Optical: systems and elements – Compound lens system – Microscope
Reexamination Certificate
2002-10-28
2004-10-26
Lavarias, Arnel C. (Department: 2872)
Optical: systems and elements
Compound lens system
Microscope
C359S368000, C359S382000
Reexamination Certificate
active
06809862
ABSTRACT:
The present invention relates to an in-situ microscope device for reactors, such as for example bioreactors.
BACKGROUND OF THE INVENTION
With such in-situ microscope devices, examinations can be carried out on specimens of the material in the inside of the reactor during ongoing operation, for example the concentration of particular cells in the medium can be monitored. The basic principles of in-situ microscopy for reactors are described in patent specification DE 40 32 002 C2.
An in-situ microscope device with the features of the precharacterizing clause of claim
1
is described e.g. in the dissertation “In-situ-Mikroskopie; Ein neues Verfahren zur Online-Bestimmung der Biomasse bei Kultivierungsprozessen” [In-situ microscopy: a new process for online determination of biomass in cultivation processes], Dr. Christoph Bittner, Hanover, 1994.
The monitoring and controlling of biotechnological processes has gained a major significance in the recent past e.g. in the chemical and pharmaceutical industries. Examples of this are the synthesis of human proteins, such as e.g. interleukin (IL-2), tissue plasminogen activator (t-PA) or antithrombin (AT-III), the preparation of which with the help of organic synthesis can be achieved only with difficulty, with the result that the manufacture of these proteins with the help of the cultivation of mammal cells is preferred. Microorganisms, here in particular yeasts, are used also in the manufacture of products of the food industry, e.g. beer, wine, cheese or bread. Further products or pharmaceuticals are produced by the cultivation of other organisms. In the case of in-situ microscopy, a microscope probe is inserted into a connection port of a fermenter (reactor) in order to monitor and control such processes. This microscope probe enables an image to be photographed directly in the culture stock. The photographed microscope image is photographed and digitized by a CCD camera connected to the in-situ microscope. The evaluation of the digitized microscopic images is carried out with the help of image-processing programs on a standard computer. Information about cell sizes and biomass, cell-size distribution, cell concentration, cell morphology and cell vitality can be obtained using the image data material obtained with the in-situ microscope and analyses applied to it. On the basis of the information, thereby obtained during ongoing operation, about the state of the system located in the reactor, process parameters can be influenced and controlled in order to achieve a desired development of the system.
An in-situ microscope for the observation of cultivation processes in yeasts is described in the above-mentioned dissertation by Bittner. The microscope has a dip tube which is inserted into a reactor connection port. In the end-section of the dip tube lying in the inside of the reactor, an inlet is provided through which the culture medium can flow freely. A microscope external tube is arranged coaxially in the dip tube and, with its lens lying at the inner end, is directed towards a specimen zone which is defined between the cover glass of the lens and a slide glass body lying opposite. Connected to the opposite end is the microscope external tube with a camera for photographing the image of the specimen zone. If the specimen zone is open, the lens cover glass and the slide glass body lying opposite lie at some distance from each other, the culture medium from the inside of the reactor being able to flow freely through this space. In order to photograph the image, the specimen zone is closed by moving the slide glass body onto the lens cover glass until a sealing ring surrounding the slide glass body comes to rest against the lens cover glass and thus creates a specimen zone with a defined volume between the slide glass body and the lens cover glass. In the known device, the specimen zone is closed by pulling the slide glass body with an illumination unit below it against the lens. This movement is achieved via a pull rod which runs in longitudinal direction in the dip tube alongside the microscope external tube, and is connected at one end to the unit of the slide glass body and at the other end to a drive outside the dip tube. Furthermore, a wiping apparatus is provided with which the lens cover glass is intended to be cleaned by wiping off if required. Such a wiping apparatus is necessary as the glass rapidly becomes dirty and another cleaning method cannot be carried out at all while cultivation is in progress and, even after the cultivation process is stopped, can be carried out only if considerable effort is expended and the microscope is completely removed from the reactor. The wiper is also driven by an external drive via a mechanical power transmission means.
The known in-situ microscope device is disadvantageous in various respects. For example, it is disadvantageous that mechanical power transmission means alongside the microscope tube must be guided through the dip tube, as this restricts the space available for the microscope tube. Furthermore, such mechanical power transmission means are costly in design terms and are incident-prone.
However, the main disadvantage of the known in-situ microscope devices is that the microscope is not accessible while cultivation is in progress because, if the reactor is opened by removing the dip tube or by pulling out the microscope, the cultivation would be contaminated. Furthermore, if the microscope was installed at the side, the reactor would first have to be emptied, which for practical use is out of the question.
The removability of the microscope is of significance not only for cleaning purposes during the operation, but also for the sterilization/autoclaving of the reactor system before commissioning, as temperatures of over 120° C. are used.
For industrial-scale applications of in-situ microscopy, microscope devices are required which are robust, flexible and easy to handle.
The object of the present invention is therefore to create an in-situ microscope device, the sensitive parts of which are accessible at any time, without having to interrupt the cultivation process or endangering it through a contamination.
The characterizing features of patent claim
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in conjunction with its precharacterizing clause serve to achieve this object. Advantageous versions of the invention are listed in the dependent claims.
BRIEF DESCRIPTION OF THE INVENTION
According to the invention, it is provided that the dip tube is guided movable in axial direction in the reactor connection port, to which a rinsing chamber with sealable openings is externally connected, through which cleaning agents can be fed in. The dip tube can be pulled back into the connection port until the inlet of the dip tube communicates with the rinsing chamber. Sealing means are provided at the dip tube in order to keep the internal space of the reactor sealed off from the rinsing chamber when the dip tube is pulled back into the rinsing chamber. In this way, the specimen zone can be cleaned, when the dip tube is pulled back into the rinsing chamber, by feeding cleaning agents into the rinsing chamber. The internal space of the reactor remains sealed off from the inside of the rinsing chamber in order that the specimen zone can also be cleaned while cultivation is in progress, without the danger of contamination.
Furthermore, the microscope external tube can be pulled out when the dip tube is pulled back into the rinsing chamber. Thereby, all parts of the microscope device which are arranged inside the dip tube can be removed and if necessary replaced or repaired, without the sterile barrier to the inside of the reactor being broken through. Thereby, changes to the design and fitting of the microscope, maintenance work and the like can also be carried out during ongoing operation of the reactor, without the cultivation process in the reactor having to be interrupted, or the danger(of contamination. Thereby, handling during ongoing operation, operational reliability and variability can be decisively improved by replacin
Behnsen Wilhelm
Frerichs Jan-Gerd
Joeris Klaus
Schaper Jorg
Scheper Thomas
Jan-Gerd Frerichs
Lavarias Arnel C.
Nixon & Vanderhye P.C.
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