Abstract:
Described is a portable confocal microscope which includes a microscope stand including a shaft having top and bottom ends, a platform connected to the shaft by a height adjustment mechanism, a base member connected to the bottom end of the shaft and first and second elongated channel members slidably and rotatably connected to the base member, an optical assembly including an objective lens including a first position adjustment mechanism for adjusting a position of the objective lens along a first axis, a confocal module transmitting light to and receiving light from a specimen to be imaged via the optical assembly, an image acquisition system recording images through the optical assembly, a specimen stage including a position adjustment mechanism for moving the stage along a second and third axis, and a computer controlling the first and second position adjusting mechanisms to generate an image for recording by the image acquisition system.

Description:
FIELD OF INVENTION 
   The present invention relates generally to confocal scanning microscopes and in particular, to a portable automated confocal microscope. 
   BACKGROUND INFORMATION 
   The confocal microscope is widely used in many areas of biological and medical research because of its ability to image subsurface features with high resolution and contrast. The operating principle of a confocal scanning optical microscope (“CSOM”) is that an illuminating beam of light is scanned across a field of view from which only light emerging from a focal plane of an objective lens contributes to image formation. This differs from conventional light microscopy, wherein light from the focus plane of the objective lens, as well as from all out of focus planes across the entire field of view, is observed. In practice, the focal plane can be positioned below the surface of an opaque material for the non-destructive imaging of microscopic details at depth, or it may be positioned on the surface of a reflective material for observing high-resolution surface detail. 
   CSOMs are generally available in two basic configurations. The first scans a high intensity laser across the field of view with a computer compiling an image from the scan. These laser CSOM devices are generally too heavy and sensitive to motion to be portable. The second configuration uses a rotating “Nipkow” disk with a series of pinholes formed therein to transmit portions of an illuminating beam of light to and from the object to compile an image in real time. The image compiled from a Nipkow CSOM is generally visible through objective eyepieces without computer compilation. 
   Nipkow disks typically contain thousands of perforations (e.g., 32,000 or more), each having a diameter of about 50 microns. These perforations are generally arranged as a series of Archimedean spirals. In operation, the disk is spun to rotate the series of precisely aligned perforations across an incident light beam to create an incident light scan with returning light directed back through the disk. One available form of a Nipkow disk CSOM uses both sides of the rotating disk, e.g., one side for light passing to the specimen and the other side for light returning from the specimen. Because the precise alignment of multiple internal mirrors is required to obtain an accurate image, these instruments are very sensitive to motion and are not easily portable. In fact, even limited motion of such devices may require time consuming realignment of the mirrors. Although Nipkow disk CSOMs using only one side of the rotating disk for both purposes are less motion sensitive, current devices achieved the required base stability only through a heavy, non-adjustable base permanently mounted to the optics. 
   SUMMARY  
   The present invention relates to a portable confocal microscope, comprising a portable microscope stand including a shaft having top and bottom ends, a platform connected to the shaft by a height adjustment mechanism so that the position of the platform along the shaft may be varied, a base member connected to the bottom end of the shaft and first and second elongated channel members slidably and rotatably connected to the base member, the first and second channel members extending in a plane substantially perpendicular to an axis of the shaft, an optical assembly including an objective lens including a first position adjustment mechanism for adjusting a position of the objective lens along a first axis, a confocal module transmitting light to and receiving light from a specimen to be imaged via the optical assembly, an image acquisition system recording images through the optical assembly, a specimen stage for supporting the specimen to be imaged including a position adjustment mechanism for moving the stage along a second and third axis, and a computer controlling the first and second position adjusting mechanisms to generate a desired image for recording by the image acquisition system. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIG. 1  shows schematically a portable confocal microscope according to the present invention. 
       FIG. 2A  shows schematically a side view of an exemplary embodiment of a portable stand according to the present invention. 
       FIG. 2B  shows schematically a top view of an exemplary embodiment of a portable stand according to the present invention. 
       FIG. 3  shows the operation of an exemplary embodiment of a Nipkow disk confocal module according to the present invention. 
       FIG. 4  shows schematically an illumination system according to the present invention. 
       FIG. 5  shows schematically an exemplary embodiment of an optical assembly according to the present invention. 
       FIG. 6A  shows schematically an isometric view of a specimen stage according to the present invention. 
       FIG. 6B  shows schematically a cross-sectional view of a specimen stage according to the present invention. 
       FIG. 6C  shows schematically a top view of a specimen stage according to the present invention. 
   

   DETAILED DESCRIPTION 
   The present invention is directed to a confocal scanning optical microscope (“CSOM”). In particular, the present invention is directed to a lightweight portable confocal microscope using single-sided Nipkow disk technology. The present invention includes a confocal module, an optical assembly, illumination system, image acquisition system, portable microscope stand, power requirements, and travel cases that may be assembled in a unit capable of transport and quickly and easily set-up in remote locations. 
     FIG. 1  shows an exemplary embodiment of a portable confocal microscope assembly  100  according to the present invention. The microscope assembly  100  includes a portable stand  102  designed to support the components of the microscope assembly  100 . An exemplary embodiment of the portable stand  102  is shown in  FIGS. 2A and 2B . The portable stand  102  may be comprised primarily of aluminum components or any other lightweight and sufficiently rigid material(s). As shown in  FIG. 2A , the portable stand  102  includes an adjustable platform  202 . The platform  202  may be removably connected to a single shaft  204  via a coupling  206  (e.g., height adjustment mechanism). The coupling  206  may include one or more locking screws  208  to lock or fasten the platform  202  in a desired position on the shaft  204 . In the exemplary embodiment, the coupling is a cylindrical sleeve containing two compressible plastic rings (not shown). The locking screws  208  may compress the plastic rings, thus conforming the plastic rings to the shaft&#39;s diameter and securing the position of the platform  202  by friction. 
   The vertical position of the platform  202  may be manually adjusted up or down along a z-axis on the shaft  204 . For example, the portable stand  102  may include a crank  210  that rotates a lead screw  212  within the shaft  204  to raise and lower the platform  202 . In the exemplary embodiment, the lead screw  212  is comprised primarily of steel and provides 6 mm of vertical adjustment for every three hundred and sixty (360) degrees rotation of the lead screw  212 . Furthermore, the platform  202  may be rotated about the shaft  204  (e.g., in a substantially horizontal plane) by rotating the coupling  206  around the shaft  204 . The platform  202  may then be locked in a desired orientation and z-axis position by turning the locking screws  208  as described above. 
   Shown in  FIG. 2B , the portable stand  102  includes a base  214 . The base  214  which is preferably removably connected to the shaft  204  via a release mechanism  224  (shown in  FIG. 2A ). As would be understood by those skilled in the art, the more components of the assembly  100  which are removable from one another, the more compactly the assembly  100  may be stored when disassembled for transport. However, more or fewer of the components may be made removable from one another to achieve an optimum travel configuration so long as set up time and effort do not become overly burdensome. 
   The base  214  is mounted on two channel members  216  and  218 , shown in  FIGS. 2A and 2B . Each of the channel members  216  and  218  may be adjustably positioned to provide optimal stability for the microscope assembly  100 . For example, each of the respective channel members  216 ,  218  is preferably rotatable about the respective connection points  220 ,  222 . In the preferred embodiment, the channel members  216 ,  218  are rotatable through three hundred and sixty (360) degrees about their respective connection points  220 ,  222 . However, as would be understood by those skilled in the art, similar results may be obtained if the members are slidable, as described below, and rotatable through one hundred and eighty (180) degrees. Of course, alternate designs with varying degrees of rotation of the members  216 ,  218  are contemplated. However, 180 degrees or more of rotation provides the highest degree of adaptability of the microscope assembly  100  to various surfaces. Further, each channel member  216 ,  218  is slidable relative to the base  214  via the respective connection points  220  and  220 . For example, the channel member  216  may be adjusted in an x 1 , direction and the channel member  218  in an x 2  direction to stabilize the portable stand  102  as shown in  FIG. 2B . Each connection point  220  and  222  may include a bearing to allow for fluid rotation of the channel members  216  and  218 , and locking screws to fasten each channel member in a desired position. 
   As shown in  FIG. 1 , the microscope assembly  100  includes a confocal module  106  supported by the adjustable platform  202 . The confocal module  106  is preferably a Nipkow disk type confocal module as these modules are less sensitive to transport. However, any other style of confocal microscope which becomes available with the required durability will work equally well with the assembly  100 . The confocal module  106  according to this embodiment uses only one side of a rotating Nipkow disk for both light passing to and returning from the specimen. The confocal module  106  may be of any manufacture known to those of ordinary skill in the art. In the exemplary embodiment, the confocal module  106  is a K2S-BIO Nipkow disk module available from the Technical Instrument Company or the Zygo Corporation. 
     FIG. 3  shows schematically the operation of another exemplary confocal module  300 . The exemplary confocal module  300  is a Nipkow disk type confocal module and includes a one-sided Nipkow disk  302 . As one of ordinary skill in the art would understand, the Nipkow disk is, for example, substantially circular, approximately one (1) centimeter in diameter and includes a series of circular perforated holes located in an Achimedes spiral. The exemplary embodiment of the confocal module  300  uses only one side of the Nipkow disk  302  for both linearly polarized light  304  passing to a specimen  308  and reflected light  306  returning from the specimen  308  and includes, for example, a linear polarizing filter  310  with a transmission axis  312 . The transmission axis  312  is an axis along which unfiltered light  314 , received from an illumination system  108  (shown in  FIG. 1 ), is allowed to pass. The exemplary confocal module  300  also includes a quarter wave plate  316  having a filter that retards or bends linearly polarized light  304  in a fast (“F”) to slow (“S”) direction, causing the light to circularly rotate, allowing polarization in all three hundred and sixty (360) degrees of rotation of the specimen  308  and creating incident right circularly polarized light  318 . Reflected incident left circularly polarized light  320  is returned from the specimen  308  back through the quarter wave plate  316  and the Nipkow disk  302 . Reflected light  306 , including the reflected incident left circularly polarized light  320 , passes through a second linear polarizing filter  322  through a transmission axis  324  to a microscope head or eyepiece, discussed in more detail below. 
   The illumination system  108  according to the present invention is shown in  FIGS. 1 and 4 . The illumination system  108  is preferably capable of operation at either 110V, 60 Hz or 220V, 50 Hz so that the device is suited for use in a wide geographic range. For example, the power input may be capable of switching automatically from 110V to 220V to be compatible with the local power supply. The illumination system  108  transmits a flat, intense beam of light at wavelengths suitable for both fluorescence and white light illumination. The illumination system  108  preferably includes a pre-aligned lamp and is constructed to be lightweight and robust. In an exemplary embodiment, the illumination system  108  is a Lambda LS Xenon Arc Lamp available from the Sutter Company in Novato, Calif. The illumination system  108  may include, for example, a 175 Watt or a 300 Watt full spectrum lamp. 
   As one of ordinary skill in the art will understand, the illumination system  108  delivers unfiltered light  314  to the confocal module  106  via a liquid light guide  110  as an intense flat beam of light. The liquid light guide  110  improves performance relative to conventional fiber optic light guides. Shown in  FIG. 4 , the illumination system  108  may include a light guide lens  402  adapted to transmit the flat beam of light via the liquid light guide  110 . The illumination system  108  may also include a cooling fan  404  and a heat dissipater  406  to control the temperature of the illumination system  108 . 
   As shown in  FIG. 1 , the exemplary embodiment of the microscope assembly  100  includes an optical assembly  112  housing at least one objective lens (not shown). For example, the optical assembly  112  may house a 10× lens (e.g., a 19 mm working height lens available from Thales Optem Inc. of Fairport, N.Y.) and a 20× lens (e.g., 20 mm working height lens available from Mitutoyo Asia Pacific Pte Ltd. of Singapore). In other embodiments according to the present invention, a 5× Thales Optem lens having a 34 mm working height may be used. 
   Another exemplary embodiment of an optical assembly  500  is shown in  FIG. 5 . The optical assembly  500  includes a coupler  502  adapted for removable connection to the confocal module  106 . The optical assembly  500  includes an objective scope  504  in which the objective lens(es) are housed. Flexibility in magnification may be achieved through the use of a 0.5× charge-coupled device (“CCD”) adapter or by converting the fixed magnification optical assembly  112  into a zoom system as would be understood by those skilled in the art. For example, a 70 XL zoom module, available from Thales Optem Inc., may be used. 
   The objective scope  504  may be focused manually or, more preferably, via a motorized device under manual input or computer control. For example, the optical assembly  500  may include a z-axis motor  506 . In this exemplary embodiment, the z-axis motor  506  is a Vexta PK 249-01AA motor available from Oriental Motor U.S.A. Corporation of Torrance, Calif. However, the z-axis motor  506  may be any low backlash motor providing sufficiently smooth start up and stopping. The z-axis motor  506  drives a focus gear system  508  to adjust the position of the objective scope  504  along a z-axis (e.g., vertical axis). As one of ordinary skill in the art will understand, adjusting the position of the objective scope  504  along the z-axis changes the position of the objective lens to alter the z-axis location of focus. 
   The z-axis motor  506  is preferably connected to a portable computer  114  (shown in  FIG. 1 ) via a peripheral component interconnect (“PCI”) bus to provide automated control via a scanning program. As would be understood by those of ordinary skill in the art, the portable computer  114  may be any computer sufficiently portable and capable of receiving PCI interface boards. In the exemplary embodiment, the portable computer  114  is a Shuttle XPC SB52G2 with a Pentium 4 Intel Processor running Windows XP, available, for example, from the Shuttle Computer Group in Los Angeles, Calif. The portable computer  114  includes a monitor  115  which is preferably thin and lightweight, and which may, for example, include wireless connectivity (e.g., a 802.11b wireless connection) and a touch screen. In the exemplary embodiment, the monitor  115  is a 15-inch Viewsonic Wireless Smart Display “Air Panel” V150p, available from Viewsonic Corporation in Walnut, Calif. The portably computer  114  may also include a flexible rubber keyboard (not shown) for increased portability and durability. The keyboard may be, for example, a “Virtually Indestructible Keyboard” available from GrandTec USA in Dallas, Tex. In other embodiments, the portable computer  114  may be a notebook or laptop computer. 
   The scanning program included in the portable computer  114  preferably controls the z-axis motor  506  and a stage  120 ,  600  (discussed below) for fully-automated x, y, and z image acquisition. In the exemplary embodiment, the scanning program includes Syncroscopy Auto-Montage and Montage Explorer software providing for fully in-focus image acquisition up to 20,000×20,000 pixel resolution in x and y (e.g., in a horizontal plane), and 1024×1024 image acquisition in z (e.g., up and down). Similar to the illumination system  108 , the power input for the portable computer  114  is preferably capable of switching automatically from 110V to 220V to enhance compatibility with local power supplies. It is further contemplated that the microscope assembly  100 , including the portable computer  110  and the illumination system  108 , may be powered by a car battery, e.g., via a cigarette lighter adapter. 
   Shown in  FIG. 1 , the microscope assembly  100  further includes a trinocular head  116  including a binocular eyepiece, allowing for real time binocular viewing, and a camera port. The trinocular head  116  may include, for example, a Nikon Instruments head fitted with 10× eyepiece objectives. In the exemplary embodiment, the trinocular head  116  may be configured to allow approximately 100% of available light to be transmitted through the binocular eyepiece, or may direct a portion of the available light (e.g., 14% of available light) to the eyepiece with the remaining light being diverted to the camera port. As would be understood by those or ordinary skill in the art, a camera  118  may be removably connected to the camera port of the trinocular head  116 . The camera  118  may include, for example, a high resolution (1360×1024 pixels) half inch (½″) color CCD. In the exemplary embodiment, the camera is a JVC KY-F1030U digital camera including 1360×1024 SXGA digital image capture and available from the JVC Company of Japan. The camera  118  is preferably connected to the portable computer  114  via, for example, a Fire Wire IEEE 1394 digital interface to allow for the transmission to and storage of images in the computer  114 . In preferred embodiments, the IEEE 1394 connection is a 6-pin connection capable of delivering power to the camera  118 , thus eliminating the need for a separate power supply. 
   Shown in  FIG. 1  and  FIGS. 2A–2B , the microscope assembly  100  includes a free standing stage  120 . The stage  120  may be of any size or shape to adequately support a desired specimen. In the exemplary embodiment, the stage is a KPL53 motorized precision micro-stepping X-Y stage, available from the Semprex Corporation in Campbell, Calif. The stage  120  is preferably freely movable relative to the stand  102  so that any desired relative position of the stage  120  with respect to the stand  102  and the optical assembly  112  may be selected. In another exemplary embodiment, the stage  120  may be movably and/or rotatably attached to the shaft  204 , for example, in a manner similar to the coupling  206  of the platform  202 . 
   Another exemplary embodiment of a stage  600  according to the present invention is shown in  FIGS. 6A–6C . The stage  600  includes a bottom plate  602  and a top plate  604 , the bottom plate  602  being of any shape or size providing adequate stability. The top plate  604  may also be of any shape and size, and may further be transparent to allow for the transmission of light therethrough. Alternatively, the top plate  604  may include a reflective surface. The stage  600  includes an x-servo  608  and a y-servo  606  mounted between the bottom plate  602  and the top plate  604  and substantially perpendicular to one another. The x-servo  608  and y-servo  606  may, for example, be DC powered and may include rotary encoders in a closed-loop system to ensure accurate positioning. As one of ordinary skill in the art will understand, the x-servo  608  may be controlled to displace the top plate  604  in either direction along an axis X 3  (as shown in  FIGS. 6B and 6C ) while the y-servo  606  may be controlled to displace the top plate  604  and the x-servo  608  in either direction along an axis y 3  shown in  FIG. 6C . The x-servo  608  and the y-servo  606  are connected to the portable computer  114 , for example, via a servo port  610 . In the exemplary embodiment, the servo port  610  is a PCI connection. 
   As discussed above, the portable computer  114  contains a scanning program. As one of ordinary skill in the art will understand, the scanning program may control the position of the objective scope  504  and the stage  120  or  600  to automatically scan a specimen on the stage  120  or  600 . For example, the microscope assembly  100  may be assembled in a desired location and a specimen may be placed on the stage  120  or  600 . The adjustable platform  202  of the portable stand  102  may be adjusted to position the optical assembly  112  an adequate distance above the specimen. For example, the adjustable platform  202  may be adjusted to have the optical assembly  112  between zero (0) and eighteen (18) inches above the specimen. The scanning program may then be engaged to control the stage  120 ,  600  to scan the specimen by, for example, controlling the position of the objective scope  504  and the stage (e.g., stage  120  or stage  600 ) to automatically scan the selected portions of the specimen and record images using the camera  118 . The microscope assembly  100  may non-destructively acquire internal micro-structural detail of a specimen (e.g., 1–100 micrometers deep to the surface) and provide two-dimensional (“2-D”) and three-dimensional (“3-D”) reconstruction for storage on the portable computer  114 . 
   The microscope assembly  100  according to the present invention is portable to allow for use at any location. For example, the microscope assembly  100  may be capable of transport to distant and/or remote locations. The microscope assembly  100  may, for example, be disassembled and packed in one or more travel cases (not shown) for transport to any number of locations. More specifically, the microscope assembly  100  may be transported in two travel cases, e.g., one 1600 model Protector Case and one 1610 model Protector Case provided by Pelican Products Inc. in Torrance, Calif. It is contemplated that the microscope assembly  100  and travel cases may weigh approximately sixty (60) kilograms or less. 
   While specific embodiments of the invention have been illustrated and described herein, it is realized that numerous modifications and changes will occur to those skilled in the art. It is therefore to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit and scope of the invention.