DE102017213200B4 - Sample Chamber, System and Method for Light Sheet Microscopy - Google Patents

Abstract

A sample chamber (1) for use in light sheet microscopy, the sample chamber (1) being fillable with an immersion liquid, comprising a top (2) having an upper opening (3) through which a sample is inserted into an interior of the sample chamber (1) for illumination and Consideration can be introduced when the sample chamber (1) is filled with the immersion liquid, a lower side (4) with a lower opening (5), a lateral wall (6) with at least one lateral passage opening (7, 7 ', 7 ", 7 "'), which is liquid-tightly closed by a lens (36', 36", 36 "'), characterized by a removable bottom plate (20) which is adapted to liquid-tightly close the lower opening (5).

Description

The invention relates to a modular sample chamber which is suitable for use in microscopy, in particular in fluorescence microscopy, such as light-sheet microscopy, but also for other microscopic imaging modalities, such as optical projection tomography.

In light sheet microscopy, a sample is labeled with a fluorescent dye or has sufficient autofluorescence. The samples are often living organisms, organoids and cells. These are positioned within a capillary and prepared for example by embedding in agarose or filling the capillary with a nutrient medium. Often, long-term observations lasting several days or weeks over which the sample must be kept alive and supplied with nutrients, ideally without interrupting the observation, taking the sample or intervening in the mechanical-optical reference system, are sought.

The sample is illuminated plane by layer with a light sheet. In order to be able to reconstruct a three-dimensional image of the sample, the sample must be brought into the focus of an optical setup and then a relative movement between the capillary and the optical structure be made possible to completely detect the sample with the light sheet. For example, the sample may be stored so that it can be rotated and translated relative to a predetermined optical configuration to fully illuminate the sample. Furthermore, the capillary should have sufficient clearance that even a slipped within the capillary sample can be moved back into the light sheet. For optimized illumination, two illumination lenses may be disposed on opposite sides of the sample. If the sample is illuminated, the occurring fluorescence is detected by means of at least one lens perpendicular to the illumination axis and imaged on a sensor.

Arrangements are known from the prior art in which a capillary containing the sample is positioned by means of a controllably movable holding device in a sample chamber filled with liquid, wherein the sample chamber has translucent windows for illumination and detection.

It comes at the transitions from the lens to the air and from the air to the window of the sample chamber to unwanted refraction, which affects the quality of the microscope images.

From the utility model DE 20 2012 007 891 U1 For example, a microscope for SPIM microscopy is known, comprising an illumination light source and an illumination beam path for illuminating the sample with a light sheet, preferably via an illumination objective, detection means for detecting light emitted by the sample, imaging optics comprising the sample via an imaging lens in an imaging beam path at least partially maps to the detection device, and a preferably liquid-filled sample chamber in the direction of illumination and / or in the detection direction has at least one translucent window. In at least one translucent window, a receptacle is provided in which at least one preferably screw-threaded insert is fitted, which has a preferably annular opening in which a sealing ring for fitting a detection lens and / or illumination objective and / or a lens holder or holder is introduced.

From the publication US 2006/0045821 A1 a microreactor for investigating material reactions and properties is shown. The microreactor comprises a core body defining a chamber which may contain one or more sample materials and has fluid passage from the chamber to the outside of the core body. The chamber is in fluid communication with an external manifold, whereby gases or liquids can be injected. Transparent windows in the core body provide continuous visual access to the chamber, allow a direct interaction of the sample beam with the sample during a reaction or observation, and an external detection of the sample beam to investigate in situ reaction processes.

From the publication US 7852554 B2 a cryogenic immersion microscope is known whose objective lens is at least partially in contact with a liquid reservoir of a cryogenic liquid in which a sample is immersed. When the cryogenic liquid has a refractive index which reduces the refraction at interfaces between the lens and the sample, overall resolution and image quality are improved. Furthermore, a sample holder coupled to an xyz platform is shown. Another problem with such arrangements is that the capillary within the sample chamber can only be moved to a limited extent due to the limited dimensions of the sample chamber and the sample may not be able to do so into the focus of the lenses, especially if the sample slips during observation. In addition, due to the dimensions of the sample chamber, the size of the sample that can be placed therein is limited.

For long-term exposures of biological samples lasting from several days to several weeks, the sample should be provided with nutrients as mentioned. At the same time, however, the sample should be moved as little as possible and, if possible, the cannula should not be taken out of the sample chamber in order to avoid damaging the sample and not having to interrupt the observation. For this, it may be helpful to have access to a lower end of the capillary, in addition to access to the upper end of the capillary, to allow for nutrient or gas delivery without having to remove the capillary from the assembly. If the sample is positioned within a sample chamber, access to the lower end of the capillary is difficult. Hoses to be attached there must be bent strongly and can therefore bend. Furthermore, the tubes may enter the optical path of the lenses when they are to be guided upwards from the lower end of the capillary.

In fluorescence microscopy also different constructions come into question: For the light sheet microscopy, two lenses can be arranged at an angle of 90 degrees to each other, wherein a lens is operated as an illumination objective and a lens as a detection objective. Furthermore, a further objective, opposite the first illumination objective, can function as the illumination objective, as well as a further detection objective, opposite to the first detection objective. In optical projection tomography, for example, an illumination objective and a detection objective can be arranged opposite one another. The arrangement of the lenses to each other is crucial for the quality of the recording. For the different microscopy procedures under consideration, or when a sample chamber has been swapped or manipulated, the optics must be readjusted each time.

The object of the present application is to at least partially circumvent the above-mentioned problems.

For this purpose, a modular sample chamber according to claim 1 is proposed. Other embodiments that help in the circumvention of the problems arise with the features of the subclaims and from the embodiments.

The functionality and the internal volume of the sample chamber according to the invention are variable by a removable bottom plate, which may be equipped with functional elements. In a particular embodiment, the bottom plate also allows access to the lower end of the capillary. The sample chamber also has lateral passage openings, which are designed such that immersion objectives can be introduced directly into the lateral passage openings and seal them in a liquid-tight manner. Thus, illumination and detection need not be through a window in the sample chamber. At the same time thereby the optical path is determined in relation to the sample chamber, thus simplifying an adjustment. However, removable side panels, which may be fitted with windows, can provide the same functionality as a conventional sample chamber.

Thus, the sample chamber opens up numerous different possibilities of microscopy, in particular the long-term microscopy of biological samples.

In one embodiment, the sample chamber has feet and / or recesses on its underside or in a lower area and / or has arms in an upper area or laterally. These feet, recesses or arms are helpful in accurately positioning the sample chamber on a fixture. By this type of positioning of the sample chamber, a fixed optical-mechanical reference system can be defined.

The feet or recesses may also be mounted laterally on the sample chamber and project down beyond them in one embodiment. The feet or recesses may be mounted in a further embodiment also on the bottom plate to be located further inside. In another embodiment, the feet are further outwardly displaced by being attached to a structure which is attached to the bottom of the sample chamber and projects laterally beyond the sample chamber. The feet or recesses should allow positioning of the chamber on a designated holding device, for example by plugging. The holding device, in some embodiments, has recesses or feet corresponding to the feet or recesses of the sample chambers so that sample chambers of different sizes can be used in the device. Furthermore, the sample chamber in the area of the feet or recesses or on the feet may comprise holes or holes for the introduction of fasteners to allow a positive and / or non-positive connection with the device for positioning. In a further embodiment, the sample chamber has arms that can be connected to a holding device. For this purpose, the arms and the holding device may have corresponding holes for the introduction of fastening means. Thus, the position of the sample chamber is preceded by the holding device fixable, which later simplifies the adjustment of the optics.

The sample chamber includes a lateral wall with through-holes, and an upper opening through which the sample can be introduced into the interior of the chamber. Furthermore, the chamber comprises a bottom plate and a lower opening, which can be closed liquid-tight by the bottom plate. Likewise, side plates are provided, with which, if necessary, the side openings can be sealed liquid-tight.

The sample chamber is preferably cuboidal. In one embodiment, the sample chamber is made integral except for the removable bottom plate. The bottom plate is integral in this embodiment, but manufactured separately. In particular, in one embodiment, the sample chamber is made of a rigid material. In one embodiment, the sample chamber is made of a biocompatible stainless steel or titanium to prevent a biological sample or live sample from being damaged by toxic substances. In one embodiment, the sample chamber is made of a non-transparent material.

The design and arrangement of the lateral passage openings, through which the illumination and detection is carried out, is of importance. They are, as mentioned, configured so that a lens can be inserted and / or pressed and the opening is thus sealed liquid-tight. At the same time, the position of the lens in its inserted or pressed state is determined in relation to the chamber and so its optical axis and its beam path, including the possible position of the light sheet and the detection plane set. In one embodiment, the passage opening tapers from the outside inwards, so that a lens can be pressed from the outside and a tight seal can be produced. Furthermore, sealing material such as a sealing ring between the wall of the through hole and the lens can be arranged. In one possible embodiment, the wall of the passage opening has a step to which the objective and the sealing ring can be pressed. The optical axis of the lens usually follows a geometric axis of the passage opening through the centers or centroids of the inlet opening of the through hole, which is located in the outer wall of the lateral wall of the chamber, and the outlet opening of the through hole, which is located in the inner surface of the lateral wall , runs. If the inlet opening and the outlet opening are arranged concentrically, the result is a geometric axis and thus an optical axis which run perpendicular to the wall. But there are also through holes possible, which have a geometric axis, which are arranged at an angle to the lateral conversion, so that lenses can be introduced obliquely. For example, to facilitate illumination and detection of a sample in light sheet microscopy as described above, two openings on opposite sides of the lateral wall are positioned so that their axes coincide. In it, the illumination lenses can be attached. In a wall lying perpendicular thereto an opening is provided at the same height, the axis of which is orthogonal to the axis of the other two openings. The axes then intersect inside the chamber. In one embodiment, the chamber and objectives are configured so that the focal points of the objectives are at or near that intersection. At this point of intersection, the sample can then be guided for illumination and observation. In optical projection tomography, on the other hand, the openings for the illumination and the detection are arranged on opposite sides, so that their axes coincide. In one embodiment, there are four openings at the same height on the four sides of a cuboid sample chamber, wherein the two opposite through holes are in alignment with each other and intersect the axes of the through holes in the center of the chamber. At this point of intersection, the sample can then be guided for illumination and observation. It is also possible, instead of or in addition to the above-described lateral through holes in the side panel, to provide through holes suitable for inserting and / or pressing lenses as above into replaceable side panels. Then can be made possible by different side plates with different through holes, the use of different lenses or the focus of the inserted lenses are moved.

Unless all the side apertures are needed for lenses, remaining apertures with preferably opaque side plates, which in one embodiment are made of the same material as the sample chamber, can be sealed in a liquid-tight manner before the chamber and objectives are positioned. In order to close the bottom opening with the bottom plate and the side openings with the side plates liquid-tight, preferably a sealing material, such as a sealing ring, is introduced into a groove provided in the bottom plate or side plates and a positive and / or non-positive connection between the bottom plate or the Side plates and the chamber allows. For this purpose, the bottom plate and the sample chamber corresponding bores or threaded holes for the introduction of, for example, screws or bolts. If the underside and sides of the sample chamber are then closed in a liquid-tight manner, an immersion liquid, for example water, can be introduced into the sample chamber. The tips of the lenses, which are preferably designed as immersion objectives, are then located directly in the immersion liquid, so that optimal conditions for illumination and detection prevail. There are also provided side plates with a transparent area, for example side plates comprising a disk. The disc may be transparent or opaque, as well as having filter properties, and be made of glass or acrylic, for example. Such disks are then used with air immersion objectives that are not inserted into the through-holes but remain outside the chamber and illuminate or detect the sample through the disks or the transparent region of the side plates.

The bottom plate also has sensors, for example, temperature sensors and / or active elements, such as elements for temperature regulation. Thus, the liquid filled into the interior of the sample chamber can be adjusted to a specific temperature via the bottom plate. For the survival of biological samples it may be necessary to be able to set the ambient temperature very accurately. This can be done with the presented version.

As mentioned, the sample can be brought to its illumination and viewing, for example, in the optical axes of the lenses and there continue to be moved. A capillary containing the sample is introduced from above into the sample chamber and held at the top with a suitably designed device which allows movement of the sample, that is, translation, lowering and lifting, and tilting and rotating of the capillary. Thus, the sample can be moved to a location suitable for microscopy within the chamber, for example in the focus of the lenses, and rotated there to microscopically record the volume of the sample, which allows a reconstruction of three-dimensional data. If the sample slips inside the capillary, readjustment is possible. Inside the sample chamber, however, the movement of the sample is not unlimited because the capillary, when moved down, abuts the bottom of the sample chamber. In one embodiment, a bottom plate is provided having a die that fits into the bottom opening of the sample chamber and establishes a flat bottom of the sample chamber. However, when using such a bottom plate, in particular, the capillary can not be lowered very far as it reaches the bottom of the sample chamber or the top of the punch. In another embodiment, therefore, a bottom plate is proposed, which has a blind hole, in which the sample can be lowered into it. Thus, the range of motion of the capillary is increased downwards and the detection of the sample is possible even if the sample slips up inside the capillary.

Furthermore, an embodiment is proposed, which has a bellows system with a hole made of a flexible material such as rubber, or a film with a hole. Through the hole in the bellows system or in the film, the capillary can be pushed through, so that it protrudes on the other side. If the capillary is thus inserted from above, the lower part of the capillary protrudes down from the bottom plate. Here, the bellows system or the film is designed so that the capillary located in the hole is tightly enclosed by the bellows system or the film, so that no immersion liquid between capillary and bellows system or film can escape to the outside. Thus, the sample chamber is still liquid-tight when the bottom plate is introduced with bellows system or film and the capillary is pushed through the hole. At the same time, however, the capillary sits in the hole so that it can continue to be moved up and down and rotated without the tightness is impaired. Thus, the sample, with appropriate simultaneous deformation of the bellows system and / or by slipping in the hole, in this configuration can be pushed particularly far down because it is passed through the bottom plate and can not abut it. Because the bellows system or the film is flexible, the capillary can still be tilted. Another characteristic of a bellows-type bottom plate or foil is that the lower end of the capillary lies outside the chamber and thus allows the connection of a flow system at the lower end of the capillary, which serves for the exchange of nutrient media or through the introduction of substances or substances is possible in order to observe the reaction of the sample to the substances.

In order to make a recording as described above, the chamber must be integrated together with lenses in an optical design. For illumination, a laser or a collimated narrow-band illumination source is needed, the beam of which is guided by mirrors into the illumination objectives. In addition, a sensor or a camera is needed to detect the detected by the detection lens signal. These and possible other units or devices, such as lenses, may in one embodiment be mounted on an optical stage or on an optical disc A grid can be positioned so that a screw connection is made possible, and the beam path can be adjusted and secured against slippage or displacement. In another embodiment, these units or devices can be compactly integrated into a microscope into which the sample chamber is inserted.

A characteristic of the configurations described is that a fixed optical-mechanical reference system can be made and the beam path to the illumination lenses and detection lens remains the same even if the chamber or its bottom plate is replaced. In order to be able to produce such a fixed optical-mechanical reference system, in one embodiment a device is provided which enables the exact positioning of the sample chamber in this reference system. This device consists for example of a base plate, which can also be screwed to the grid of an optical table or an optical disk, and a base on which the sample chamber can be plugged and an outer frame, so that the device as an outer chamber for receiving the sample chamber is formed. The outer frame is designed to allow positioning of the lenses. The outer frame has a passage in which the lens can be inserted or through which a light beam or laser beam can penetrate. In addition, guide rails can be fastened to the outer frame, to which in turn objective plates can be attached. The lens plates have an opening with a thread, so that a lens can be screwed into the lens plate. Then, the lens plate together with the lens along the attached to the outer frame of the outer chamber guide rails can be moved. Thus, when using immersion objectives, an immersion objective along the guide rail can be introduced and pressed into the lateral passage opening of the sample chamber or, when using air immersion objectives, the air immersion objective can be displaced along the guide rails, thus shifting its focal point. In order for the immersion objective to be inserted into the lateral passage opening as described, or for a specimen located in the chamber to be illuminated or viewed through a disk by means of an air immersion objective, the position of the specimen chamber must be determined in relation to the outer chamber and the lateral frame of the outer chamber his.

This is accomplished in one embodiment by attaching the socket to a specific position on the base plate and designing the socket so that the chamber can be plugged on and can not slip and the plugged chamber also has a certain height, such as the Immersionsobjektive are exactly at the height of the lateral passage openings or the Luftimmersionsobjektive are equal to the windows of the side panels. To allow the chamber to be fitted, the chamber has at least one foot or recess at the bottom. Characterized in that the base has a structure in its upper region, which is complementary to the foot / feet or the recess / the recesses of the sample chamber, a slipping of the sample chamber is prevented. In addition to the feet or recesses or instead of the feet or recesses, the base and the sample chamber may also have holes, tapped holes or holes for the introduction of fasteners such as screws or bolts, so that slippage is also prevented.

The socket also has, in one embodiment, a recess arranged to expose a portion of the underside of the bottom plate of the plugged-on sample chamber. This has the effect that when using the base plate with bellows system or with foil with inserted capillary, the lower portion of the capillary protrudes into the recess of the base and does not abut the base and can also be moved within the recess without abutting. In one embodiment, the recess is large enough to allow the connection of a flow system, for example a hose, to the lower end of the capillary.

In one possible embodiment, the sample chamber is held by the inserted lenses at a defined position, so that the base is not needed.

In another possible embodiment, to allow accurate positioning of the sample chamber within the outer chamber, instead of the pedestal or in addition to the pedestal, the sample chamber is provided with arms which can be connected to the lateral frame of the outer chamber. The arms are configured to hold the associated sample chamber at a defined position and, as above, for example, bring the lateral through openings at the level of the lenses. In one embodiment, the sample chamber is not on the base plate, but has a defined distance from the base plate, so that for example, located on the underside of the bottom plate tubes of a flow system place.

Thus, access to the bottom plate is guaranteed in all the above embodiments, so that flow systems, sensors or active Elements can be connected laterally or down to the bottom plate.

Furthermore, in the abovementioned embodiments with the aid of the arms or feet, a fixed arrangement of sample chamber and outer chamber can be produced, in which the position of the sample chamber is predetermined by the outer chamber, so that a mechanical-optical reference system is defined. With the help of the arms or feet, it is possible to reliably restore the same optical conditions for each new attempt, or after a possible intervention in the system, by aligning the optics and / or sample chamber with the, preferably stationary, outer chamber.

Moreover, in the last two embodiments where the sample chamber is held directly by the lenses or by means of the arms, it is possible to use widely differing floorboards, for example much larger floorboards which do not fit on the pedestal.

The outer chamber may, in one embodiment, have recesses in the outer frame through which a connected flow system and / or the cabling of the sensors or the active elements of the bottom plate may be passed.

• 1 zeigt eine perspektivische Ansicht der Probenkammer mit ihren Öffnungen;
• 2 zeigt die Unterseite der Probenkammer;
• 3 zeigt einen Querschnitt durch die seitliche Umwandung und eine Durchgangsöffnung;
• 4a zeigt eine perspektivische Ansicht der Unterseite einer Bodenplatte mit Balgensystem;
• 4b zeigt eine perspektivische Ansicht der Oberseite einer Bodenplatte mit Balgensystem;
• 4c zeigt eine Seitenansicht der Bodenplatte mit Balgensystem wie in 4a und b, mit einer Probe;
• 5a zeigt eine perspektivische Ansicht der Oberseite einer Bodenplatte mit Stempel;
• 5b zeigt eine perspektivische Ansicht der Unterseite einer Bodenplatte mit Stempel;
• 6a zeigt eine perspektivische Ansicht der Oberseite einer Bodenplatte mit Sackloch;
• 6b zeigt eine perspektivische Ansicht der Unterseite einer Bodenplatte mit Sackloch;
• 7 zeigt eine perspektivische Explosionsdarstellung eines Mikroskopaufbaus;
• 8 zeigt eine Seitenansicht einer Grundplatte mit Sockel und seitlichem Rahmen;
• 9 zeigt eine Draufsicht auf eine Grundplatte mit Sockel und seitlichem Rahmen;
• 10a zeigt eine Seitenansicht der Grundplatte mit Sockel und auf dem Sockel montierter Probenkammer mit montierter Bodenplatte;
• 10b zeigt eine Explosionsdarstellung einer Seitenansicht der Grundplatte mit Sockel, Probenkammer und Bodenplatte;
• 10c zeigt eine perspektivische Ansicht der Grundplatte mit Sockel und auf dem Sockel montierter Probenkammer mit montierter Bodenplatte;
• 11 zeigt einen Schnitt durch eine perspektivische Ansicht der Grundplatte mit Sockel und auf dem Sockel montierter Probenkammer mit montierter Bodenplatte mit Sackloch;
• 12 zeigt eine perspektivische Ansicht der Grundplatte mit Sockel und auf dem Sockel montierter Probenkammer größerer Größe, mit montierter Bodenplatte;
• 13a zeigt eine perspektivische Explosionsdarstellung der äußeren Kammer, einer Probenkammer mit Armen und einer Bodenplatte; und
• 13b zeigt eine perspektivische Darstellung der äußeren Kammer, einer Probenkammer mit Armen, und einer Bodenplatte in montiertem Zustand.

Below is a brief description of the figures.

• 1 shows a perspective view of the sample chamber with its openings;
• 2 shows the bottom of the sample chamber;
• 3 shows a cross section through the lateral conversion and a passage opening;
• 4a shows a perspective view of the underside of a bottom plate with bellows system;
• 4b shows a perspective view of the top of a bottom plate with bellows system;
• 4c shows a side view of the bottom plate with bellows system as in 4a and b, with a sample;
• 5a shows a perspective view of the top of a bottom plate with stamp;
• 5b shows a perspective view of the underside of a bottom plate with stamp;
• 6a shows a perspective view of the top of a bottom plate with blind hole;
• 6b shows a perspective view of the underside of a bottom plate with blind hole;
• 7 shows an exploded perspective view of a microscope assembly;
• 8th shows a side view of a base plate with base and side frame;
• 9 shows a plan view of a base plate with base and side frame;
• 10a shows a side view of the base plate with base and mounted on the base sample chamber with mounted bottom plate;
• 10b shows an exploded view of a side view of the base plate with base, sample chamber and bottom plate;
• 10c shows a perspective view of the base plate with base and mounted on the base sample chamber with mounted bottom plate;
• 11 shows a section through a perspective view of the base plate with base and mounted on the base sample chamber with mounted bottom plate with blind hole;
• 12 shows a perspective view of the base plate with base and on the base mounted sample chamber of larger size, with mounted bottom plate;
• 13a shows an exploded perspective view of the outer chamber, a sample chamber with arms and a bottom plate; and
• 13b shows a perspective view of the outer chamber, a sample chamber with arms, and a bottom plate in the assembled state.

1 shows a sample chamber 1 , It is cuboid and preferably made of a biocompatible stainless steel or titanium. The sample chamber 1 points at the top 2 an upper opening 3 through which a sample can be introduced into the interior of the sample chamber. The sample chamber 1 also has lateral passage openings 7 through which the sample can be illuminated or light emitted by the sample can be detected. The passage openings 7 are formed here round, so that, for example, a lens can be introduced into the opening. In the example shown here are four through holes 7 shown, all at the same height on the four sides of the cuboid sample chamber 1 are located, with two opposite passage openings in alignment with each other. The sample chamber 1 has holes 8th in their lateral transformation 6 on so that side plates for closing the through holes 7 can be screwed. Furthermore, the sample chamber has 1 at least one foot 11 or a recess 10 in one embodiment several feet 11 and recesses 10 which serve to position the sample chamber on a dedicated device. Furthermore, holes can 9 at the sample chamber 1 be present, which make it possible to screw the chamber in turn on a dedicated device.

2 shows a view of the bottom 4 from the sample chamber 1 , The underside has a, in this example round, lower opening 5 and drilling 12 on. Furthermore, a possible arrangement of feet 11 and recesses 10 shown.

3 shows a cross section through the lateral conversion 6 and a lateral passage opening 7 , the passage opening tapers here from an inlet opening 15 in the outer surface 13 the lateral conversion 6 lies, towards an exit opening 16 in the inner surface 14 the lateral conversion is located. Through the centroid 15 ' the entrance opening 15 and the centroid 16 ' the outlet opening 16 is an axis 19 defines that of the lateral passage opening 7 assigned. In this example, the axis is orthogonal to the surfaces 13 . 14 the lateral conversion. Are the side through holes arranged as in 1 Thus, the axes of all lateral passage openings intersect at a point inside the sample chamber. The wall 17 the lateral passage opening has a step 18 on. The stage 18 is configured so that an object can be pressed from the outside, for example a lens with a sealing ring.

4 to 6 show various floor panels, with the reference numerals 20 ' . 20 " . 20 "' are provided, and specific embodiments of a general base plate in the sense of the application, with the reference numeral 20 is provided.

4a shows a perspective view of an embodiment of a bottom plate 20 ' from below, down to the sample chamber 1 can be attached. 4b a perspective view of the bottom plate 20 ' from above, and 4c is a side view, wherein in 4c continue a capillary 48 is shown, which may contain a sample. The bottom plate 20 ' can drilling 21 included, which allow the introduction of fasteners, such as screws or bolts, and thus to establish a positive and / or positive connection with the sample chamber 1 by means of the holes 12 on the bottom 4 the sample chamber 1 enable. The bottom plate is with a bellows system 22 made of a flexible material, which has a passage opening 23 through which a capillary with a sample 48 can be inserted through it. The flexible material of the bellows system is liquid-tight at the capillary 48 on, leaving no liquid on the outside of the capillary 48 can escape past the chamber. The lower end 49 the capillary 48 sticks out of the chamber. The bellows system 22 is made of a flexible material so that the capillary 48 can be tilted with the sample. In addition, the capillary can 48 be rotated or moved up or down without the sealing effect of the liquid-tight connection is impaired. An upper area of the capillary 48 , in which the sample is located, protrudes at the top of the bellows system out into the interior of the chamber and can be there illuminated and microscoped. By manufacturing the bellows system from a flexible material, such as rubber, a liquid-tight seal of the sample chamber may also be provided 1 with attached base plate 20 ' be achieved. Furthermore, the bottom plate 20 ' lateral recesses 24 in which sensors or active elements, such as heating elements, can be introduced.

5a shows the top of another embodiment of a bottom plate 20 "and 5b the underside of the bottom plate 20 " , The bottom plate 20 " also has holes 21 for introducing fasteners on a recess 24 for introducing active elements or sensors. In 5a is one at the top of the bottom plate 20 " attached stamp 26 to see. The stamp is formed here round, so that he in the lower opening 5 the sample chamber 1 fits and preferably has a height chosen so that the sample chamber 1 with inserted bottom plate 20 " has a flat bottom. The sample chamber 20 " further comprises a groove 25 into which sealing material, such as a sealing ring, can be introduced around the sample chamber 1 with inserted bottom plate 20 " be able to close liquid-tight.

6a shows the top of another embodiment of a bottom plate 20 " ' and 6b the underside of the bottom plate 20 " '. It also has holes 21 for introducing fasteners and in one embodiment a recess 24 for introducing active elements or sensors. In 6a is the top of the bottom plate 20 " 'shown. There is the opening of a blind hole 27 to see that when the bottom plate 20 " 'to the sample chamber 1 is attached, making sure that the bottom through the bottom plate 20 " 'limited volume of the sample chamber 1 is enlarged. This allows, for example, one from the top of the sample chamber 1 introduced and held from above capillary 48 continue to move up and down than, for example, the bottom plate 20 " is possible.

7 shows an exploded view of a microscope assembly. A floor plate 20 comes with a sealing ring 28 provided and from below between the feet 11 of the sample chamber 1 introduced so that the recess 24 for introducing active elements or sensors due to recess 10 in the sample chamber 1 remains freely accessible. A side passage opening is provided with a side plate 29 and a sealing ring 30 sealed liquid-tight by using the holes 8th the sample chamber 1 , with the sample chamber 1 is screwed. The side plate 29 can either be solid or be designed so that a disc 31 , formed transparent or opaque, can be introduced. A base plate 32 with a side frame 33 forms an outer chamber 34 , The sample chamber 1 is then inside the outer chamber 34 positioned and can be fixed there to make a fixed optical-mechanical reference system. The side frame 33 , and the outer chamber 34 are designed in such a way that different sized sample chambers 1 Find a place. Through guide rails 37 become objective plates 35 on the side frame 33 slidably mounted. In the lens plates 35 can lenses 36 ' . 36 " . 36 "' , preferably designed as immersion objectives, are screwed in. In the embodiment presented here, the chamber is designed for up to four objectives. Along the guide rails, the lens plates can be 35 with the screwed-in lenses 36 ' . 36 " . 36 "' , so move them in the side openings 7 the sample chamber 1 can be introduced. This is done by the guide rails for different sized sample chambers 1 allows. A sealing ring 41 is arranged by lenses 36 ' . 36 " . 36 "' , to the stage 18 the Wall 17 a passage opening 7 can be pressed to the passage opening 7 close liquid-tight. So are all the side openings 7 either by means of a side plate 29 or by means of lenses 36 ' . 36 " . 36 "' , and the lower opening 5 , by means of a base plate 20 , liquid-tight, can be an immersion liquid such as water in the sample chamber 1 be filled and then a capillary 48 through the upper opening 3 into the sample chamber 1 introduced and held from above. The tips of the lenses, preferably designed as immersion objectives 36 ' . 36 " . 36 "' , are then in the immersion liquid, without a disc between lens and sample is necessary, so that optimum quality of the image or the illumination can be achieved. In light-sheet microscopy, two opposing lenses can also be used as illumination lenses, so that both sides of the sample are illuminated equally well. Another, perpendicularly arranged lens serves as a detection lens. The base plate can have recesses 50 so that the base plate can be mounted on a substrate, for example on an optical table or in a microscope. Is the base plate 32 using the recesses 50 screwed to an optical table, on the table with lasers, mirrors, sensors and other elements, an optical assembly can be set up with a beam path that allows the illumination and detection of the sample and is set to the configuration of the outer and inner chamber ,

8th shows a side view of the base plate 32 and the side frame 33 , On the side frame 33 are holes 42 for attachment of guide rails 37 and a passage 43 for inserting a lens 36 ' . 36 " . 36 "' , visible, as well as further drilling 44 used to attach the lens plate 44 can be used. Through a material recess 45 in the lateral frame 33 is one on the base plate 32 mounted pedestal 46 visible. The base 46 is complementary to the feet 11 and recesses 10 the sample chamber 1 designed so that a sample chamber 1 can be plugged on it and not slip. Such is the position of the sample chamber 1 on the base plate 32 and within the side frame 33 defined and thus defines an optical-mechanical reference system. The base 46 also has a recess 47 , so that even when using different sized floor panels 20 the sample chamber 1 can be plugged. Furthermore, the socket may have holes so that the sample chamber 1 using the holes 9 can be screwed to the base. Becomes a bottom plate 20 ' with bellows system 22 used, so the lower end protrudes 49 the capillary 48 in the recess. The lower end 49 The capillary is thus free, so there, for example, a device for nutrient supply to the sample inside the capillary 48 can be connected. The side frame may also include material recesses through which an attached flow system and / or the cabling of the sensors or the active elements of the bottom plate 20 can be performed.

9 shows a top view of the outer chamber 34 with base plate 32 and side frame 33 , as well as the pedestal 46 , The outer frame offers space inside for different sized sample chambers 1 , There are notches on the base plate 50 to see, which make it possible by introducing fasteners, the base plate 32 , for example, on an optical table or inside a microscope, to screw.

10a shows a side view, wherein the sample chamber 1 , with mounted side plate 20 on the bottom plate 32 attached pedestal 46 is mounted. The bottom plate 20 takes place in the recess 47 of the pedestal 46 place and the recess 10 the sample chamber 1 is designed so that recess 24 the bottom plate 20 is accessible to the introduction of active elements or sensors, even if flatter bottom plates 20 as in the figure be introduced shown. Furthermore, the recess 47 big enough that the bottom 4 the bottom plate 20 Exposed and provides space for a river system. By positioning the sample chamber 1 on the pedestal 46 a fixed arrangement is made and defines a mechanical-optical reference system that is easy and reproducible to produce and secured against slipping.

10b shows an exploded view of a side view of the base plate 32 with pedestal 46 , Sample chamber 1 and bottom plate 20 as in 10a shown.

10c shows a perspective view of the base plate 32 with pedestal 46 and on the base mounted sample chamber 1 with mounted base plate 20 as in 10a , In addition, the recesses 50 visible, which serve to fix the arrangement on an optical table or in a microscope.

11 shows a section through a perspective view of the base plate with base and mounted on the base sample chamber with mounted bottom plate as in 10 , The cut runs through the sample chamber 1 , the bottom plate 20 and the pedestal 46 , On average, you can see that the bottom plate 20 here as a base plate 20 "' with blind hole 27 is trained. The blind hole 27 is so under the lower opening 5 the sample chamber 1 positioned that the volume of the sample chamber 1 which can be filled with liquid enlarged and a lower end 49 a capillary 48 in the blind hole 27 can be lowered.

12 shows a perspective view of the base plate 32 with pedestal 46 and on the pedestal 46 mounted sample chamber 1 with mounted base plate 20 , The sample chamber 1 is significantly larger in this example than in the previous examples, for introducing a larger sample. The feet 11 are further inside so arranged that the sample chamber 1 can be positioned on the pedestal and a bottom plate 20 the same size as in the previous examples between the feet 11 can be mounted.

13a shows an exploded perspective view of the outer chamber 34 , a sample chamber 1 with arms 51 and a bottom plate 20 , The poor 51 can by means of the holes 52 and the holes 53 in the side frame 33 be connected to the outer chamber by introducing a fastener. By attaching the arms 52 On the outer chamber, in turn, a fixed arrangement is made and defined a mechanical-optical reference system that is simple and reproducible to produce and secured against slipping.

13b shows a perspective view of the outer chamber 34 with a sample chamber 1 with arms 51 and a bottom plate 20 as in 13a , in assembled condition. Through the recesses 45 in the lateral frame 33 can be used for the sensors, active elements or river systems of the bottom plate 20 required cables or hoses are led to the outside.

LIST OF REFERENCE NUMBERS

1.

sample chamber

Second

Top of the sample chamber

Third

Upper opening of the sample chamber

4th

Bottom of the sample chamber

5th

Lower opening of the sample chamber

6th

Lateral conversion

7th

Lateral passage opening

7 '.

First lateral passage opening

7 ".

Second lateral passage opening

7 " '.

Third lateral passage opening

8th.

Bore on the chamber to secure the side panels

9th

Bore on the chamber to secure the chamber to the base

10th

Recess at the bottom of the sample chamber

11th

Foot of the sample chamber

12th

Bore on the chamber for fixing the bottom plate to the chamber

13th

Outer surface of the lateral conversion

14th

Inner surface of the lateral transformation

15th

Entry opening of the lateral passage opening

15 '.

Centroid of the inlet opening of the lateral passage opening

16th

Outlet opening of the lateral passage opening

16 '.

Centroid of the outlet opening of the lateral passage opening

17th

Wall of the lateral passage opening

18th

Stage in the wall of the lateral passage opening

19th

Axis of the lateral passage opening

20th

baseplate

20 '.

Base plate with bellows system

20 ".

Base plate with stamp

20 ' ".

Bottom plate with blind hole

21st

Bore in the bottom plate for fixing the bottom plate to the chamber

22nd

bellows system

23rd

Through opening in the bellows system for introducing a sample

24th

Recess in the bottom plate for sensors or active elements

25th

Groove in the bottom plate for inserting a sealing ring

26th

Stamp on the bottom plate

27th

Blind hole in the bottom plate

28th

Sealing ring for the bottom plate

29th

side plate

30th

Sealing ring for the side plate

31st

side window

32nd

baseplate

33rd

Side frame

34th

Outer chamber

35th

lensboard

36 '.

First lens

36 ".

Second lens

36 " '.

Third lens

37th

guide rail

38th

Hole in the lens plate for attaching a guide rail

39th

Passage in the lens plate for inserting a lens

40

Through hole in the lens plate for attaching the lens plate

41st

Sealing ring for insertion between the lens and the side passage opening

42nd

Hole in the side frame for attaching a guide rail

43rd

Passage in the side frame for inserting a lens

44th

Through hole in the side frame for attaching the lens plate

45th

material cut

46th

base

47th

Recess in the base

48th

Capillary with sample

49th

Lower end of the capillary

50th

Recess in the base plate for introducing a fastener

51st

Arms of the sample chamber

52nd

Bore in the arms of the sample chamber

53rd

Hole in the side frame for attaching the arms

Claims (15)

A sample chamber (1) for use in a light sheet microscopy, the sample chamber (1) being fillable with an immersion liquid comprising a top (2) having an upper opening (3) through which a sample is inserted into an interior of the sample chamber (1) for illumination and viewing is feasible, when the sample chamber (1) is filled with the immersion liquid, a lower side (4) with a lower opening (5), a lateral wall (6) with at least one lateral passage opening (7, 7 ', 7 ", 7 "'), which is liquid-tightly closed by a lens (36', 36", 36 "'), characterized by a removable bottom plate (20) which is adapted to liquid-tightly close the lower opening (5). Sample chamber (1) after Claim 1 , characterized in that the bottom plate (20) non-positively and / or positively connected to the sample chamber (1) is connectable. Sampling chamber (1) according to one of the preceding claims, characterized in that through a center of gravity (15 ') of an inlet opening (15) of each lateral passage opening (7) located in an outer surface (13) of the lateral walling (6) and a centroid (16 ') lying in an inner surface (14) of the lateral walling (6) outlet opening (16) of each lateral passage opening (7) an axis (19) is defined, wherein the through a first lateral passage opening (7') defined axis and by a second passage opening (7 ") defined axis intersect in the interior of the chamber, preferably at a right angle. Sample chamber after Claim 3 , characterized in that the axis defined by a third lateral passage opening (7 "') coincides with the axis defined by the first lateral passage opening (7'). Sample chamber (1) according to one of the preceding claims, characterized in that the bottom plate (20) has a groove (25) for introducing sealing material. Sample chamber (1) according to one of the preceding claims, characterized in that the bottom plate has recesses (24) for the introduction of sensors or active elements, for example designed for temperature regulation or for temperature measurement. Sample chamber (1) after Claim 6 characterized in that the sample chamber has a recess (10) in a lower region such that a lateral portion of the bottom plate (20) containing the recesses (24) for insertion of sensors or active elements is exposed for the cable routing of the sensors or active elements. Sample chamber (1) according to one of the preceding claims, characterized in that the bottom plate (20) has a region of a flexible material with an opening (23) which is configured such that a capillary (48) passes through the opening (23). can be pushed and the flexible material liquid-tight against the capillary (48), wherein a rotation, translation or inclination of the capillary (48) is made possible, the flexible region, for example designed as bellows system (22) or as a film. Sample chamber (1) according to one of the preceding claims, characterized in that it comprises feet (11) and / or recesses (10) on the underside and / or has arms (51) in an upper or a lateral region. A system comprising a sample chamber (1) Claim 9 and an outer chamber (34) comprising a base plate (32) and a side frame (33), the sample chamber (1) being positionable in the outer chamber (34) and frictionally and / or positively locking with the outer chamber (34) is connectable, wherein a base (46) is provided at a fixed position on the base plate, as far as feet (11) and / or recesses (10) on the underside of the sample chamber are present, to the feet (11) and / or Recesses (10) has complementary structures, on which the sample chamber is positionable, and the base (46) recesses (47), so that at least a portion of the bottom plate (20) of the positioned sample chamber (1) is exposed, and / or wherein the lateral Frame (33), as far as arms (51) of the sample chamber are present, with the arms (51) of the sample chamber (1) is connectable, and the arms (51) are configured so that the positioned sample chamber (1) a fixed distance to the base plate (32) un d the lateral frame (33). System after Claim 10 , additionally comprising at least one objective (36 ', 36 ", 36"') and guide rails (37) which can be attached to the lateral frame (33) and lens plates (35) with threads, so that the at least one objective ( 36 ', 36 ", 36"') can be screwed to the lens plates (35) and together with the lens plates (35) along the guide rails (37) is displaceable. System after Claim 11 in which the lateral passage openings (7) of the sample chamber (1) and the guide rails (37) and objective plates (35) are configured so that a screwed-on objective (36 ', 36 ", 36"') can be inserted into a lateral passage opening (7). can be introduced and wherein this lateral passage opening (7) by the insertion of the lens (36 ', 36 ", 36"') is sealed liquid-tight. System after Claim 12 comprising a plurality of sample chambers (1) of different sizes Claim 9 wherein the base (46) and the side frame (33) are configured such that, depending on sample size and application area, another sample chamber (1) can be positioned on the pedestal (46). Use of a sample chamber (1) according to one of Claims 1 - 9 or a system according to one of Claims 10 to 13 in a microscopy method, preferably in a light-sheet microscopy method, wherein a capillary (48) is inserted through the upper opening into the chamber, a first objective (36 ') is operated as an illumination objective, and into a first passage opening (7') of the at least one lateral Through hole (7, 7 ', 7 ", 7"') is introduced and this liquid-tight, a second lens (36 ") is operated as a detection lens and in a second passage opening (7") of the at least one lateral passage opening (7, 7 ', 7 ", 7"') and liquid-tightly closes it, and an optional third lens (36 "'), which is operated as an illumination objective, into a third passage opening (7"') of the at least one lateral passage opening (7, 7 ', 7 ", 7"') is inserted and this liquid-tight closes. Use of a sample chamber or system in a method according to Claim 14 wherein the microscopy method is a long-term microscopy method, wherein the capillary remains permanently in a holder and in the sample chamber during the microscopy operation and is rotated, tilted and displaced to produce a multi-dimensional recording.

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