient 34 decreasing distance between the chamber walls an increasing capillary action is formed, through which a liquid in the sample chamber to the observation area 30 can be pulled. In the observation area 30 In this design, no gradient is formed to enhance the optical properties of the chamber. On the lower chamber wall 12 is in 3 as tempering example, a transparent heating layer 12.1 (eg ITO layer).

The behavior of a liquid in the capillary force potential is described in more detail in the 4 and 5 shown. 4A illustrates the top view of the sample space of a sample chamber 100 analogous to 1 , where in 4A only the positions of the media connections 13 . 14 and the gas connections 15 and the course of the capillary force gradient 33 are shown. 4B schematically illustrates the z. B. capillary force potential formed by a coating along a reference line through the media connections 13 . 14 leads. If a liquid medium through the media connection 13 is fed into the sample chamber, the liquid medium first follows the gradient of increasing capillary force (decreasing capillary force potential) until it reaches the extended observation area 30 achieved with the minimum of capillary force potential. Upon further fluid delivery, the area covered by the capillary force potential is filled until the fluid is the second fluid port 14 reached, through the one through the sample chamber 100 flowing sample is derived. Corresponding is between the media connections 13 . 14 the liquid line 31 (hatched drawn) formed in the environment in the areas with a higher capillary force potential gaseous medium (dotted line) is arranged.

4 illustrates that the sample chamber according to the invention advantageously represents a self-adjusting system in which any change in the amount of liquid (eg, increase, decrease, change in flow, evaporation, and the like) compensates redistribution first near the media ports 13 . 14 and the adjacent capillary force gradient, while the liquid medium in the observation area 30 remains essentially unchanged. Furthermore, gas bubbles that may be formed randomly in the chamber during the liquid supply to the edge of the liquid line 31 and not in the observation area 30 guided.

5 analogously illustrates a modified variant of a sample chamber 100 in the observation area 30 a gel area 36 is provided in the through a separate gel connection 37 a gel layer, e.g. B. can be introduced from collagen. According to 5B the capillary force potential has maxima at the gas connections 15 , lower values for the media connections 13 . 14 for a liquid medium and a minimum in the gel area 36 on.

According to the invention, alternatively, a multiphase system having a plurality of liquid phases can be formed, in which at the potential minimum, instead of the gel, a first liquid, along the liquid line 31 between the media connections 13 . 14 a second liquid and otherwise a gas in the sample chamber are arranged.

The 6 and 7 show a modified embodiment of a closed sample chamber preferred for practical applications of the invention 100 , The sample chamber 100 comprises a chamber body 22 with a central through opening 23 , at its inner edge on the top of the chamber body 22 a step profile 21 for positioning the chamber walls 11 . 12 is formed. On the bottom of the chamber body 22 owns the opening 23 a cone-shaped expansion 24 through which a microscope objective of the lower chamber wall 12 can be approximated.

The chamber body 22 is made in one piece from a sufficiently stable carrier material. For multiple applications, it consists of an autoclavable material, such. B.

Steel, titanium, glass or silicone rubber. If the chamber body is intended for a single use, it may for example consist of a plastic or also of glass or silicone rubber. The chamber body 22 can be made by injection molding. Advantageously, the chamber body 22 be made so compact that its external dimensions correspond to those of a standard carrier glass (76 mm x 26 mm x 1.5 mm).

The steps of the step profile 21 form the support elements for holding and alignment of the chamber walls 11 . 12 , As shown, the step profile may comprise a plurality of stages as carrier elements, For example, have three or four stages of different heights, depending on the desired clear width of the sample space 10 different sized chamber walls can be placed.

The chamber walls 11 . 12 are rectangular, transparent glasses (d = 150 microns) with optical quality, on the respective stages on the chamber body 22 are glued with an inert, non-toxic silicone adhesive. The distance between the chamber walls 11 . 12 in the design according to the 6 and 7 is 100 μm. The generally protruding edge of the larger (upper) chamber wall 11 on which this on the step profile 21 rests, has a width of about 3 to 4 mm. In addition to the said edge, the upper chamber wall projects beyond 11 on the sides where in the chamber body 22 the media connections 13 - 15 are formed, the lower chamber wall 12 so that the contact area 35 is formed. At the contact area 35 In general, one of the chamber walls is exposed to a media connection.

The upper chamber wall 11 is to form the liquid line 31 and the open spaces 32 coated with a hydrophobic material, for example PTFE or silicone adhesive. The hydrophobic character decreases from the edge to the middle, so that in the middle of the liquid line 31 and in the region of least hydrophobic nature of the observation area 30 is formed (see 5 ). From the media connections 13 . 14 to the observation area 30 Gradients of decreasing hydrophobic character are formed while to the open spaces 32 there is a sharp coating limit. The coating can be formed on both chamber walls or only on a chamber wall.

Through the chamber body 22 lead a first fluid media line 51 and two gas media lines 52 . 53 from external connections 25 to the media connections 13 and 15 , On the opposite side are accordingly another liquid media line 54 and gas media lines 55 . 56 provided for the discharge of liquid or gaseous media. Both sides are the media lines through the locking device 60 made of highly elastic silicone rubber.

At the mouths of the fluid media lines 51 and 54 are in the chamber body 22 funnel-shaped depressions 26 , These advantageously have a dual function. First, they serve as a reservoir for liquid media. Second, they form a distance gradient 34 that with the capillary force gradient 33 interacts on the chamber walls. In this design, so the distance gradient 34 on the inner surface of the sample space 10 on the surface of the chamber body 22 educated.

If over the liquid media line 51 an aqueous dissolved medium in the sample space 10 is guided, the medium collects on the liquid line 31 while the room over the open spaces 32 remains free of liquids. This area is over the gas media lines 52 . 53 filled with a gas that is selected depending on desired process conditions. For culturing cells, for example, air with a content of 5% to 10% CO _{2 is introduced} .

The 8th and 9 illustrate an injection device 70 for injecting gases and liquids into the sample chamber 100 according to the 6 and 7 , The injection device 70 owns a base part 71 three injection cannulas 72 , which correspond to the orientation of the media lines 51 to 55 in the chamber body 22 are arranged. The ends 73 the capillaries 72 are pointed and closed to pass through the locking device 60 to facilitate. At a distance from the end 73 owns every cannula 72 an outlet opening 74 , which is arranged laterally on the cannula. By this lateral arrangement of the outlet opening is a violation of the material of the locking device 60 avoided. When inserting and executing the material of the locking device 60 not to hurt, are the inner edges of the outlet openings 74 preferably rounded. Due to the elasticity of the silicone rubber remains the locking device 60 even after removal of the injection needles 72 tight.

The loading of the sample chamber 100 with biological cells according to a first variant, for example by the upper chamber wall 11 is grown in a culture vessel with the cells. After formation of an adherent cell layer along the fluid line 31 with a thickness of z. B. 10 to 20 microns, the chamber wall 11 in the chamber body 22 glued in and further nutrient medium rinsed in. Alternatively, the cells are flushed into the composite sample chamber to access the fluid line 31 adhere to grow. According to a further alternative, cells can be arranged on the lower chamber wall and further cultured after a first growth in the closed sample chamber. The inventors have found that cells in the sample chamber 100 without intermediate ventilation for 2 to 3 days and with perfusion 2 to 3 weeks could be cultivated.

In the sample chamber, the biological samples can be kept alive or cultured and simultaneously subjected to a microscopic examination. For this purpose, the sample chamber is located in the beam path of a microscope, so that its optical axis perpendicular to the chamber walls through the observation area 30 runs.

10 Illustrates by way of example further embodiments of inventive sample chambers. In these examples, a chamber body 22 provided in one piece as an integral component z. B. is made of a transparent plastic. This embodiment is particularly well suited as a disposable product. The above-described media ports are provided in this embodiment at the front and rear sides of the chamber body by a fitting part (not shown) formed to match the cross-sectional shape of the sample space. At the connection part, the media and possibly gas or gel connections are aligned so that the respective substance is supplied to the appropriate areas in the sample space. For multiple use sample chambers according to the invention with correspondingly shaped chamber bodies can be made of a material that can be sterilized without loss of stability and shape, such. As corrosion-resistant steel, titanium, glass or thermostable plastic or rubber. In these examples, if necessary, a transparent chamber wall is provided for the optical examination of samples in the sample space.

According to 10A is the fluid line 31 provided with the Kapillarkraftgradienten in the middle of the sample space. According to 10B become two fluid lines 31A . 31B formed, the liquid so to the observation area 30 (dashed lines) lead to just that a phase boundary in the observation area 30 is arranged. 10C illustrates a chamber body 22 with a layer-shaped sample space whose inner walls each carry a coating in which the desired Kapillarkraftgradient is formed. 10D illustrates a more complex sample space with different subregions in which, for example, gel-liquid or liquid-liquid phase boundaries can be investigated.

10E shows a widening fluid line 31 that are sideways of interior walls 38 is limited by a partially permeable membrane. Further illustrated 10E schematically an electrode device 80 with one or more electrodes 81 , The electrodes 81 may be connected to an external voltage source (not shown) and connected to voltages for generating fields for manipulation of particles in the liquid line or for measuring electrical quantities. The electrodes 81 are fixed on one of the chamber walls as illustrated, or they are inserted through openings in the sample chamber.

10F shows a further variant with a complex structure of the sample space for the investigation of phase boundaries between liquid and gaseous media, also with electrodes 81 ,

Since the capillary force potential formed according to the invention in the sample space can be shaped differently depending on the specific requirements, the sample chamber according to the invention has a large variability in the distribution z. B. liquid and gaseous phases, as exemplified in 11 is shown. The partial images of the 11 show the sample space according to the invention sample chambers in a schematic plan view, wherein the liquid-filled area is black and the gas-filled area dotted or white drawn. The variants shown serve primarily the investigation of the phase boundary between the liquid and the gaseous medium. The lower part of the picture in 11 shows the separation of the phases with an inner wall 38 analogous to 10E ,

12 schematically illustrates various variants of flow systems using the sample chamber according to the invention 100 in connection with a liquid conveyor 90 generally comprising one or more common or separate liquid and gas reservoirs 91, 92, one or more connecting conduits 93 and one or more pumps 94 . 94A . 94B includes. The reservoirs 91 . 92 are known per se containers for receiving the respective desired liquids or gases. The connection lines 93 are, for example, pipe or hose lines, as they are known from laboratory technology. As a pump 94 For example, a peristaltic or membrane pump in one of the connecting lines 93 arranged.

According to 12A leads a first connection line 93 from a common liquid and gas reservoir 91 over the pump 94 to the first media connection 13 the sample chamber 100 , The pump 94 leads the liquid into the chamber, where the liquid is drawn to the observation area in the capillary force potential. When the liquid line in the sample chamber 100 is filled, the further liquid supply leads to a displacement through the second media connection 14 from which the liquid as illustrated in the reservoir 91 is returned. When the pumping direction is reversed, the fluid may leak out of the sample chamber 100 be discharged, under the action of capillary forces a sample is held in the observation area. In this state, a gas exchange in the sample chamber 100 respectively.

According to 12B are two pumps 94A . 94B intended. The first pump 94A leads fluid from the reservoir 91 into the sample chamber 100 while the second pump 94B Liquid from the sample chamber 100 with a larger power than the first pump 94A sucks. Since the capillary forces hold the liquid sample in the observation area, only the amount of liquid coming from the first pump 94A is delivered from the sample chamber 100 deducted. 12B also shows two pressure equalization lines 95 with the open spaces in the sample chamber 100 are connected and the pressure equalization in the sample chamber 100 serve.

According to 12C are two reservoirs 91 . 92 provided with the sample chamber 100 each separate gas and liquid circuits form, with the pumps 94A . 94B operate.

The in the foregoing description, drawings and claims Features of the invention can both individually and in combination for the realization of the invention be significant in their various embodiments.

Claims (58)

Sample chamber ( 100 ), comprising: - two spaced-apart chamber walls ( 11 . 12 ), - a sample room ( 10 ) passing through the chamber walls ( 11 . 12 ) and in which a sample ( 1 ) in an observation area ( 30 ) is accessible to an investigation, and - a media connection ( 13 . 14 ), which is connected to the sample space ( 10 ) and introducing a liquid medium into the sample space ( 10 ), wherein - at least one of the chamber walls ( 11 . 12 ) at least in the observation area ( 30 ) is formed from an optically transparent material, characterized by - at least one capillary force gradient ( 33 . 34 ) located between the media port ( 13 . 14 ) and the observation area ( 30 ) is formed such that the liquid medium along the at least one Kapillarkraftgradienten a to the observation area ( 30 ) directed capillary force and between the media connection ( 13 . 14 ) and the observation area ( 30 ) at least one fluid line ( 31 ) is formed, wherein - in the observation area ( 30 ) between the chamber walls ( 11 . 12 ) a maximum capillary force is exerted on the liquid medium. A sample chamber according to claim 1, wherein said at least one capillary force gradient ( 33 ) along the liquid line ( 31 ) through a to the observation area ( 30 ) is increasingly formed liquid-attracting and / or decreasing liquid-repellent surface. A sample chamber according to claim 2, wherein the at least one capillary force gradient ( 33 ) along the liquid line ( 31 ) through a to the observation area ( 30 ) increasingly hydrophilic and / or decreasing hydrophobic surface is formed. Sample chamber according to at least one of the preceding claims, in which the at least one capillary force gradient along the liquid line is defined by a distance gradient ( 34 ) formed by a reduction of the clear width of the sample space ( 10 ) between the media connection ( 13 . 14 ) and the observation area ( 30 ). A sample chamber according to claim 4, wherein on at least one of the chamber walls along the distance gradient ( 34 ) one to the observation area ( 30 ) is increasingly formed liquid-attracting and / or decreasing liquid-repellent surface. Sample chamber according to claim 4 or 5, wherein in a first, to the media connection ( 13 . 14 ) adjoining subarea of the distance gradient ( 34 ) and in a second, to the observation area ( 30 ) adjoining partial area as capillary force gradient ( 34 ) one to the observation area ( 30 ) is increasingly formed liquid-attracting and / or decreasing liquid-repellent surface. Sample chamber according to at least one of the preceding claims, wherein the at least one capillary force gradient ( 34 ) is spatially and / or temporally adjustable. Sample chamber according to at least one of the preceding claims, in which both chamber walls ( 11 . 12 ) at least in the observation area ( 30 ) are formed of an optically transparent material. Sample chamber according to at least one of the preceding claims, wherein at least one of the chamber walls ( 11 . 12 ) at least in the observation area ( 30 ) is made of glass or plastic and / or carries a scale. Sample chamber according to at least one of the preceding claims, in which the chamber walls ( 11 . 12 ) at least in the observation area ( 30 ) each have a thickness less than 200 microns. Sample chamber according to at least one of the preceding claims, wherein at least one of the chamber walls ( 11 . 12 ) at the observation area ( 30 ) has a wettability maximum. Sample chamber according to at least one of the preceding claims, which has at least two media connections ( 13 . 14 ) having. A sample chamber according to claim 12, wherein the at least two media connections ( 13 . 14 ) relative to the observation area ( 30 ) on different sides of the sample chamber ( 10 ) are arranged and the liquid line ( 31 ) with the capillary force gradient between the observation area ( 30 ) and each media connection ( 13 . 14 ) is formed. Sample chamber according to at least one of the preceding claims, which has at least one gas connection ( 15 ) for gas supply or removal and / or pressure equalization. Sample chamber according to at least one of the preceding claims, wherein between the chamber walls ( 11 . 12 ) Spacers are arranged. Sample chamber according to at least one of the preceding claims, wherein the liquid line is at least on one side of an inner wall ( 38 ) between the chamber walls ( 11 . 12 ). Sample chamber according to claims 15 and 16, in which the spacers are separated by the inner wall ( 38 ) are formed. Sample chamber according to Claim 16, in which the inner wall ( 38 ) is porous or partially permeable. Sample chamber according to Claim 16, in which the inner wall ( 38 ) has a liquid attracting and / or liquid repelling surface. Sample chamber according to claim 19, in which the inner wall ( 38 ) has an increasing liquid attracting and / or decreasing liquid repellent surface along the liquid line. Sample chamber according to at least one of the preceding claims, wherein the width and / or shape of the liquid line between the media connection ( 13 . 14 ) and the observation area ( 30 ) changed. Sample chamber according to at least one of the preceding claims, wherein in the sample space ( 10 ) a gel area ( 36 ) is provided for receiving a gel. Sample chamber according to claim 22, in which the liquid line ( 31 ) in the gel area ( 36 ) has a Kapillarkraftmaximum or a part of the liquid line ( 31 ) the gel area ( 36 ) goes through. A sample chamber according to claim 22, wherein the gel region ( 36 ) is bounded at least on one side by an inner wall. Sample chamber according to at least one of the preceding Claims, their size is the same the size of one Standard cover glass for the Microscopy is. Sample chamber according to at least one of the preceding claims, in which the chamber walls ( 11 . 12 ) have different sized surfaces, wherein the larger chamber wall completely covers the smaller chamber wall and at least one edge with a contact area ( 35 ) surmounted. A sample chamber according to claim 26, wherein the media port ( 13 . 14 ) is arranged on one of the chamber walls and to the contact area ( 35 ) opens out. Sample chamber according to at least one of the preceding claims, in which a lateral covering device ( 20 ) is provided with the sample space ( 10 ) is closed on the side. Sample chamber according to Claim 28, in which the covering device ( 20 ) a step profile ( 21 ), on which the chamber walls ( 11 . 12 ) rest on the side. Sample chamber according to Claim 28, in which the covering device ( 20 ) comprises an elastic material. Sample chamber according to at least one of the preceding claims, comprising a chamber body ( 22 ) having a holder for the chamber walls ( 11 . 12 ). Sample chamber according to claim 31, in which the chamber body ( 22 ) is integrally formed. A sample chamber according to claim 31 or 32, wherein the chamber body ( 22 ) is made of a transparent material such as plastic. Sample chamber according to at least one of claims 31 to 33, in which the chamber body ( 22 ) a through opening ( 23 ), in which a step profile ( 21 ) at the inner edge of the opening ( 23 ) is formed. Sample chamber according to claim 34, wherein the opening ( 23 ) of the chamber body ( 22 ) at least on one side a conical widening ( 24 ) for approaching an optical measuring device to the sample space ( 10 ) having. Sample chamber after at least one of Claims 31 to 35, wherein the chamber body ( 22 ) at least one media line ( 51 - 55 ) at the at least one media port ( 13 . 14 ) directly into the sample space ( 10 ) opens. A sample chamber according to claim 36, wherein the chamber body ( 22 ) at least one liquid media line ( 51 ) and at least one gas media line ( 52 - 55 ) contains. Sample chamber according to claim 37, in which the media lines ( 51 - 55 ) are arranged coaxially. Sample chamber according to at least one of claims 31 to 38, wherein the at least one media connection ( 13 . 14 ) in a wall of the chamber body ( 22 ) is arranged, which has a funnel-shaped depression to form the distance gradient. Sample chamber according to at least one of claims 36 to 39, in which the at least one media line ( 51 - 55 ) a locking device ( 60 ), with which the at least one media line ( 51 - 55 ) is closable. Sample chamber according to claim 40, in which the blocking device ( 60 ) comprises an elastic material. Sample chamber according to at least one of the preceding claims, provided with an injection device ( 70 ), which is in the sample room ( 10 ) is insertable. Sample chamber according to Claim 42, in which the injection device ( 70 ) at least one hollow injection cannula ( 72 ) having. Sample chamber according to Claim 43, in which the injection cannula ( 72 ) at one free end a tip and at a distance from the free end of a lateral outlet opening ( 74 ) having. Sample chamber according to at least one of the preceding claims, provided with an electrode device ( 80 ) Is provided. Sample chamber according to claim 45, in which the electrode device ( 80 ) Comprises electrodes on at least one of the chamber walls ( 11 . 12 ) are arranged. Sample chamber according to claim 45, in which the electrode device ( 80 ) Comprises electrodes through openings in at least one of the chamber walls ( 11 . 12 ) in the sample room ( 10 ) protrude. Sample chamber according to at least one of the preceding claims, comprising a tempering device ( 12.1 ) having. Sample chamber according to at least one of the preceding claims, in which the chamber body ( 22 ) is formed as a disposable chamber body. Sample chamber according to at least one of the preceding claims, in which the chamber body ( 22 ) is formed as a reusable chamber body, wherein the chamber body ( 22 ) preferably from sterilizable, z. B. by autoclaving or UV irradiation, material such as. Plastic, titanium, glass or stainless steel. Injection cannula, in particular for fluid transport into or out of a sample chamber according to at least one of the preceding claims, which has a tip at a free end and a lateral outlet opening at a distance from the free end (US Pat. 72 ) having. Method for receiving a liquid medium in a sample chamber according to at least one of the preceding claims, with the following steps: provision of the liquid medium at the media connection ( 13 . 14 ) for feeding into the sample space ( 10 ), and - movement of the liquid medium under the action of the capillary force towards the observation area ( 30 ). The method of claim 52, wherein a flow of the liquid medium through the sample space ( 10 ) is provided. A method according to claim 52 or 53, wherein in the sample space ( 10 ) a biological sample is arranged. The method of claim 54, wherein the biological sample is prior to assembly of the sample chamber on one of the chamber walls. 11 . 12 ) is arranged. The method of claim 54, wherein the biological Sample with the liquid Medium is introduced into the sample chamber. Method according to claim 55 or 56, with the further step: - cultivation of the biological sample in the observation area ( 30 ). Method according to at least one of claims 52 to 57, with the further step: - microscopic examination of the liquid medium or of the biological sample at least in the observation area ( 30 ).