Abstract:
An assembly for viewing cells inside tissue of a living organism. The assembly includes a confocal microscope having an objective for magnifying an image positioned at a focal plane of the objective and a light source adapted to direct light through the objective. The assembly also includes a rigid elongate tube extending from the objective to a tip sized and shaped for penetrating the tissue of the living organism. The tube has a hollow interior aligned with the objective. The interior of the tube is free of fiberoptic bundles and cover glasses. The assembly also includes a unitary cylindrical lens positioned in the hollow interior of the tube for transmitting light from the light source to the cells inside the tissue of the living organism adjacent the tip to illuminate the cells. The lens has a focal plane adjacent the tip positioned at a location corresponding to the illuminated cells, an image plane opposite the tip positioned at the focal plane of the microscope objective and a sufficient resolution for transmitting an image of the illuminated cells positioned at the focal plane of the lens to the focal plane of the objective.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This is a continuation of U.S. patent application Ser. No. 09/150,113 filed Sep. 9, 1998 now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to microscopes, and more particularly, to methods and apparatus for viewing and manipulating cells inside living tissue with a microscope. 
     Before tissue can be viewed using conventional microscopes, it must usually be removed from its host organism, especially when features below the surface of the tissue are viewed. However, living tissue cannot survive long after removal from its host without sophisticated support equipment. Further, the tissue may change if the support equipment does not precisely duplicate its natural environment. Although a few conventional microscopes (e.g., surgical microscopes) have been designed to view living tissue without removing it from its host, these microscopes have limited resolution. Therefore, small features cannot be seen with these microscopes. 
     Due to the inherent limitations of conventional microscopes, many features of living tissue have not been viewed directly. For instance, physical changes in the human brain resulting from internal processes have not been viewed at the cellular level. As a result, information such as how quickly connections (e.g., synapse connections) are made and lost within the brain is unknown. Further, viewing removed brain tissue does not permit clear understanding of these processes because complex behavior (e.g., speech or learning) cannot be studied when the tissue is removed from its host. The inability of conventional microscopes to view brain cell connections is particularly frustrating because it is envisioned that viewing these connections could answer questions concerning the causes of brain dysfunction such as Alzheimer&#39;s disease. 
     One of the reasons tissue must be removed from its host before it may be viewed by most conventional microscopes is that the tissue must be highly illuminated to be seen through the microscopes. Confocal optical microscopes eliminate this problem by illuminating the tissue with a laser aimed at the tissue through the lens of the microscope. These microscopes make it possible to view an object without an external illumination source. However, conventional confocal optical microscopes cannot view more than a very short distance (i.e., about 200 nm) below the surface of the tissue. Thus, deeper tissue cannot be viewed without separating the tissue from the living organism. 
     Conventional microscopes and methods of use have other disadvantages which limit their usefulness when viewing tissue inside a host. For instance, stains are ordinarily applied to tissue before being viewed with microscopes to improve the optical attributes of features within the tissue. However, the amount of stain used to produce suitable optical attributes frequently kills or injures cells in the tissue and sometimes harms the host. Therefore, conventional methods of applying stain are generally not appropriate when examining living tissue inside a host organism. 
     SUMMARY OF THE INVENTION 
     Among the several objects and features of the present invention may be noted the provision of a microscope attachment capable of viewing internal features of tissue without removing the tissue from its host; the provision of a microscope which enables small amounts of stains and/or other fluids to be precisely directed toward a particular site in tissue within the field of view of the microscope; the provision of a microscope capable of precisely positioning instruments for manipulation of tissue within the field of view of the microscope; and the provision of a microscope having a fluid delivery system which delivers fluid to a site within the field of view of the microscope in amounts which are effective and substantially nontoxic. 
     Briefly, apparatus of this invention is an assembly for viewing cells inside tissue of a living organism. The assembly includes a confocal microscope having an objective for magnifying an image positioned at a focal plane of the objective and a light source adapted to direct light through the objective. The assembly also includes a rigid elongate tube extending from the objective to a tip sized and shaped for penetrating the tissue of the living organism. The tube has a hollow interior aligned with the objective. The interior of the tube is free of fiberoptic bundles and cover glasses. The assembly also includes a unitary cylindrical lens positioned in the hollow interior of the tube for transmitting light from the light source to the cells inside the tissue of the living organism adjacent the tip to illuminate the cells. The lens has a focal plane adjacent the tip positioned at a location corresponding to the illuminated cells, an image plane opposite the tip positioned at the focal plane of the microscope objective and a sufficient resolution for transmitting an image of the illuminated cells positioned at the focal plane of the lens to the focal plane of the objective. 
     In another aspect, the invention includes an attachment for use with a microscope. The attachment includes a rigid elongate tube having a hollow interior extending to a tip having a width of less than about three millimeters to permit the tip to be inserted into tissue of a living organism. The attachment further comprises a unitary cylindrical lens positioned in the hollow interior of the tube for transmitting an image of a specimen positioned at a focal plane of the lens adjacent a front end thereof to an image plane of the lens adjacent a rear end thereof opposite said front end. The lens is free of fiberoptic bundles and cover glasses between the focal plane of the lens and the image plane of the lens. The lens has a sufficient resolution to permit cells of the tissue to be viewed through the lens with a microscope. The attachment also has a mount for mounting the tube and lens on a microscope in a position wherein the image plane of the lens corresponds with a focal plane of the microscope objective. 
     In yet another aspect, the invention includes a method of viewing cells inside tissue of living organisms. The method comprises the steps of mounting a cylindrical lens adjacent an objective of a microscope and adjusting the lens so an image plane of the lens corresponds with a focal plane of the microscope objective. The lens and the objective are simultaneously moved as a unit to penetrate the tissue and position the lens so the cells lie within a focal plane of the lens. 
     Other objects and features of the present invention will be in part apparent and in part pointed out hereinafter. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is an elevation in partial section of a microscope attachment of the present invention; 
     FIG. 2 is a bottom plan of the attachment in partial section; 
     FIG. 3 is an elevation of the attachment shown inserted into a brain of a patient; 
     FIG. 4 is a detailed cross section of the attachment shown introducing a fluid into a brain of a patient; and 
     FIG. 5 is a detailed cross section of a second embodiment of the attachment shown guiding an instrument into a brain of a patient. 
    
    
     Corresponding reference characters indicate corresponding parts throughout the several views of the drawings. 
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Referring now to the drawings and in particular to FIG. 1, an attachment for use with a microscope is designated in its entirety by the reference numeral  10 . The attachment  10  generally comprises a lens assembly, a mount and a focusing mechanism, generally designated  12 ,  14  and  16 , respectively. 
     The lens assembly  12  includes a unitary cylindrical lens or microlens  20  such as a Selfoc® microlens having a small diameter, (e.g., less than about 3.0 mm). Selfoc is a federally registered trademark of Nippon Sheet Glass Co., Ltd. of Osaka, Japan. Selfoc® microlenses are available through NSG America, Inc. of Somerset, N.J. The microlens  20  transmits an image of a specimen (not shown) positioned at a focal plane FP of the microlens adjacent its front or lower end  22  to an image plane IP of the microlens adjacent a rear or upper end  24 . As will be understood by those skilled in the art, the microlens is a unitary cylindrical lens having a diameter less than about 3 mm, and more preferably less than about 0.5 mm, having an objective lens portion  26  (FIG. 4) and a rlay lens portion  28  (FIG.  4 ). 
     As shown in FIG. 4, a tube or sleeve  30  surrounds the microlens  20  to protect it from damage. The tube  30  has a hollow interior  32  extending downward from an upper end  34  to a tip  36  adjacent the front (lower) end  22  of the microlens  20 . The tip  36  is sufficiently narrow (e.g., less than about 3 mm, and more preferably less than about 0.5 mm) to permit the tube  30  and microlens  20  to be inserted inside living tissue without severely damaging the tissue. Although other materials may be used without departing from the scope of the present invention, the tube of the preferred embodiment is an  18  gauge stainless steel tube. Alternatively, it is envisioned that the tube may be made of glass, plastic or other insulating material. The microlens  20  is adhesively bonded inside the hollow interior  32  of the tube  30  in the preferred embodiment, but it is envisioned that other means of attachment may be used and that the microlens  20  may be made removable from the hollow interior  32  of the tube  30  without departing from the scope of the present invention. The tube  30  includes a radial flange forming a collar  38  about midway between the upper end  34  and the tip  36  for engaging the focusing mechanism  16  as will be explained in further detail below. The tube  30  is held by a thimble-shaped connector  40  having a central opening  42  which slidably receives the tube. The connector  40  includes a set screw  44  for joining the connector to the mount  14 . A spring  46  surrounds the tube  30 . The upper end of the spring  46  abuts the collar  38  and lower end of the spring rests against the inside of the bottom of the connector  40  to bias the tube  30  upward toward an objective O of the microscope and against the focusing mechanism  16 . 
     As illustrated in FIG. 1, the mount  14  includes a cylinder  50  having an inner sleeve  52  sized for receiving a microscope objective O (shown in phantom in FIG.  1 ). It is envisioned that sleeves  52  having differing inner diameters may be provided to accommodate different microscope objectives O. Set screws  54  extending through the cylinder  50  and sleeve  52  engage the objective O to releasably mount the attachment  10  on the microscope. These screws  54  may have soft tips (e.g., Teflon® polymer tips) to avoid marring the objective O. (Teflon is a federally registered trademark of E.I. duPont de Nemours and Company.) As shown in FIGS. 1 and 2, an opening  56  is provided in the side of the cylinder  50  for accessing the focusing mechanism  16  as will be explained below. An end wall  58  extends across the bottom of the cylinder  50  to form the lower end of the mount  14 . The wall  58  has an open segment  60  aligned with the opening  56  in the side of the cylinder  50  for providing additional access to the focusing mechanism  16 . In addition, the wall  58  has a central aperture  62  (FIG. 1) for receiving a portion of the focusing mechanism  16  and one or more peripheral openings  64  for receiving ancillary systems which are used in combination with the attachment  10 . As illustrated in FIG. , a lug  66  extends down from the end wall  58  below the central aperture  62  for engagement by the connector  40  when connecting the lens assembly  12  to the mount  14 . A threaded hole  68  extends vertically through the lug  66  for receiving the focusing mechanism  16 . 
     The focusing mechanism  16  includes a tubular adjustment screw  70  having a thumb wheel  72  at its upper end. The screw  70  extends downward through the threaded hole  68  of the lug  66  and engages the collar  38  on the tube  30  surrounding the microlens  20 . As will appreciated by those skilled in the art, when the thumb wheel  72  is turned, the screw  70  rotates and moves either up or down with respect to the mount  14 . Because the lower end of the screw  70  engages the collar  38  of the microlens  20 , the lens assembly  12  also moves up or down along a longitudinal axis A of the microlens  20  but does not rotate. Therefore, rotation of the screw  70  effects vertical axial translation of the microlens  20  with respect to the objective O without rotating the tube  30  with respect to the mount  14 . An annular pad  74  attached to the upper side of the thumb wheel  72  protects the microscope objective O from damage when the attachment  10  is mounted on the objective and when the focusing mechanism  16  is adjusted. Since the position of the microlens  20  may be adjusted independently of the mount  14 , the microlens may be focused so that the image plane IP of the microlens corresponds with the focal plane of the microscope objective O. Further, the microlens  20  may be focused without moving the mount  14  relative to the objective O. As a result, the microlens  20  may be focused with respect to the objective O and the microlens may be moved to the precisely desired site in the tissue without affecting the focus. Further, both these adjustments may be performed without changing the position of the mount  14  on the objective O. 
     As previously mentioned, the attachment  10  may also include ancillary systems. For instance, the attachment  10  may have one or more fluid delivery systems, generally designated by  80  in FIG.  4 . Each fluid delivery system  80  comprises a micropipette  82  and flexible tubing  84  sized for receiving an inlet end of the micropipette. An upstream end of the tubing  84  is connected to a fluid source  85  and the downstream end is connected to the micropipette  82 . The tubing  84  extends through one of the peripheral openings  64  in the mount  14  to hold the tubing in position. As shown in FIG. 2, a slot  88  may be provided in the connector  40  for holding the micropipette  82  in position with respect to the lens assembly  12 . Although other means of attachment are envisioned as being within the scope of the present invention, the micropipette  82  of the preferred embodiment is adhesively bonded to the outside of the lens assembly tube  30 . As will be appreciated by those skilled in the art, the micropipette  82  is somewhat compliant so it can bend as shown in FIG.  1 . In addition, the flexibility of the tubing  84  permits the tubing to follow the micropipette  82  as the focusing mechanism  16  moves the microlens  20  up or down with respect to the mount  14 . The outlet end  90  of the micropipette  82  is positioned adjacent the front end  22  of the microlens  20 . Moreover, the outlet end  90  is angled as shown in FIG. 1 to direct fluid F (FIG. 4) toward the field of view of the microlens  20  and to provide a pointed tip for improving the ease with which the attachment  10  may be advanced into tissue. The fluid delivery system  80  may be used to deliver a preselected amount of fluid F to a desired site within the field of view of the microlens  20 . For instance, a liquid medicant can be injected into the tissue so its effects can be studied through the microscope, or a stain can be applied to the tissue to improve the contrast of features of the tissue. In addition, more than one fluid delivery system  80  may be coupled with the attachment  10  for delivering more than one fluid to the site. 
     Other ancillary systems are also envisioned. For example, as shown in FIG. 5, the attachment  10  may include an instrument guidance system, generally designated  100 , for guiding instruments (e.g., an electrode E) toward the site adjacent the front end  22  of the microlens  20 . Although other configurations are envisioned as being within the scope of the present invention, the instrument guidance system  100  shown in FIG. 5 comprises flexible tubing  102  adhesively bonded to the outside of the lens assembly tube  30 . The tubing  102  extends upward through a slot  88  provided in the connector  40 . In addition, the tubing  102  may extend through one of the peripheral opening  64  in the mount  14  to hold the tubing in position. Depending upon the particular instrument intended to be carried by the tubing, the diameter of the tubing may vary. Because the lens assembly  12  does not rotate as the focusing mechanism  16  is adjusted, the angular position of the ancillary systems does not change with respect to the microlens  20  as the microlens is focused. As a result, the ancillary systems do not become twisted around the lens assembly  12  as the microlens  20  is focused. 
     The attachment  10  of the present invention is used to view a desired site in living tissue of a host organism as shown in FIG.  3 . The site is prepared by making an incision in the skin and soft tissue of the organism and removing any bone in a conventional manner. The mount  14  is positioned on a microscope objective O and the screws  54  are tightened to hold the mount in place. Once the screws  54  are tightened, the focusing mechanism  16  is adjusted by turning the thumb wheel  72  so the image plane IP of the microlens  20  corresponds with the focal plane of the microscope objective O. After the microlens  20  is focused, the microscope stage (not shown) may be adjusted to move the microlens and the objective O as a unit until the site on the living tissue lies within the field of view of the microlens at its focal plane FP. A guide needle (not shown) can be advanced in front of the microlens  20  to allow easier penetration of dense tissue. Ancillary systems may be used to introduce fluids or guide instruments to the site. 
     As will be appreciated by those skilled in the art, the attachment  10  of the present invention allows use of the microscope focusing mechanism (not shown) to micromanipulate the attachment into position. Further, the attachment  10  allows sites on both the exterior and interior of the tissue to be viewed while the tissue remains in the organism. Because the fluid delivery system directs fluid to the specific site of interest, small amounts of fluid, which are effective at the site but non-toxic to tissue surrounding the site, can be used. Moreover, the lens assembly  12  may be removed from the attachment  10  to change lens elements. Because the lens assembly  12  is removable, it may be discarded after use to prevent infecting others with infectious diseases (e.g., Jacob-Creutzfelt disease) which may be present in the tissue. 
     As will be further appreciated by those skilled in the art, the attachment  10  of the present invention is particularly useful when used with a conventional confocal optical microscope (e.g., a single or dual photon confocal microscope) as described in the Background of the Invention so light is directed from a light source  104  (FIG. 3) through the microlens  20  to illuminate the specimen. Because a confocal microscope does not require external lighting, only a small opening (about the size of the tube  30 ) need be made in the tissue to accommodate the attachment  10 . Thus, the tissue is subjected to less mechanical and optical trauma than it would otherwise be. The image of the illuminated specimen is transmitted back through the microlens  20  to the image plane IP of the microlens. Because the image plane IP of the microlens  20  corresponds to the focal plane of the objective O, the microscope magnifies the image so cells and other structures in the tissue may be viewed. Moreover, because light can be transmitted through several hundreds of microns of tissue and the focal plane FP of the microlens  20  can be positioned below the surface of the tissue, the attachment  10  may be used to view sub-surface portions of the tissue such as the extracellular matrix, the cells and the intracellular matrix. Further, the attachment  10  may be used to view the matrices and cellular membranes without penetrating and thereby damaging the extracellular matrix. For example, as the attachment  10  of the present invention is inserted into the extracellular matrix, arteries ahead of the microlens  20  can be viewed so the user can alter the path of the attachment as it is advanced before causing permanent and irreversible global tissue damage (e.g., brain herniation from intracranial hemorrhage). Those skilled in the art will further appreciate that the attachment  10  of the present invention may be used with a conventional epiflourescence microscope. 
     In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained. 
     As various changes could be made in the above constructions without departing from the scope of the invention, it is intended that all matter contained in the above description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.