Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1998-08-05
1999-12-07
Leary, Louise N.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
435 405, 4352831, 435 4052, 435 968, 435973, C12Q 100, C12Q 108
Patent
active
059981296
DESCRIPTION:
BRIEF SUMMARY
The invention concerns a process and apparatus for micro injection, sorting and harvesting of single biological objects. The objects are disposed side by side on a fixed planar carrier. This process is suitable for the micro injection of specific substances into individual biological objects, for example cells, and subsequently to sort them. Further with this process single objects can be spacially separated from a very great number of objects (e.g. 10.sup.5 to 10.sup.9) and singled out. The separation of heaped up cells/cell clumps as a unit is also possible. Also the process can be used for the separation of specific cells from a tissue sample. A precondition for this sorting process is the prior recognition and selection of the objects concerned on the basis of specific (qualities (e.g. by color, fluorescence marking or by radioactive marking). Within tile term "biological objects" in the context of the present application is meant mainly live or trapped biological cells or parts of cells.
For injection of material into livinig cells usuially micro capillary tubes were used that were usually controlled by usually a pneumatic or hydraulically moveable micro manipulator. The desired substances are injected into the individual cells under great mechanical stress. The manufacture of sterile micro capillary tubes is tine consuming and expensive.
Tsukakosli et al. (1974) and Tao et al. (1987) used a focused laser beam to drill small self healing holes without mechanical contact in a cell membrane. The short opening time is sufficient for the material that is dissolved in the surrounding fluid to penetrate the cell. A larger efficiency in laser micro injection of genetic material was achieved when the laser blasts the hole directly into the cell.
The problem with this method is that for a precise laser micro injection in the submicron region the target objects have to be approached with an accuracy of the order of nanometers in the lateral, that is in the X/Y direction, and also in the vertical, that is in the Z direction. For an automated micro injection the relevant target cells have to be recognized via an image recognition process, then positioned in the line of fire of the laser and most importantly then exactly focused in the Z direction.
A further problem is to isolate or to prepare the successfully injected cells from the other cells for further tests.
For the separation of single biological objects there exist optical methods, such as the optical tweezer in which the object moves in an aqueous solution (K. Schutze, A. Clement-Sengwald, Nature 667 (vol. 368) 1994). On account of the very small transfer of force this method is limited to objects that are able to move freely in the solution. As the sorted and unsorted objects are in the same solution a separate cultivation is only achievable with extra expenditure of effort. For a separate cultivation of cells they have to be separated or sucked apart by another method, for example, with micro capillary tubes. Adherently growing cells or trapped cells on a dissected sample can be separated with a fine needles that are moved by means of a micro manipulator. In this situation the cells are contacted directly and thereby can be mechanically stressed. In addition there is the danger of contamination by undesired cell material. Both methods are comparatively time consuming such that they are not suitable for the manipulation of a large number of objects.
For the separation of single cells from a large number (>10.sup.6) dispersed in a fluid there are commercially available devices for separating and sorting biological objects. While in the fluorescence activated cell sorter (FACS) electro static principles for the spacial separation are used the magnetic activated cell sorter (MACS) operates with magnetic forces. In these systems the cells are however, not disposed side by side on a planar carrier. In addition both of these methods have the disadvantage that many of objects can only be separated to a limited extent (FACS) or even not be separated from each
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Schutze Karin
Schutze Raimund
Leary Louise N.
P.A.L.M. GmbH
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