Abstract:
A fused silica micropipette having a complex shape, and method of manufacture, are disclosed. The fused silica micropipette may be used for microinjection applications, such as assisted conception, wherein the micropipette penetrates a cell membrane prior to injection. A high temperature, laser powered microforge capable of working with fused silica to make the complex structures of the micropipette of the present invention is also disclosed.

Description:
FIELD OF THE INVENTION  
       [0001]     The present invention is related to pipettes, and is particularly directed to a fused silica micropipette for cellular applications.  
       BACKGROUND OF THE INVENTION  
       [0002]     Very fine diameter pipettes, or micropipettes, are used in various scientific applications for research or clinical procedures at the cellular level. As used herein, the term “micropipette” is used to mean a pipette having a tip diameter of less than about 150 microns. Micropipettes may be used, for example, to penetrate the wall or membrane of a living cell in order to inject something into the cell, or to act as a recording electrode, without damaging the cell. Likewise, a micropipette may be used to apply suction to hold a cell for observation or further manipulation during the course of an experiment or procedure.  
         [0003]     Typically, micropipettes are made from glass capillaries that are heated and then stretched to reduce the diameter of a portion of the tubing to a suitable dimension. Exemplary methods and apparatus for making fine diameter pipettes with high precision are disclosed in U.S. Pat. Nos. 4,600,424 and 5,181,948. The foregoing patents, however, are limited to methods and apparatus for making micropipettes which are linear or straight, i.e., wherein the lumen defines a single axis.  
         [0004]     There is a demand for micropipettes having more complex structures that are tailored to the specific applications for which they are used. Such complex structures include micropipettes having tips with specific beveled profiles, having tips with spikes or other added surface features, or having one or more bends near the tip. Techniques for forming glass micropipettes having complex structures using a microforge are described in Chapter 10 “Microtool Manufacture” of the text “Micromanipulation in Assisted Conception” (Flaming, S. D. and King, R. S., Cambridge University Press, 2003). As noted in this chapter, glass micropipettes are commercially available in a variety of different configurations for specific applications.  
         [0005]     One of the most important clinical uses of micropipettes is in the field of assisted conception. In one well-known procedure, a first micropipette is used to aspirate and inject sperm cells directly into an oocyte which is held in position by a second micropipette. Each of the micropipettes used in this procedure is designed with its specific function in mind. Thus, the micropipette used for injection, sometimes referred to as a microinjection pipette, has a different structure than the holding micropipette. Another important application is in the field of embryonic stem cell research.  
         [0006]     Heretofore, available microforges capable of making micropipettes with complex structures have been limited to working with glass. Specifically, previously available microforges have been incapable of working with fused silica (quartz), because of their inability to provide the much higher temperature needed to soften fused silica so that it can be worked. As a result, micropipettes having complex shapes, such as bends, spikes, etc., have heretofore been made of glass, and such glass micropipettes have been considered adequate for use in assisted conception and other cellular microinjection applications. Thus, there are no known fused silica micropipettes with complex structure in the prior art, nor is there any reason articulated in the prior art to make one. 
     
    
     BRIEF DESCRIPTION OF THE DRAWING  
       [0007]      FIG. 1  is a side view of an exemplary embodiment of the fused silica micropipette of the present invention.  
         [0008]      FIG. 2  is a partial view of the tip of an exemplary micropipette of the present invention.  
         [0009]      FIG. 3  is a schematic diagram of a microforge for making the micropipettes of the present invention. 
     
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS  
       [0010]     Quartz, or fused silica, is known to have properties which are useful in certain specific micropipette applications. These properties include strength, low electrical noise, good optical clarity and chemical purity. Thus, for example, the low noise properties of quartz is important for micropipettes used as intracellular recording electrodes. However, fused silica is substantially more difficult to work due to its relatively very high melting point. Thus, heretofore, fused silica micropipettes have generally not been employed unless a specific property of the material was required for the specific application. None of the known beneficial material properties of fused silica has previously been considered important for micropipettes used for microinjection and, therefore, to date there has been no impetus to use fused silica micropipettes in assisted conception or other microinjection procedures.  
         [0011]     Heretofore, microforges capable of heating fused silica to a working temperature have not been available and, therefore, it has been impossible to fashion micropipettes having complex structures out of fused silica. This has not been a perceived problem because glass micropipettes have been considered adequate for those applications requiring complex structures. However, the present inventor has discovered, after developing a high temperature microforge capable of working with fused silica, that fused silica micropipettes have unexpected advantages when used in certain microinjection applications such as assisted conception.  
         [0012]     While applicant has discovered that the fused silica micropipettes of the present invention produce superior results in assisted conception, the reasons why they are superior is not fully understood. The superior results appear to be unrelated to any of the known reasons for using fused silica, i.e., there is no apparent relation between the superior results discovered by the applicant and the greater strength, lower noise, optical properties, etc., of fused silica. Specifically, the applicant has discovered that the fused silica micropipettes of the present invention more readily penetrate through cell membranes and are, therefore, easier for clinicians and researchers to work with than glass pipettes for conducting procedures such as microinjection. Applicant hypothesizes that the greater apparent ease with which fused silica penetrates into cellular tissue is related to the surface chemistry of the quartz which makes the surface less “sticky” relative to the membrane materials. It is believed that this could be related to the fact that fused silica has fewer hydroxyl (OH—) groups on its surface than glass.  
         [0013]      FIG. 1  depicts a fused silica micropipette  10  in accordance with a preferred embodiment of the present invention, which is useful in connection with clinical assisted conception procedures. The illustration of  FIG. 1  is not drawn to scale. Fused silica micropipette  10  comprises a shaft  20  which constitutes the major portion of the micropipette. At one end of shaft  20  the micropipette reduces diameter at a shoulder region  25  which leads to a shank  30  of reduced diameter. Shank  30  may either be substantially constant in diameter or slightly tapered. Shank  30  has a bend  35  formed therein, as described below, defining a tip portion  40  of micropipette  10 . As depicted in  FIG. 2 , tip portion  40  has an end  45  having an opening  50  therein and a spike  60 . End  45  is preferably beveled. Various types of bevels may be used, depending on the application and the preferences of the user. Opening  50  communicates with the capillary lumen, permitting micropipette  10  to aspirate or inject materials, such as sperm cells, embryonic stem cells, etc. In other embodiments, such an opening may be used to apply slight suction to hold a cell in position. However, a micropipette used for holding a cell usually does not have a spike. Spike  60  enhances the ability of micropipette  10  to penetrate a cell wall membrane.  
         [0014]     In a preferred embodiment, shaft  20 , shoulder  35  and the portion of shank  30  between the shoulder and bend  35  are all coaxial, define a single axis  15 . Likewise, tip  40  defines an axis  55  which intersects axis  15  at bend  35 . According to one embodiment of the present invention, the angle θ between axes  15  and  55  is preferably in the range of about 30° to about 60°. The cellular level applications which use micropipettes generally require the use of microscopes and micromanipulators to observe and conduct the procedures. Frequently more than one tool is in use at a time within the relatively small area in the microscope&#39;s field of view. Therefore, other bends may be made in micropipette  10 , as required, in order to facilitate use of the tool in a crowded space. Thus, the angle θ may simply be viewed as the angle of bend  35 , without regard to the axial alignment of the remaining structure of micropipette  10 .  
         [0015]     A high temperature micropipette puller capable of processing fused silica, such as the P-2000 laser-based system available from Sutter Instrument Company of Novato, Calif., may be used in a traditional way to stretch a fused silica capillary to reduce its diameter to a desired dimension. Capillaries having a starting diameter of 1 mm or more may be used. As is known, the stretched capillary is scored and broken to form a tip which is then beveled using a suitable grinding wheel or other abrasive surface. After this procedure, shank  30  preferably has a diameter less than about 150 μm. The desired tip diameter depends on application for which the micropipette is to be used. In one preferred embodiment the tip diameter is less than about 10 microns. The pulling and subsequent processing produces a micropipette (not shown) having shaft  20 , shoulder  25 , shank  30 , beveled tip  45  and opening  50 . Further working of the tool requires the use of a microforge.  
         [0016]      FIG. 3  is a schematic diagram of a novel high temperature microforge, developed by the present inventor, for making the fused silica micropipettes of the present invention. Micropipette  10  is held in the microforge by micromanipulator  330  or other suitable micropositioning device. The tip of the micropipette is positioned in the field of view of microscope  340 , where it is heated using a beam from laser  350 , under the control of laser control electronics  360 . In a preferred embodiment, laser  350  is a CO 2  laser. Adjustable focusing optics  355  concentrate the laser beam to a desired spot size within the field of view of microscope  340 . The position at which the focused laser beam strikes the micropipette may be adjusted by micromanipulator  330 . Beam detector  370  provides feedback to laser control electronics concerning the beam intensity. While beam detector  370  is depicted in  FIG. 3  between laser  350  and focusing optics  355 , it could, alternatively, be positioned after focusing optics  355 . One or more of these devices, e.g., the laser controller, the micromanipulator, the focusing optics, etc., may be under the programmable control of a computing device (not shown) such as a personal computer.  
         [0017]     In order to create a bend in the micropipette, such as bend  35 , the laser beam is focused onto the shank of the micropipette at the point where the bend is to be made. The laser power output and duration of beam application can be adjusted to provide the correct amount of heat necessary to create the bend. The temperature differential arising from the fact that the laser beam is incident only on one side of the micropipette is usually sufficient to cause spontaneous bending. If desired, however, a mechanical force can be used after the tool has been heated to assist the bending operation. While one bend  35  is shown in the preferred embodiment of  FIG. 1 , multiple bends may be created in a single micropipette in the same manner.  
         [0018]     Spike  60  may be formed on the tip of the micropipette by using the laser to heat a small ball of fused silica until it is molten, then moving the tip of the micropipette into contact with the molten ball using micromanipulator  330 . Alternatively, the molten ball may be moved into contact with the tip of the micropipette. After contact is made, the tip and the ball are quickly separated using the micromanipulator, resulting in the formation of spike  60 . By adjusting the power output of the laser, the viscosity of the molten ball can be varied, thereby providing the ability to control the length of spike  60 . Micromanipulator  330  may be programmed to withdraw a set distance with a desired velocity profile in order to provide reproducible results.  
         [0019]     The embodiments described above are illustrative of the present invention and are not intended to limit the scope of the invention to the particular embodiments described. Accordingly, while one or more embodiments of the invention have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit or essential characteristics thereof. For example, while the construction of the preferred embodiment of the present invention has been described in connection with a capillary, those skilled in the art will appreciate that a solid fused silica rod my be used for tools not requiring a lumen. Accordingly, the disclosures and descriptions herein are intended to be illustrative, but not limiting, of the scope of the invention, which is set forth in the following claims.