Chemistry: molecular biology and microbiology – Apparatus – Mutation or genetic engineering apparatus
Reexamination Certificate
2000-06-14
2001-06-26
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Mutation or genetic engineering apparatus
C310S036000, C310S328000
Reexamination Certificate
active
06251658
ABSTRACT:
DESCRIPTION
1. Field of Invention
This invention relates to an inertial impact drill, and is especially suitable for use in cytological applications, as a clinical or scientific instrument for making microscopic holes in biological tissue.
It is a feature of this invention to provide an instrument that is able to puncture a cell wall with ease and enter the cell with a minimum damage thereby to preserve its viability.
2. Background
There is an increasing need for being able to inject into biological cells and their nuclei, genetic and other materials. Such procedures are used in cloning, in-vitro fertilization, genetic research, and in developing methods for treating cancer and genetically caused diseases. During procedures of this type a micropipette is used, guided by a micromanipulator, to penetrate the cell wall and in many cases then to enter the cell nucleus. In scientific experiments involving biological cells, microelectrodes are also frequently used to measure changes of electrical potentials or to apply electrical potentials to a cell.
Many methods and instruments are available now to facilitate cell wall puncture. All of them involve micromanipulators, which are used to force the tip of the micropipette through the cell wall. Some of these methods tend to severely deform the cell and frequently to cause damage, making the cell unusable for a particular task. Furthermore, the use of some of these instruments requires considerable skill. Representative examples of such methods and instruments may be found in the following patents and literature.
U.S. Pat. No. 3,835,338 of Sep. 10, 1974 discloses an electrically controlled ultra-micromanipulator that utilizes three sets of electrostrictive actuators orthogonally arranged to move and position a probe in small incremental steps.
U.S. Pat. No. 4,894,579 of Jan. 16, 1990 describes apparatus that uses a piezoelectric/electrostrictive element driving an inertia member to produce, through inertia, a fine net incremental displacement of a moving member.
U.S. Pat. No. 5,225,750 of Jul. 6, 1993 discloses a device that uses a piezoelectric or an electrostrictive element to generate rotary motion to, in turn, drive a plunger in controlled very small steps. The plunger moves a liquid to be injected through a microsyringe.
U.S. Pat. No. 5,229,679 of Jul. 20, 1993 describes a micromanipulator for biological applications that uses piezoelectric or electrostrictive actuators to drive an inertial mass to control, through impact, the incremental displacement of an aim in very small steps, facilitating the positioning in three axes of a miniature instrument attached to the arm. In the cases of using the device to puncture cell walls, the arm with the miniature instrument, e.g., a micropipette, advances in small steps due to the impacts through the cell wall. As in all systems of this type the incremental forward motion may cause damage to the cell.
An article by T. Higuchi of the University of Tokyo, Japan (“Innovative Actuators for Micromanipulation and Microinjection in Biotechnology”, Proc. of the 4th Toyota Conf., 21-24 October 1990, Elsevier Science Publishers, 1994).
Prime Tech, a Japanese company, sells the PMM micromanipulator device, which uses a piezoelectric element to produce fine linear incremental forward motion of a micropipette used for injection of materials into cells.
Narishige, another Japanese company, sells a micromanipulator in conjunction with a micropipette to inject matter into biological cells. The device generates vibration using ultrasonic vibration for the penetration of the cell wall as the micromanipulator moves the micropipette forward.
Japanese patent, Publication Number 03166079 A (1991) discloses a means for eliminating vibration at the tip of a micro-instrument driven by a piezoelectric element and for micro-motion of this instrument in a straight line.
Japanese patent, Publication Number 04207982 A (1992) describes a piezoelectrically actuated micro-motion device in which two orthogonally placed piezoelectric actuators are driven by sinusoidal voltages.
Japanese patent, Publication Number 04041187 (1992) shows apparatus similar to that of U.S. Pat. No. 5,229,679, with a foot pedal controller.
W J. Lederer in his paper “Piezoelectric translator. A simple and inexpensive device to move microelectrodes or micropipettes small distances rapidly” (Pflugers Archives, v. 399, No. 1, September 1983, pp. 83-6) describes the use of any piezoelectric buzzer in conjunction with a simple mechanism to provide a rapid linear displacement over a distance of up to 15 nm of microelectrodes or micropipettes.
R. Hengstenberg discusses in his article “A piezoelectric device to aid penetration of small nerve fibers with microelectrodes” (J. Neurosci. Methods, v. 4, no. 3, October 1981, pp 249-55) a means for quickly penetrating the cell walls by a rapid advance to insert microelectrodes.
Burleigh Instruments, Inc., of Fishers, N.Y., USA, sells a piezoelectric linear stepper motor, the LSS-1000 Inchworm® System, for positioning over a distance of many millimeters microelectrodes and micropipettes for in-vitro and n-vivo electrophysiological procedures. The Inchworm (U.S. Pat. Nos. 3,902,084 and 3,902,085) produces sub-micrometer size steps with high acceleration and high velocity that enables the tip for the microelectrode or micropipette to penetrate many millimeters of biological tissue. This device, however, may cause damage to cells, especially the ova, because it produces large indentations in cell walls.
Burleigh Instruments, Inc. also sells a piezo-electric actuator (the LSS-2000 Cell Penetrator System) for rapidly advancing microelectrodes and micropipettes in rapid programmable steps of up to 200 micrometers. This product has been successfully used, for example, to penetrate frog muscle fibers (Charleton and Robitaille, J. of Neuroscience, January “92, pp 297-305). However, this product does not work for cells that have thick walls, such as the ova.
All of these prior art devices achieve or facilitate penetration of cell walls by way of controlled net forward displacement of a micropipette or a microelectrode. Such displacement that forces the tip of the micropipette or microelectrode through the cell wall can lead to gross permanent distortion of cell wall with the resulting damage to the cell.
SUMMARY OF THE INVENTION
An inertial impact drill according to our invention facilitates the penetration of cell walls with a minimum damage by imparting inertial impact on the tip of an insertion element micropipette or a microelectrode without any net displacement of the tip. The smooth penetration is further facilitated by the mechanical oscillation of the tip of the micropipette or microelectrode at their resonant frequencies that are significantly higher than the repetition rate of the impact pulses. Any forward motion that might be required, as the cell wall is being gradually eroded to make an opening, is provided by a separate micromanipulator to which the drill is attached. In many cases the micromanipulator is only required to advance the assembly of the drill and the micropipette or the microelectrode sufficiently far to press the tip against the cell wall forming a small indentation. The impact action of the drill then erodes the cell wall generating an opening without further advancement by the micromanipulator. In the drill of the invention, actuators are energized by triangular electrical pulses that approximate impulses and thus are constituted of a fundamental frequency and a very large series of harmonics with frequencies greater than 1 KHz. Consequently, one of these harmonics will be close to the natural resonant frequency of the micropipettes or microelectrodes and thus will excite mechanical oscillation at the natural resonant frequency, which is higher than the repetition rate of the impulses.
Preferably the drill provided by the inventor has opposing actuators attached to opposite ends of an inertial mass. The actuators are biased with a dc voltage such that they are maintained at about one half of t
Culhane Robert L.
Fasick, III John C.
Friedrich Edward H.
Henderson David A.
Burleigh Instruments, INC
Lukacher M.
Redding David A.
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