Patent Application
20060096358
The present invention is related to optical systems and, more particularly, to optical systems for extended depth-of-field imaging through a cylindrical specimen container.
An optical tomographic device is intended to produce three-dimensional reconstructions of specimens contained in a capillary tube by providing a multitude of “shadowgrams.” A shadowgram, also known in the art as a projection, is a measure of light attenuation along a set of ray paths through the specimen.
To obtain a three-dimensional representation of an object, a microscope objective is axially scanned such that its plane of focus scans through the specimen's thickness. The focal plane of the objective lens can be moved through the specimen while the detector is located in the microscope's image plane. Thus, a projection image can be compiled from a set of discrete focal planes within the specimen, and this compilation is called a pseudo-projection.
Another method for obtaining shadowgrams is to utilize an optical system with an extended depth of field, such that most or all of the object is in focus in a single projection image. A number of methods exist, such as x-ray computerized tomography, that permit the creation shadowgrams from true-projections.
In order to obtain more complete three dimensional information, rotation of a cylindrical specimen container is used to present multiple viewing perspectives. Unfortunately, known mechanisms do not provide a rotational joint that suitably allows high rotational speed combined with ease of use and stability, especially in the case where the cylindrical specimen container comprises a fragile device such as a micro-capillary tube.
Some example descriptions of discrete focal-plane scanning are provided by N Ohyama et al., in U.S. Pat. No. 5,680,484 issued Oct. 21, 1997, entitled “Optical Image Reconstructing Apparatus Capable of Reconstructing Optical Three-Dimensional Image Having Excellent Resolution and S/N Ratio”; by E A Swanson et al., in U.S. Pat. No. 5,321,501 issued Jun. 14, 1994, entitled “Method and Apparatus for Optical Imaging with Means for Controlling the Longitudinal Range of the Sample”; by R E Grosskopf, in U.S. Pat. No. 4,873,653 issued Oct. 10, 1989, entitled “Microscope System for Providing Three Dimensional Resolution”; and by A D Edgar, in U.S. Pat. No. 4,360,885 issued Nov. 23, 1982, entitled “Micro-Optical Tomography.” However, all these methods suffer from low throughput rates due to the stopping and restarting of the moving parts. Another method using true projections is provided by A C Nelson, in U.S. Pat. No. 6,522,775 issued Feb. 18, 2003, entitled Apparatus and Method for Imaging Small Objects in a Flow Stream Using Optical Tomography. This method is inherently high throughput where motion uniformity and control become even more critical.
In overcoming the deficiencies in the state of the art, the present invention takes advantage of the development of polymer grippers. Polymer grippers have been developed for use as holding devices for optical elements such as optical fiber, planar chips, GRIN lenses and filters. Polymer grippers provide self-aligning, snap-in holding with three points of contact. However, known uses for the polymer gripper are believed to be limited to statically holding optical elements in place, without rotation, for splicing, holding within other devices, pre-positioning fibers during manufacture and similar uses.
In contrast to known uses and constructions, the present invention discloses for the first time a system and method for using polymer grippers as a rotational joint in combination with a microcapillary tube. The present invention provides a method and apparatus for using at least one pair of polymer grippers in a system for continuously scanning the focal plane of pseudo-projection or a true-projection optical imaging system along an axis perpendicular to said image plane through the thickness of a specimen during a detector exposure. The process is repeated from multiple perspectives, either in series using a single illumination/detection subsystem, or in parallel using several illumination/detection subsystems, or some combination of series and parallel acquisition. In this way, a set of shadowgrams is generated, which can be input to a tomographic image reconstruction algorithm (such as filtered backprojection) to generate a three-dimensional image. The apparatus described has greater speed and higher signal-to-noise than the prior art described above while providing a means for 3D reconstruction by computer-aided tomographic techniques.
The present invention provides a rotational system including a cylindrical container with a cylindrical container axis. The cylindrical container is inserted into at least one pair of opposing polymer grippers. A motor, or other driving mechanism, is coupled to rotate the cylindrical container.
The invention is described herein with respect to specific examples relating to biological cells; however, it will be understood that these examples are for the purpose of illustrating the principals of the invention, and that the invention is not so limited. For illustrative purposes, an object such as a biological cell may be labeled with at least one tagged molecular probe, and the measured amount and location of this probe may yield important information about the disease state of the cell, including, but not limited to, various cancers such as lung, colon, prostate, breast, cervical and ovarian cancers, or infectious agents.
Referring now to
It will be recognized that the curved surface of the cylindrical container will act as a cylindrical lens and that the resulting focusing effect may not be desirable in a projection system. Those skilled in the art will appreciate that the bending of photons by the cylindrical container can be eliminated if the spaces between (a) the illumination source 11 and the cylindrical container and (b) between the cylindrical container surface and the detector 12 are filled with a material 10 whose index of refraction matches that of the cylindrical container and that the cylindrical container can be optically coupled, with oil or a gel, for example, to the space filling material. When index of refraction differences are necessary, for instance due to material choices, then at minimum the index of refraction difference should only exist between flat surfaces in the optical path. Illumination source 11 and detector 12 form a source-detector pair 14. Note that one or more source-detector pairs may be employed.
Consider the example of cells packed into a cylindrical container. The cells may preferably be packed single file so that they do not overlap. The maximum density of packing whole cells of about 100 microns in diameter into a cylindrical container, such as, for example, a microcapillary tube with inside diameter of 100 microns or less, can be roughly 100 cells per centimeter of cylindrical container length. For bare nuclei of about 20 microns in diameter, the packing can be roughly 500 nuclei per centimeter of cylindrical container length where the cylindrical container diameter is proportional to the object size, about 20 microns in this case. Thus, within several centimeters of cylindrical container length, a few thousand non-overlapping bare nuclei can be packed. To move in the z-direction, the cylindrical container may be translated along its central axis 4. Or conversely, the objects can be caused to flow in the z-direction through the capillary tube. Moving the cylindrical container in the x, y-directions relative to an objective lens allows objects within the tube to be centered, as necessary, in the reconstruction cylindrical container of the optical tomography system. By rotating the cylindrical container around its central axis 4, a multiplicity of radial projection views can be produced.
One advantage of translating a cylindrical container filled with cells, that are otherwise stationary inside the cylindrical container, is that objects of interest can be stopped, and then rotated, at speeds that permit nearly optimal exposure for optical tomography on a cell-by-cell basis. That is, the signal to noise ratio of the projection images can be improved to produce better images than may be usually produced at constant translational speeds and direction typical of flow systems. Objects that are not of interest can be moved out of the imaging system swiftly, so as to gain overall speed in analyzing cells of interest in a sample consisting of a multitude of cells. Additionally, the ability to stop on an object of interest, and then rotate as needed for multiple projections, nearly eliminates motion artifacts. Still further, the motion system can be guided at submicron movements and can advantageously be applied in a manner that allows sampling of the cell at a resolution finer than that afforded by the pixel size of the detector. More particularly, the Nyquist sampling criterion could be achieved by moving the system in increments that fill half a pixel width, for example. Similarly, the motion system can compensate for the imperfect fill factor of the detector, such as may be the case if a charge-coupled device with interline-transfer architecture is used.
In another embodiment, the cylindrical container 3 may be replaced with a solid medium in a cylindrical shape, and having cells embedded within such as described with reference to
Referring now to
A piezoelectric transducer (PZT) 57 is used to move an objective lens 60 an axial distance of about 40 microns or more. In one useful embodiment, a micro-objective positioning system provides a suitable actuator 57, which is driven up and down along the z axis of coordinate system 6. In this embodiment, it may be used with a high numerical aperture objective, mounted on an standard transmission microscope 64 with a video camera 43 attached and a computer-controlled light source and condenser lens assembly 61. The computer-controlled condenser and light source 50 may advantageously be a light source including one or more incandescent bulbs, an arc lamp, a laser, or a light emitting diode. Computer control signals 70 are linked to the computer-controlled condenser and light source 50 for controlling light modulation.
The output from the camera 43 is stored in a computer memory 72. A specimen assembly 65 can be translated along the x or y axes of coordinate system 6. In addition, a cylindrical container 3, as for example a microcapillary tube, containing the specimen can be rotated about its “θ” axis 49, via a motor 5 that can be computer-controlled. As used herein microcapillary tube is defined as a capillary tube having a diameter where the field of view for microscopic imaging is comparable to the capillary tube diameter. A gripping apparatus comprising a plurality of pillars 80 is schematically indicated. Since the gripping apparatus is described in more detail below, the entire apparatus has not been shown in order to simplify the figure for understanding of the main components. In an example embodiment the motor 5 is controlled by control signals 71 as provided by the computer 7. For high speed applications other controls may be added in order to reduce vibrations during an axial scan.
Referring now to
With the fixed optical point source 21, in conjunction with an opposing detector 23 mounted around a circumference of the tube, it is possible to sample multiple projection angles through the entire cell 1 as it flows past the sources when the tube is being rotated. By timing of the emission or readout, or both, of the light source and attenuated transmitted and/or scattered and/or emitted light, each detected signal will coincide with a specific, known position along the axis in the z-direction of the flowing cell at a particular rotation angle. In this manner, a cell 1 flowing with known velocity along a known rotating axis perpendicular to a light source that is caused to emit or be detected in a synchronized fashion, can be optically sectioned with projections through the cell that can be reconstructed to form a 2D slice in the x-y plane. By stacking or mathematically combining sequential slices, a 3D picture of the cell will emerge. It is also possible to combine the cell motion with the positioning of the light source (or sources) around the flow axis to generate data that can be reconstructed, for example, in a helical manner to create a 3D picture of the cell. Reconstruction can be done either by stacking contiguous planar images reconstructed from linear (1D) projections using fan-beam reconstruction algorithms, or from planar (2D) projections directly using cone-beam reconstruction algorithms.
Referring now particularly to
One useful type of polymer gripper is commercially available from Corning Incorporated, Corning N.Y., USA and is made of an environmentally stable polymer, which utilizes a photolithographic process to provide sub-micron accuracy. The polymer adheres to many materials including various glasses, crystals, ceramics, metals and polymers. Any patterns, curves, fan-outs, or squares, capable of being made using a photolithographic mask can be transformed onto a specified substrate.
The optical gel 15 surrounding the tube may advantageously be the same optical gel 10 in which the cells are embedded and/or chosen to match the refractive index of the cylindrical container 3. This allows the optical characteristics of the medium to remain substantially constant, even as the perspective presented to the objective 60 is varied. Thus, the tube is juxtaposed between the glass substrate 54 and a thin top coverslip 55 resulting in index matching between the two flat parallel surfaces. The index matching allows a nearly distortion free image to be acquired while still allowing the specimen to be easily rotated by turning the cylindrical container 3 at one or both ends using the motor 5. Immersing the cylindrical container 3 in the index matching material 15 also provides lubrication during rotation about the “θ” axis 49.
Index matching materials are commercially available (e.g. commercial sources include Nye Optical Gels, Dymax Corp, and Cargille Labs) and include, for example optical gels, oils and fluids of varying indices of refraction for reducing light reflection at optical interfaces. Optical gels are particularly useful where higher viscosity is desired and may comprise a medium of oil, gel, polymer epoxy, or other optically transparent materials that matches refractive indices of the surroundings. Specimens can be held in index-matching epoxy, embedding media, or plastic polymer as well as index-matching gels and viscous fluids.
The motor 5 may comprise any motor and/or motor and gear combination capable of precise speed control such as an electronic motor, a stepper motor or equivalent devices. In some embodiments the motor 5 comprised a microstepper motor that was used for its accuracy in angular positioning of a cell mounted in a microcapillary tube. However, the microstepper motor rotation is relatively slow and cumbersome though it provides better than 0.001 degree accuracy. In another useful embodiment, a small 6% noncumulative error in the full step of a stepper motor can be accepted without resorting to microstepping. Using a 5-phase stepper motor allows a full step size of about 0.72 degrees, resulting in an acceptable expected non-cumulative error of 0.0432 degrees. A 0.72 degree step size yields 250 projections for total rotation of 180 degrees. It is also possible to run the stepper motor at half-steps of 0.36 degrees if desired, though the position inaccuracy remains the same 0.0432 degrees. One commercially available 5-phase stepper motor, available from Nyden Corporation, CA, USA, is model PS533A which can be run at over 100 rpm, giving a 0.72 degree step time of 3.3 msec.
In another useful embodiment of the invention, continuous rotation of a cylindrical container, such as a microcapillary tube, has been found to be particularly advantageous. Continuous rotation of a cylindrical container adjusts for a tradeoff between the precision of rotation (i.e. how closely the tube rotates around an ideal, fixed axis) and any friction due to rotation of the tube relative to the pair of opposing polymer grippers. Some cases using a stepper motor exhibit friction that may cause a stick-slip motion such that the cylindrical container doesn't necessarily move the same amount for each step of non-continuous motion of the rotation stage. Such stick-slip motion leads to an angular error in reconstruction that can be overcome by employing continuous rotation, so that friction has only a dynamic component. Since the dynamic coefficient of friction is lower than the static friction coefficient, there is less friction in a continuous rotation case.
Another consideration while running in continuous rotation is possible rotational blurring of the image (pseudoprojection). It is estimated that 25% of the minimum system resolution of 0.5 micron (=0.125 micron) produces an acceptable 10% loss of contrast. Therefore, in one example, a rotational speed is selected such that the angle of rotation during the exposure time to form the pseudoprojection is as follows: acceptable angle of rotation=inv tan((0.125 micron)/(radius of pseudoprojection sweep=25 micron)=0.286 degrees. Using an exposure time of about 20 msec allows rotation at a speed of 0.286 degree/20 msec=14 degrees/sec. An exposure time of 1-2 msec, will yield a rotation speed of >180 degrees/sec.
In another useful embodiment, a sinusoidal velocity function may advantageously be employed for tube rotation. Using a sinusoidal velocity function, the rotational velocity never reaches zero, but oscillates between a low velocity and a higher velocity. The sinusoidal velocity function need not be a continuous motion, but may be regulated to sinusoidally vary the velocity so as to avoid stiction. The sinusoidal velocity function allows some slower movement to avoid rotational blur that may be increasingly evident as rotational velocity increases. Note also that there will inherently be a discrepancy between the drive function and the response of the tube. A microstepper motor may be used to produce a smooth sinusoidal rotational velocity function that overcomes such inertial effects.
Referring now to
The slurry may be in a container 115 that is coupled to an injection device 117, wherein the container 115 may advantageously be a disposable container and the injection device 117 is a conventional injection molding device or equivalent. A linear polymer medium 103, comprising particles 101 emerges from the molding tube 118 and is cured by heat curing or ultra-violet absorption into a solid cylindrical container of polymer having embedded particles. In one embodiment of the apparatus of the invention, the injection device 117 operates to regulate the spacing between each object along the length of the linear polymer medium 103. The polymeric solution preferably comprises a polymer selected to be substantially transparent to visible light and provide, upon solidification and curing, a matching of its index of refraction with the index of refraction of a portion of the particles contained in the slurry 116.
The invention has been described herein in considerable detail in order to comply with the Patent Statutes and to provide those skilled in the art with the information needed to apply the novel principles of the present invention, and to construct and use such exemplary and specialized components as are required. However, it is to be understood that the invention may be carried out by specifically different equipment, devices and algorithms, and that various modifications, both as to the equipment details and operating procedures, may be accomplished without departing from the true spirit and scope of the present invention.
