Optical: systems and elements – Compound lens system – Microscope
Reexamination Certificate
2000-10-31
2003-12-09
Spector, David N. (Department: 2873)
Optical: systems and elements
Compound lens system
Microscope
C435S283100, C435S286200, C359S398000
Reexamination Certificate
active
06661575
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to micromanipulation operations in the fields of microtechnology and nanotechnology. More specifically, the invention relates to methods and apparata for positioning micro-
anoobjects, e.g., cells, genetic material, and molecules, in a user-defined orientation.
BACKGROUND OF THE INVENTION
The micromanipulation of biological material such as cells is of extreme importance to modern biological investigation and biotechnology. Generally, a microscope, usually an inverted phase contrast microscope, is used to visualize the biological material to be manipulated. A 1, 2, or 3 axis micromanipulator is then used to manipulate and position the biological material within the view of the microscope. Usually such micromanipulators consist of a joystick and a microdrive which is moved in response to the user's joystick actuation. Current microdrives use pneumatic, hydraulic, mechanical (e.g., screw), electromechanical (e.g., stepping motor), electromagnetic, electrostatic, piezoelectric, and magnetostrictive principles of operation to provide movement, with examples being provided in U.S. Pat. Nos. 3,835,338; 4,139,948; 4,270,838; 4,367,914; 4,610,475; 4,679,976; 4,694,230; 4,700,584; 4,749,270; 4,836,244; 4,894,579; 4,901,446; 5,325,010; 5,456,880; 5,677,709; 5,831,166; 5,845,541; 5,973,471; and 6,055,859.
Microtools, such as glass micropipettes or microcapillaries, are attached to the microdrives. The size, shape, and other characteristics of the microtool depend on the operations to be performed; for example, a micropipette for fixing (holding) oocytes generally has a round diameter of about 50-120 microns, whereas the diameter of a micropipette for fixing other cells could be less than 50 microns, or as great as 120 microns (or sometimes greater). Two or more microtools are often used simultaneously on the same microdrive. One microtool (generally a micropipette), termed the holder and denoted by the reference numeral
12
in
FIG. 1
, is used to fix/grasp the biological material (e.g., an oocyte) so as to effect its micromanipulation. The fixing of an oocyte
100
at the tip of the holder
12
involves supplying negative medium pressure inside the holder
12
. The negative pressure can be supplied by several sources, such as a microsyringe or a vacuum device connected to the holder
12
by tubing. A vacuum device, such as in U.S. Pat. No. 5,456,880, is preferred because it provides a desired precise amount of pressure, and constant negative or positive pressure can be maintained even if medium leakage owing to non-tight contact with the oocyte at the holder tip occurs.
In some applications, precise manipulation of biological material can be of critical importance. For example, operations on oocytes—such as removal of genetic material from an oocyte, transferring somatic cells under an oocyte's zona pellucida in cloning technology, and injection of genetic material into an oocyte's pronucleus for production of transgenic animals—all present problems of oocyte orientation. Oocytes must also be carefully manipulated into a precise orientation when performing IVF (in vitro fertilization) or other assisted reproduction operations such as ICSI (intracytoplasmic sperm injection), PZD or ZD (partial or full zona dissection), SUZI (sperm under zona injection). In most species, an oocyte is comprised of an animal and vegetal component, and the location of the oocyte's first polar body identifies the animal pole. For example, referring to
FIG. 1
, ICSI into an oocyte
100
involves orientation of the matured oocyte
100
with the holder
12
to situate the polar body
102
at a 6 o'clock or 12 o'clock position. Otherwise, a micropipette or other microtool acting on the oocyte
100
from at or near the horizontal plane could damage the meiotic spindle and metaphase plate during ICSI.
The micromanipulation of biological cells as described in the above-referenced patents uses a combination of weakly-controlled cell rotation and translational cell motion, and generally only allows for partial (i.e., less than 360°) rotation. Usually, after being coarsely oriented in generally the desired orientation, the oocyte
100
is non-firmly fixed in the holder
12
so that it may be finely oriented by the use of other micropipettes. Finally, once the oocyte
100
is positioned as desired, it is firmly fixed on the holder
12
so further microoperations may be performed e.g., injection of spermatazoa in ICSI. Often, difficulties are encountered because the non-spherical shape of the oocyte
100
leads to undesired oocyte movement and orientation, particularly when fine orientation is being performed. Additional problems occur because the injected cells tend to stick to the surfaces of the holder
12
and.or other microtools, especially when somatic cells or spermatozoa are inside the micropipette or other injection apparatus. Thus, orientation of oocytes is often a repetitive, time-consuming, trial-and-error process, and leads to significantly decreased efficiency in operations such as cloning, IVF and the like. Time is wasted in attaining the proper orientation, and the oocyte
100
is meanwhile experiencing time damage because it is resting in a non-native environment. This is a significant factor in causing the failure of such operations.
Apart from orientation problems in biological fields, similar problems exist in the field of scanning probe microscopy (SPM) and in nanotechnology. In such fields, it is necessary to rotate and otherwise orient microparticles, nanoparticles and molecules in order to observe or manipulate them, and thereby create nanostructures and nanodevices. See, for example, U.S. Pat. Nos. 5,606,162 and 5,760,300.
SUMMARY OF THE INVENTION
The invention, which is defined by the claims set forth at the end of this document, is directed to methods and apparata for manipulating micro-
anoobjects which at least partially alleviate the aforementioned problems. A basic understanding of some of the preferred features of the invention can be attained from a review of the following brief summary of the invention, with more details being provided elsewhere in this document.
The invention involves methods and apparata for manipulating micro-
anoobjects wherein a microtool exerts an attractive force on the micro-
anoobject, and a vibrator coupled to the microtool generates oscillating motion in the microtool (and more preferably orbital motion in the microtool) in at least one plane. It has been found that so long as the attractive force on the micro-
anoobject is not too high, the oscillating motion of the microtool will cause rotation of the micro-
anoobject, with the “weak” attractive force of the microtool maintaining the micro-
anoobject adjacent the microtool during such rotation. In essence, the microtool oscillation drives the rotation of the micro-
anoobject, while at the same time it helps to avoid sticking of the micro-
anoobject to the microtool. Once the micro-
anoobject has been rotated to a desired orientation, the attractive force of the microtool on the micro-
anoobject can be increased to such a level that the micro-
anoobject will be firmly fixed to the microtool. Such fixation is more easily accomplished if the orbital motion of the microtool is ceased after the micro-
anoobject is rotated to the desired orientation.
Orbital motion of the microtool can be achieved by providing linear oscillating inputs to the microtool in two different directions (e.g., in two different orthogonal directions), and providing a phase difference in the oscillations. In effect, the oscillating inputs to the microtool are tailored to have the microtool move in a Lissajous pattern. Orbital motion may occur in one or more planes, i.e., in one or more degrees of freedom, to rotate micro-
anoobjects in these planes. As an example, if the vibrator includes three oscillators affixed to the microtool which respectively cause microtool oscillation in the X, Y, and Z directions, coordination of the oscillations
DeWitt Ross & Stevens S.C.
Fieschko, Esq. Craig A.
Spector David N.
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