The present invention relates generally to the field of specimen preparations and, more particularly, to a method for preparing cells in transport meddium such as a thixotropic gel or a polymer medium for use in three dimensional image acquisition.
For some imaging applications, it is desirable to generate optical information in three dimensions from a thick specimen. Three-dimensional optical information can be generated using the techniques of computed tomographic image reconstruction, in which successive projection images are acquired from a number of perspectives. The perspectives usually form an arc of substantially 180 degrees about the specimen. For three-dimensional imaging, it is important that each perspective receive light in approximately the same manner, without large alterations in the transmitted light due to the optical characteristics or dimensions of the sample container. For this reason, methods such as placing the samples on a flat surface, such as a microscope slide, are not suitable, as the optical thickness of the slide and of the cover-glass (if one is used) will vary significantly as the slide is rotated by 180 degrees about one of its lateral dimensions.
One example of embedding specimens within a standard flat microscope slide format has been published by Reymond and Pickett-Heaps (1983), entitled “A Routine Flat Embedding Method for Electron Microscopy of Microorganisms Allowing Selection and Precisely Orientated Sectioning of Single Cells by Light Microscopy,” Journal of Microscopy, Vol. 130, Pt. 1, April 1983, pp.79-84. Reymond and Pickett-Heaps describe a molding technique for making thin slides of embedding material containing cells for optical sample preparation for electron microscopy. Unfortunately, variations from multiple perspectives when viewing a slide can produce large optical aberrations, as well as a large degree of scattering and absorption. Such large optical aberrations may render the projections taken unusable, especially if taken from a perspective close to the plane of the slide.
A more effective type of sample container should have approximately equivalent optical thickness about an arc of 180 degrees. Geometries that may meet this requirement include hollow tubes having concentric inner and outer walls, or tubes with concentric polygonal inner and outer walls Examples of a sample chamber design for optical applications are shown in Schrader, “Sample Arrangement for Spectrometry, Method for the Measurement of Luminescence and Scattering and Application of the Sample Arrangement,” U.S. Pat. No. 4,714,345, issued Dec. 22, 1987; and Gilby, “Laser Induced Fluorescence Capillary Interface,” U.S. Pat. No. 6,239,871, issued May 25, 2001.
When a specimen comprises individual biological cells, or other material with spatial dimensions of roughly 100 microns or less, there may be additional requirements for the chamber. Because of the small sizes involved, it may prove difficult to insert the cells into, for example, a small capillary tube. Glass capillaries tend to be brittle, and hence easily broken. If the sample to be examined includes a large number of cells, strung out along a long length of glass capillary tubing, then their storage and transport can be very difficult. The alternative method of using a large number of short tubing segments is equally unappealing. Further, if the mechanism for insertion makes use of capillary rise, then the method may be subject to constraints imposed by the chemistry related to the capillary rise. This can be a particular problem when the cell preparation and presentation medium have specific requirements of their own, which may be incompatible with the requirements of the glass-solvent interfacial chemistry.
One drawback of immobilizing the cells within a tube, using such means as injecting epoxies or other optical adhesives into the tube, often results in empty spaces within the tube due to volume change upon curing or upon evaporation of the epoxy's solvent. Further, curing may not be possible due to the enclosed, unventilated volume within the tube. Thus the cells may not be fully immobilized, and the presence of empty spaces, such as bubbles, may contribute to spurious scattering effects during image acquisition. Yet another issue arises due to the possible mismatch between the refractive indices of the sample container, the medium within which the cells are suspended, and the cells themselves. A mismatch between the first two can result in undesirable lensing effects and aberrations of the light rays. At the same time, for some biomedical applications it may be desirable to examine the cell nuclei, while excluding the cell cytoplasms from consideration. Thus, in using a glass tube with a suspending medium, it may become necessary to match the refractive indices of three materials, namely, the tube walls, the suspending medium, and the cell cytoplasm. An example of refractive-index matching is described by Albert et al., in “Suspended Particle Displays and Materials for Making the Same,” U.S. Pat. No. 6,515,649, issued Feb. 4, 2003.
Another issue arises when a chain of custody is required, as may be the case in a biomedical screening application. See, for example, the article by Nicewarner-Peña et al., entitled “Submicrometer Metallic Barcodes,” Science 294, 137 (2001).
In contrast to conventional methods and to overcome the problems noted hereinabove, one method of the present invention uses polymeric materials that are less brittle than glass, and thus easier to handle. Polymeric materials can be made flexible, allowing a single length to be wrapped into a compact roll for convenient handling and storage. Further in contrast to conventional methods, the method of the present invention does not require entrapment of polymers inside a small volume, and permits a uniform, homogeneous medium in which cells are presented. By using the same material as both the sample container and as the suspending medium, the method of the present invention reduces the problem to matching the polymer's refractive index with that of the cytoplasm. If it is desirable to also image the cytoplasm, then refractive-index matching is not required. In the present invention, chemical interactions between the sample and its container play a less significant role.
The present invention provides a method for embedding particles in a solid structure including the steps of extruding a slurry of particles and a polymeric solution into a linear polymer medium having particles embedded into a polymer portion; and curing the polymer portion of the linear polymer medium.
While the novel features of the invention are set forth with particularity in the appended claims, the invention, both as to organization and content, will be better understood and appreciated, along with other objects and features thereof, from the following detailed description taken in conjunction with the drawings.
The method and apparatus of the invention is here described with reference to specific examples that are intended to be illustrative and not limiting. Generally, a specimen to be examined is embedded, or encapsulated, in a homogeneous, optically clear medium, such as a polymer. The suspension comprising the specimen and the medium can be shaped to provide a desired geometry. Upon making the medium into a solid, either by curing or by evaporating the solvent, a flexible, optically clear solid suspension is formed. The solid suspension can be used as a means for supporting, presenting, handling, and storing the specimen. The method and apparatus of the invention is amenable to additional features such as matching of the refractive indices of the materials in the solid suspension and the inclusion of microscopic barcodes to facilitate identification of the specimen. The components used can be made as inexpensive, disposable items, as is necessary when the specimens are biomedical samples.
The medium may be formed by extrusion and subsequent curing of a slurry composed of cells and polymers in solution; by micromolding and subsequent curing of a such a slurry; or by forcing such a slurry into a microcapillary tube, followed by curing. The method disclosed may be useful in applications requring high throughput of cells as part of a three-dimensional imaging system. The manufacturing method can be extended by forming distinct droplets of unpolymerized polymer to form individual spheres encapsulating an individual cell.
Referring now to
The slurry may be in a container 15 that is coupled to an injection device 17, wherein the container 15 may advantageously be a disposable container and the injection device 17 is a conventional injection molding device or equivalents. A linear polymer medium 3, comprising particles 1 emerges from the molding tube 18 and is cured by heat curing or ultra-violet absorption into a solid cylinder of polymer having embedded particles. In one embodiment of the apparatus of the invention, the injection device 17 operates to regulate the spacing between each object along the length of the linear polymer medium 3. 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 16.
Referring now to
Referring now to
An alternative method for embedding particles in a solid structure includes micromolding a slurry including particles and a polymeric solution; and curing the polymer portion of the slurry to form a solid specimen carrier. The step of micromolding may advantageously include using a disposable mold. The step of micromolding may advantageously also include an intermediate step of using an injection device to regulate the spacing between each object along the length of solid specimen carrier. Other combinations of steps and elements may be carried out as described above.
Another alternative method in accordance with the principles of the present invention for embedding particles in a solid structure, includes the steps of pressurizing a slurry including particles and a polymeric solution to force the slurry into a microcapillary tube, and curing the polymer portion of the slurry to form a solid specimen carrier. Other combinations of steps and elements may be carried out as described above.
Referring now particularly to
Referring now to
It will be recognized that the curved surface of the linear polymer medium will act as a cylindrical lens and that this focusing effect may not be desirable in a projection system. Those skilled in the art will appreciate that the bending of photons by the linear polymer medium can be eliminated if the spaces between (a) the illumination source 11 and the linear polymer medium and (b) between the linear polymer medium surface and the detector 112 are filled with a material whose index of refraction matches that of the linear polymer medium and that the linear polymer medium 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 112 form a source-detector pair. Note that one or more source-detector pairs may be employed.
Consider the present example of cells embedded into a linear polymer medium. The cells may preferably be embedded single file so that they do not overlap. The density of embedding whole cells of about 100 microns in diameter into a linear polymer medium with diameter less than 100 microns can be roughly 100 cells per centimeter of linear polymer medium length. For bare nuclei of about 20 microns in diameter, the embedding can be roughly 500 nuclei per centimeter of linear polymer medium length where the linear polymer medium diameter is proportional to the object size, about 20 microns in this case. Thus, within several centimeters of linear polymer medium length, a few thousand non-overlapping bare nuclei can be embedded. By translating the linear polymer medium along its central axis 4, motion in the z-direction can be achieved. Moving the linear polymer medium in the x, y-directions allows objects within the linear polymer medium to be centered, as necessary, in the reconstruction cylinder of the optical tomography system. By rotating the linear polymer medium around its central axis 4, a multiplicity of radial projection views can be produced. Moving the linear polymer medium in the z-direction with constant velocity and no rotation simulates the special case of flow optical tomography.
One advantage of moving a linear polymer medium filled with cells that are otherwise stationary inside the linear polymer medium is that objects of interest can be stopped, 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 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 using 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.
Cell Preparation for Step Flow Actuation of Cells
An alternate method for cell preparation is described hereinbelow for step flow actuation of cells. Step flow actuation of cells requires that cells be embedded in a highly viscous, preferably thixotropic, liquid, for example, having a typical viscosity>1 million centipoises (cps). Unlike flow cytometry, where non-viscous fluids are used to transport cells, and the parabolic velocity profile is used for hydrodynamic focusing to center cells in the tube, step flow has a flat velocity profile. Because of the high viscosity of the carrier medium, cells remain stationary when the medium has zero velocity. Using this type of medium for transport, cells can be actuated into the field of view for measurement, but then stopped so that images of the cell can be acquired without blurring. Furthermore, the cell can be rotated around one axis in a stepwise manner for tomographic imaging purposes.
Herein is described a method for preparing cells and embedding them into a suitable high viscosity gelatinous medium, a method for actuation of the cells embedded in the high viscosity gelatinous medium, and the manner in which the method allows detailed high resolution imaging of the cell.
The method for preparation of cells for embedding in a high viscosity medium suitable for imaging involves transfer of cells into a suitable solvent which does not chemically react with the carrier medium, in this example the solvent is xylene, and centrifugation of the resulting cell/solvent mixture into an optical gel such as, for example, Nye OC431A. Nye OC431A optical gel advantageously has high viscosity so that cells remain stationary when desired, and a refractive index matched to the silica microcapillary tube that serves as the conduit for cell actuation. Refractive index matching both inside the tube, and outside the tube between two flat parallel surfaces is employed for high resolution imaging in order to minimize optical distortions. Since it is likely that the solvent is retained within the fixed stained cell after centrifugation into the optical gel, the solvent also may affect refractive index matching of the interior of the cell to the optical gel (or other carrier medium). Thus, the solvent used may preferably be selected to match the surface refractive index.
As noted above, a conventional flow cytometer uses a very low viscosity carrier medium, typically water having a dynamic viscosity=1 centipoise (cps). In contrast, a step flow system and method constructed in accordance with the present invention uses a moderate-to-high viscosity carrier medium. One objective of the step flow system is to ensure registration of multiple images taken sequentially on a specimen. In the case of optical tomography, for example, a sequence of images is acquired from multiple angles. Registration is important, especially for doing 3D tomographic reconstruction from such a data set. In order to keep acceptable registration, the viscosity of the carrier medium may be determined from the following relationship,
In order to prevent loss of registration between multiple images, the specimen cannot move more than a specified distance d over the period of time it takes to acquire all images. The maximum acceptable distance d can be defined to be 0.25 of the desired image resolution. In one example, the maximum acceptable distance d equals 0.25(0.5 microns)=0.125 microns. Time T for acquisition of a data set comprising 250 images typically ranges from 250 msec to 60 sec. Thus the maximum sedimentation velocity
Inserting these values, the dynamic viscosity η of a useful medium is >37 centipoise (cps) for T=250 msec. For a time interval T=60 sec, η is >8800 cps. The density of the medium itself may also be altered to yield an acceptably low sedimentation rate over the time period T. However, in considering acceleration and deceleration of the carrier medium, it is advantageous to have the density of the specimen similar to the density of the carrier medium so that movement of the specimen relative to the carrier medium is minimized.
Higher viscosities may be useful, though higher viscosities limit the throughput rate of specimen processed by the instrument, as well as limiting the acceleration and deceleration of the carrier medium during actuation. If other external forces, such as that due to centripetal acceleration caused by spinning the microcapillary tube around its axis, are present, the viscosity of the carrier medium may be increased to keep specimen positional stability to an acceptable level.
In the case of a step flow system using a moderate-to-high viscosity carrier medium, hydrodynamic focusing is unnecessary for particle positional stability over the total measurement time T. Hydrodynamic focusing may be employed to improve centration of the cell specimen with the microcapillary tube axis, but is not critical for positional stability. In the case where the carrier medium exhibits non-Newtonian behavior, a flattened velocity profile may occur, in which case it becomes even more necessary to employ increased carrier medium viscosity for specimen positional stability.
Example Cell Staining Protocol Method Using Medium Strength Hematoxylin Such as, for Example, Gill's #2 Hematoxylin.
Cells are typically prepared in ethanol and are purified or cultured using standard procedures prior to the following steps:
The process of centrifugation of cells into an optical gel medium is as follows.
Once the cells are embedded in the high viscosity gel (herein called “cells-in-gel”), high pressure such as, in one example, greater than 1000 psi, using air, preferably with water vapor removed, or using mechanical pressure by applying a syringe plunger, will actuate the cells-in-gel through a microcapillary tube. Some useful microcapillary tubes have inner diameters of about 40-50 microns.
Imaging of Cells
Cells-in-gel are actuated through the microcapillary tube until a single cell appears in the field of view of the imaging system. Pressure is removed, and thus flow is stopped. The cylindrical shape of the cell medium in the microcapillary tube (or cells embedded in polymer threads, also cylindrically-shaped) allows access around 360 degrees normal to the cylinder axis; 180-degree access is critical for tomographic 3D imaging. For any view of the cell within the cylindrically shaped container, the carrier medium's refractive index is well matched throughout a volume between two flat parallel windows. This feature allows rotation and access for imaging through 360 degrees of rotation, but without significant optical distortion. Index matching using, for example, the average over visible wavelengths, between the Nye OC431A optical gel and the surrounding structures is within about 0.02 and produces a nearly-distortion free image as if there were no cylinder present. Only a few microns of the image on the inside of the microcapillary tube remain distorted.
Example Method for Cell Preparation for Buccal Scrapes in 3-D Visualization
General Sample Collection
An alternate embodiment of the method of the invention for buccal scrapes is described hereinbelow. Scrapings of the internal aspects of the oral cavity, that is, buccal surfaces of the cheek, are obtained as by using a plastic scraper or the like. Care should be taken to avoid abrading so vigorously as to cause bleeding. After scraping both left and right buccal surfaces, the scraper is placed into a container of isotonic solution for preservation of cytology specimens and for the liquefication of mucus. Mucoliquefying transport fluid for the collection and transport of fresh cytological specimens such as Mucolexx® available from Thermo Electric Corp., Pittsburgh, Pa., US, is used to cover the area containing the scrapings. The scraper is agitated very briskly for 20-30 seconds to dislodge any cellular material, then the scraper is removed and discarded.
The following steps are then carried out:
Once the sample is shaken and syringed, it may be stored at room temperature for up to a week or more. If additional buccal samples from the same patient are being collected, they may be added to this container, followed by the required shaking period, and the combined sample may be kept at room temperature without cell deterioration.
Sample Concentration:
A method for increasing the sample concentration is carried out using the following steps:
A sample staining procedure using Hematoxylin is carried out using the following steps:
Cell Insertion into an optical system, such as a micro-capillary tube, is carried out using the following steps:
Referring now jointly to
where ρ is density, <v> is average (characteristic) flow velocity, D is characteristic length and μ is (absolute) viscosity. In the case of a circular cross-section tube, the characteristic length D is the inner diameter of the microcapillary flow tube 64.
In order to embed cells in any medium, the cells are concentrated in the medium using centrifugation, with the average density of the cells nearly equal to that of the medium. This is necessary so that the cells are neutrally buoyant in the carrier medium. The cells quickly sediment out of the solvent, however, they must not sediment through the medium quickly, or the concentration of cells may not be increased. The rate of sedimentation of cells through the solvent must be much higher than the rate of sedimentation of cells through the medium in order to achieve increased cell concentration.
Referring now to
If a non-curing media such as optical gel (e.g. Nye OC-431A or OC-431A-LVP), is used in place of a polymer as described above, a resultant cell-media mixture does not exit the tube and is not subject to a heating/curing assembly 65. The cell-gel mixture is instead actuated through the microcapillary tube 64 for viewing in an optical tomography system or other imaging system. The centration of the cells within the tube helps to retain contrast in pseudoprojection because it enables the range of objective scanning to be reduced. Improved centration also allows the total number of acquired projections to be reduced while still retaining the same resolution in a tomographically reconstructed 3D image.
In the case of 3D imaging of cells in a flow cytometer, a number of additional difficulties occur. Many images are acquired in series, and the registration of these images must be more accurate than the desired resolution of the system. For a 3D image to have a 0.5 micron resolution, the registration must be better than 0.5 micron (a 25% error is acceptable, that is, about 0.125 micron). This means that the rotational and translational motion of the cell must be very small, barring that motion along the flow axis. Using higher viscosity media with a flow system can reduce translational and rotational errors to an acceptable level, especially with symmetrically shaped cells that experience no stabilizing force that might prevent rotation. However, use of higher viscosity media necessitates a few changes from that used in standard flow cytometry. The focusing effect found with a single stream is due to the gradient of flow velocity, with an ideal laminar flow of an incompressible liquid yielding
Thus a parabolic velocity profile aids in focusing cells in a flow cytometer. However, as viscosity is increased, or if non-Newtonian fluids are used for transport, then the velocity gradient is reduced. Non-Newtonian fluids like a Bingham fluid may exhibit “plug flow” where the velocity profile is flat, having no gradient within a central region. When this occurs, hydrodynamic focusing using multiple input streams must be employed to achieve focusing, and hence centration of the cells.
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.
This application claims the benefit of the priority date and is a continuation-in-part of co-pending U.S. patent application Ser. No. 10/126,026, filed Apr. 19, 2002, of Nelson entitled “VARIABLE-MOTION OPTICAL TOMOGRAPHY OF SPECIMEN PARTICLES,” the disclosure of which is incorporated herein by this reference. This application is also related to concurrently filed application to Fauver et al. entitled, “IMPROVEMENTS IN OPTICAL PROJECTION TOMOGRAPHY MICROSCOPE,” attorney docket no. 60097US that is assigned to the same assignees as the present application and the disclosure of which is hereby incorporated by reference.
Number | Date | Country | |
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Parent | 10126026 | Apr 2002 | US |
Child | 10968645 | Oct 2004 | US |