DE102006007197B4 - Arrangement for optical observation devices for the examination of cell cultures - Google Patents

Abstract

Arrangement for optical observation devices for the examination of cell cultures, with at least physiological sensors, which are arranged close to a receiving area of the cell cultures around the optical axis (9) of the observation device, consisting of an upwardly open sample vessel (10) for receiving the cell cultures and nutrient media , which is arranged horizontally displaceable to the receiving area lying in the optical axis of the observation device, a curved upper chamber part (3) that can be lowered over the sample vessel (10), in its wall sensors (4) and possibly manipulators and possibly input and output Outlet openings are let in and aligned with the receiving area, wherein the upper chamber part (3) with the sensors (4) can be positioned on a lower chamber part (7) in a closed functional position in which the sensors (4) have a fixed position with respect to the receiving area occupy around the optical axis (9), and the upper chamber part (3) a B. eobachtungsfenster in the optical axis (9) and is connected to means for raising and lowering on the stand of the observation device.

Description

The invention relates to an arrangement for optical observation devices for the examination of cell cultures according to the preamble of claim 1.

From the DE 44 17 078 A1 For example, an apparatus for microscopying test cells, tissue slices or the like organic material is known. The device is a substantially closed culture chamber having a receiving area for the organic material. The chamber has at least one observation window arranged transversely to the viewing direction for an optical observation device, such as a microscope or camera, and at least one inlet opening and at least one outlet opening for a nutrient fluid. Characteristic of the device is that at least one physiological sensor is provided in the chamber, which is arranged at least close to the receiving area.

To introduce the material, the device must be opened and closed again. For this purpose, a screw ring is provided, which holds two spaced apart by an elastic spacer ring housing parts. The screw cap may interfere with easy accessibility of the chamber.

From the DE 44 17 079 A1 is a microscope slide, camera or the like known. The slide has a receiving area for test cells, tissue sections or the like organic material, and is provided with a smaller observation area for the organic material than the receiving area, wherein the slide consists of a transparent material at least in the observation area. Within the receiving area, at least one sensor for measuring physiological parameters is arranged adjacent to the observation area.

The DE 197 53 598 C1 describes a device for measuring physiological parameters bounded by two semiconductor chips spaced by a seal surrounding a test chamber. The cohesion of the device takes place by the semiconductor chips on the outside cross-brackets. The measurement is carried out with the aid of sensors applied to the chips. The device is unsuitable for microscopic observation.

From the DE 197 49 218 A1 is a high-temperature vacuum sample holder for light microscopes known. A high temperature heater is provided with a heating plate mounted on thermally insulating ceramic feet in a flat water cooled housing. This housing has at the bottom a microscope adapter, which can be clamped instead of the slide disc of a sample manipulator table in the corresponding recess. A glass plate on the top allows the coupling of light or the observation of the substrates.

In the DE 198 03 551 C1 a tempering cell for heating or cooling a sample to be examined with a microscope is described, which has a viewing window to the sample space, which is adjustable by electrical heating elements and coolant flowed through cooling channels to a desired temperature. The tempering cell can be mounted on a sample table or table top of a commercial microscope. The tempering is designed high pressure resistant, including the viewing window is glued from the inside of the sample chamber forth on a secured by lock nut window seat and sealed by a self-reinforcing metal gasket. A sample change is carried out via a separate, sealable feed opening.

From the DE 198 15 696 A1 a temperature-controlled overflow apparatus for biological samples is known, which consists of a sample carrier and at least one inlet and one outlet. The inlet is directed obliquely to the sample. The formation of the chamber is not described further.

Furthermore, in the document Wonka, F .; A. Hofer; J. Hoffmann; F. Gölfert; H.E. Krinke; M. Thümmler; R. H. W. Funk: Development of a multifunctional cell chamber for vital microscopy Poster presentation: 32nd Annual Conference of Dtsch. Society for Biomedical Engineering, Dresden, 08.-12.09.1998, published in: Biomedical Engineering, Volume 43, Supplementary Volume 1, 1998: 422-423 a chamber system for receiving and supplying cell cultures known that can be tempered during microscopic observation. The interface between the microscopes used for observation and the chambers is a receiving frame. The main body of the chambers is a plate which is mountable with the slide in the receiving frame.

Also in Hofer, A .; Nagel, F .; Wonka, F .; Krinke, H. E .; Gölfert, F .; Funk, R.H.W .: A novel arrangement of cell chambers is described by means of video-enhanced microscopy Medical & Biological Engineering & Computing / Cellular Engineering, Vol. 37 (1999): 667-669. On a frame in the dimensions of a slide plate two cell chambers are formed, which are closed by a respective cover plate. The cover plates are with the. Bolted frame.

A significant disadvantage of these embodiments is that the frame receiving a base body can only be used on special microscope stages (eg Leica). Furthermore, some problems with regard to tightness, tempering dynamics, biocompatibility, robustness and handling have occurred.

A disadvantage of the above-mentioned prior art that the introduction of the cultured carrier element in the receiving device sometimes requires a lot of experience in handling and often very time consuming (loosening of screw, exact positioning of a large number of sub-elements, etc.). The geometry has an unfavorable effect on optimal optical observability (resolution, etc.); on the one hand, too large a volume of nutrient medium in the culture environment, associated with poor perfusion of the culture, which should be permanent and full-coverage, or on the other hand too small volume, which leads to flow-induced shear forces, which represent an unacceptable mechanical stress on the cultures.

The publication EP 16 30 586 A1 describes a culture chamber with sample vessel and upper, lowerable chamber part for increased sample throughput in a microscope. The sample vessel can be introduced into or removed from the culture chamber after opening. A sensor arrangement with at least physiological sensors in the upper chamber part close to a cell culture to be observed is not described. The document contains an indication that it is desirable to locate a temperature sensor within the sample chamber at a position as close as possible to the sample vessel. However, there is the problem of observability of the sample.

From the DE 102 59 251 A1 a closed culture chamber emerges, in the upper part of which sensors are introduced closely adjacent to the optical axis. Although in such culture chambers, the positioning of the active tips of the sensors as close as possible to a cell culture to be observed, however, closed culture chambers for the exchange of cell cultures by removing the sample vessel can not be easily and quickly open, then close again and then the active tips again in the required position. The sensors are only for temperature measurement.

The publication US 2005/0248836 A1 shows an incubator whose upper part receives sensors close to the optical axis. The active tips of the sensors are fed laterally through the chamber housing and positioned near the cell culture to be observed. The chamber upper part is screwed to the lower chamber part, so that here too there is a closed chamber arrangement which can not be easily opened.

The JP 2005 010 258 describes as a priority document to the already mentioned document EP 16 30 586 A1 also a culture chamber with sample vessel and upper, lowerable chamber part with the disadvantages described.

The object of the invention is to provide a very simple and quick-to-use arrangement for optical observation devices for the investigation of cell cultures, which allows the preservation or targeted influencing of the vitality of both animal and plant cell or tissue cultures and at the same time a good microscopic Observability guaranteed.

According to the invention the object is achieved by the features mentioned in claim 1. Advantageous embodiments emerge from the features mentioned in the subclaims.

According to the invention, the physiological sensors with their active tips, optionally present inlet and outlet openings and further possibly existing manipulators form a planar boundary for the receiving area, wherein the limit of the arrangement of the means in the chamber upper part and is determined by its position , At least in the functional position, the chamber upper part assumes a fixed position with respect to the optical axis, which is determined by the position of the objective and the condenser.

The field of observation is transverse to the optical axis of the observation device and detects the receiving area of the cell culture to be examined. In a lateral displacement of the chamber bottom part of a new recording area is detected in the observation field, the limitation remains the same by the totality of the means for observation.

In contrast to the closed chambers, above all, an improvement in terms of the reception range of the cell culture is achieved. In a change of the receiving area by lateral displacement of the chamber lower part to the optical axis of the observer, the set close distance of the means for supplying and monitoring the cell culture is maintained in each case. This has advantages in the evaluation of the entire cell culture and their care during observation. In addition, the arrangement can be easily and quickly opened and closed and set up for automatic operation.

The invention provides a half-open chamber which, in its functional position for observation of the cell culture, can be closed in a gastight manner like a closed chamber.

Advantageously, the chamber upper part can be fastened to the condenser of an inverted microscope by means such as sensors, inlet and outlet openings and micromanipulators. The chamber upper part is bell-shaped and encloses the lower chamber part with the cell culture and the nutrient medium gas-tight. The sensors pierce the wall of the chamber upper part obliquely to the optical axis and are arranged with their active tips approximately circularly about the optical axis in a height. The positioning of the active tips is such that, in the functional position, there is a narrow boundary for the receiving area and the active tips of the sensors are immersed in the nutrient medium without touching the cell culture. Due to the arrangement of the bell-shaped chamber upper part on the condenser, the chamber upper part can be raised and lowered and quickly brought into the functional position.

According to a variant of the invention, the bell-shaped upper part is arranged on the stand of a microscope and can be raised and lowered by means of an additional device. The lifting and lowering takes place along the optical axis of the observation device.

Advantageously, the upper part can be swung out of the optical axis for automation purposes.

Furthermore, the object is achieved by a method having the features mentioned in claim 10.

For adjusting sensors, inlet and outlet openings and micromanipulators in the arrangement according to the invention, the chamber upper part is positioned with prepositioned sensors, inlet and outlet openings and micromanipulators with respect to a means for determining spatial coordinates, set a maximum allowable nominal dimension for the spatial coordinates, and Sensors, inlet and outlet openings and micromanipulators adjusted by means of the brackets located in the wall of the chamber upper part with respect to the position of their active tips until an actual size is reached below the allowable nominal size.

The nominal dimension is determined by spatial coordinates X, Y, Z and ensures minimum distances from the bottom of the sample vessel and the optical axis of the observer.

The invention will be explained in more detail with reference to exemplary embodiments. in the drawings show:

1 a schematic representation of an inverted microscope with attachment of the chamber upper part of the condenser

2 a schematic representation of an upright microscope with attachment of the chamber upper part of the lens

3 a schematic representation of the inventive arrangement with removed condenser in a three-dimensional view

1 schematically shows an inverted microscope. Shown is a microscope stage 6 with lens 1 below the microscope stage 6 and condenser 2 above the microscope stage 6 , The microscope stage 6 has an observation field 8th on, through which the optical axis 9 runs.

Also shown is a chamber upper part 3 moving in the vertical direction on the condenser 2 is appropriate. The chamber top 3 is bell-shaped and lies in the so-called functional position on the lower chamber part 7 on. The chamber lower part 7 lies on the microscope stage 6 on and can be moved on this in the plane.

Above the observation field 8th in the microscope stage 6 is in the chamber lower part 7 a recess provided in the a sample vessel 10 is used. The sample vessel 10 can be with the chamber lower part 7 above the observation field 8th transverse to the optical axis 9 move.

In the bell-shaped chamber upper part 3 openings are provided in the brackets 5 are fitted. The brackets 5 are for receiving sensors 4 and possibly existing or required inlet and outlet openings provided.

In the holder shown on the left in the drawing 5 is a sensor 4 inserted, with its active tip into the sample vessel 10 protrudes. The active tip is positioned close to the cell culture so that it is not destroyed and the field of view for microscopic observation is not compromised.

In a conventional plurality of sensors (see 3 ) form the active tips of the sensors 4 an annular arrangement around the field of observation 8th so that observation and examination of the cell culture remains possible.

In place of the active tips of the sensors 4 For example, the ends of inlet and / or outlet ports may be placed to supply the cell culture. Likewise, the brackets can 5 be equipped with in the dimensions of the sensors same micromanipulators.

The chamber top 3 lies with lowered condenser 2 on the lower part of the chamber 7 on. In the chamber lower part 7 is in the field of the observation field an opening for a suitable for this purpose sample vessel 10 available. condenser 2 , Chamber upper part 3 and chamber lower part 7 form with inserted sample vessel 10 in the so-called functional position a quasi-closed chamber. This quasi-closed chamber can now be set by tempered gas supply and slight overpressure in a separate atmosphere. The games in the fits on the clashing items are all kept small, so that only a small gas loss arises.

The sensors are arranged in a ring around the optical axis and close to the observation window. The chamber lower part 7 has a twisted seat, which is a stop for the chamber upper part 3 offers, so that a damage of the sensors with a lateral displacement of the chamber lower part 7 is prevented.

The fast interchangeability of the sample vessel 10 is made possible by the chamber top 3 with all sensors 4 and possibly provided inlet and outlet openings with the condenser 2 from the chamber base 7 is lifted, after which the sample vessel 10 from the chamber base 7 can be removed.

After a sample vessel 10 in the chamber lower part 7 has been brought to the intended position, the chamber upper part 3 with all sensors 4 and possibly provided inlet and outlet openings with the lowering of the condenser 2 on the chamber lower part 7 and the active tips of the sensors 4 take back their predetermined position around the observation window 8th one.

2 schematically shows an upright microscope. Shown is a microscope stage 6 with a condenser underneath 2 and a lens above 1 , The chamber top 3 is axially displaceable (in the direction of the optical axis) on the lens 1 attached.

Shown again is the so-called functional position, in which the chamber upper part 3 on the lower part of the chamber 7 is attached. In the opening of the chamber bottom 7 is a sample vessel 10 used. In the holder 5 (left side in the 2 ) is a sensor 4 admitted. For gas-tight completion of the chamber is still an assignment of the holder 5 (right side in 2 ) required by another sensor 4 or can be done by approximately identical inlet or outlet openings.

3 schematically shows an inventive arrangement with microscope stage 6 and chamber lower part 7 , On the chamber lower part 7 sits the spatially represented chamber upper part 3 on. The chamber top 3 is essentially a bell-shaped body whose outer wall has eight flat surfaces. In the chamber upper part are four sensors 4 arranged, which are aligned to the observation field. The sensors 4 are by means of the brackets 5 adjusted in their position. In the chamber upper part 3 is still a closable window 11 provided for visual observation in addition to microscopic observation.

LIST OF REFERENCE NUMBERS

1

lens

2

condenser

3

Plenum top

4

sensor

5

bracket

6

microscope stage

7

Chamber-base

8th

observation field

9

optical axis

10

sample vessel

11

window

Claims (10)

Arrangement for optical observation devices for the examination of cell cultures, comprising at least physiological sensors which are close to a receiving area of the cell cultures about the optical axis ( 9 ) of the observation device are arranged, consisting of an upwardly open sample vessel ( 10 ) for receiving the cell cultures and nutrient media, which is arranged so as to be horizontally displaceable relative to the receiving area located in the optical axis of the observation device, via the sample vessel ( 10 ) lowerable arched chamber top ( 3 ), in whose wall sensors ( 4 ) and possibly manipulators and optionally inlet and outlet openings and are aligned to the receiving area, wherein the chamber upper part ( 3 ) with the sensors ( 4 ) on a chamber lower part ( 7 ) is positionable in a closed functional position, in which the sensors ( 4 ) a fixed position with respect to the receiving area about the optical axis ( 9 ), and the chamber top ( 3 ) an observation window in the optical axis ( 9 ) and is connected to lifting and lowering means on the stand of the observation device. Arrangement for optical observation devices according to claim 1, characterized in that, when the observation device is designed as an inverted microscope, the chamber upper part ( 3 ) has an opening in which a condenser ( 2 ) is admitted. Arrangement for optical observation devices according to claim 1, characterized in that, when the observation device is designed as an upright microscope, the chamber upper part ( 3 ) has an opening in which a lens is recessed. Arrangement for optical observation devices according to claim 1, characterized in that in addition to the means for raising and lowering a means for pivoting the chamber upper part ( 3 ) from the optical axis ( 9 ) is provided. Arrangement for optical observation devices according to one of claims 1 to 4, characterized in that the chamber lower part ( 7 ) on the microscope stage ( 6 ) is designed aufleggbar and a bearing surface for the chamber upper part ( 3 ) having. Arrangement according to one of claims 1 to 5, characterized in that in the chamber upper part ( 3 ) Brackets ( 5 ) are inserted into the sensors ( 4 ), Micromanipulators and / or inlet and outlet openings can be inserted by means of suitable counterparts. Arrangement according to one of claims 1 to 6, characterized in that the inside of the chamber upper part ( 3 ) is lined with a flexible membrane, wherein the membrane at the sensors ( 4 ) and possibly other existing micromanipulators and media lines sealingly. Arrangement according to one of claims 1 to 7, characterized in that the bearing surface on the chamber lower part ( 7 ) has a raised edge, which has a stop for the chamber upper part ( 3 ). Arrangement according to one of claims 1 to 8, characterized in that the bearing surface between the chamber upper part ( 3 ) and chamber lower part ( 7 ) is remunerated. Method for adjusting sensors, micromanipulators and media lines in an arrangement for optical observation devices according to one of Claims 1 to 6 or 8 to 9 and Claim 7, characterized in that the chamber upper part ( 3 ) with prepositioned sensors ( 4 ), Micromanipulators and media lines is positioned opposite a means for determining spatial coordinates, a maximum allowable nominal dimension for the spatial coordinates is set, and the sensors ( 4 ), Micromanipulators and media lines by means of the holders ( 5 ) located in the wall of the chamber upper part ( 3 ) are adjusted with respect to the position of their active tips until an actual dimension below the permitted nominal dimension is reached.

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