Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/47—Scattering, i.e. diffuse reflection
  • G01N21/4795—Scattering, i.e. diffuse reflection spatially resolved investigating of object in scattering medium
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1434—Optical arrangements
  • G01N15/1436—Optical arrangements the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
• • G—PHYSICS
  • G06—COMPUTING OR CALCULATING; COUNTING
  • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
  • G06T11/00—2D [Two Dimensional] image generation
  • G06T11/003—Reconstruction from projections, e.g. tomography
  • G06T11/006—Inverse problem, transformation from projection-space into object-space, e.g. transform methods, back-projection, algebraic methods
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1434—Optical arrangements
  • G01N2015/144—Imaging characterised by its optical setup
  • G01N2015/1445—Three-dimensional imaging, imaging in different image planes, e.g. under different angles or at different depths, e.g. by a relative motion of sample and detector, for instance by tomography
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N2021/178—Methods for obtaining spatial resolution of the property being measured
  • G01N2021/1785—Three dimensional
  • G01N2021/1787—Tomographic, i.e. computerised reconstruction from projective measurements

Definitions

• the present invention relates to optical tomographic imaging systems in general, and, more particularly, to optical projection tomography for 3D microscopy, in which a small object, such as a biological cell, is illuminated with ultraviolet radiation for pseudoprojection imaging and reconstruction into a 3D image.
• Processing in an optical tomography system begins with specimen preparation.
• specimens taken from a patient are received from a hospital or clinic and processed to remove non-diagnostic elements, fixed and then stained.
• Stained specimens are then mixed with an optical gel, inserted into a microcapillary tube and images of objects, such as cells, in the specimen are produced using an optical tomography system.
• the resultant images comprise a set of extended depth of field images from differing perspectives called "pseudoprojection images.”
• the set of pseudoprojection images can be reconstructed using backprojection and filtering techniques to yield a 3D reconstruction of a cell of interest.
• the ability to have isometric or roughly equal resolution in all three dimensions is an advantage in 3D tomographic cell imaging, especially for quantitative image analysis.
• the 3D reconstruction then remains available for analysis in order to enable the quantification and the determination of the location of structures, molecules or molecular probes of interest.
• An object such as a biological cell may be labeled with at least one stain or tagged molecular probe, and the measured amount and location of this biomarker may yield important information about the disease state of the cell, including, but not limited to, various cancers such as lung, breast, prostate, cervical, stomach and pancreatic cancers.
• the present disclosure allows an extension of optical projection tomography to live cell imaging and is expected to advance cell analysis, drug development, personalized therapy, and related fields.
• live cell microscopy has traditionally been done by non-labeling 2D imaging techniques such as phase contrast, DIC, and polarization contrast microscopy.
• DUV illumination for live cells
• native DNA and protein absorb the light at 260nm and 280nm, respectively, without the use of any photochemical label that must permeate the cell membrane and sometimes the nuclear membrane of the cell, which is in a non-normal state.
• the label or stain is only an intermediary step toward the measurement of target protein or nucleotide (DNA) which adds a large degree of variability in this measurement. Elimination of such exogenous species would potentially improve the accuracy of a quantitative measure of protein or nucleotide (DNA), as well as reduce time, effort and complexity by eliminating steps in the sample preparation.
• DUV illumination has demonstrated phototoxicity in the past, due to the high dose of radiation required to stimulate a strong signal.
• LEDs DUV light-emitting diodes
• the present disclosure describes a new, novel and surprisingly effective 3D imaging system that provides solutions to long felt needs in the field of DUV 3D imaging of cells, and more particularly, live cells.
• a method for 3D imaging of cells in an optical tomography system including moving a biological object relatively to a microscope objective to present varying angles of view.
• the biological object is illuminated with optical radiation having a spectral bandwidth limited to wavelengths between 150 nm and 390 nm.
• Radiation transmitted through, scattered by, or secondarily emitted by the biological object and captured by the microscope objective is sensed with a camera to record images from a plurality of differing view angles.
• a plurality of pseudoprojection images of the biological object from the sensed radiation is formed and the plurality of pseudoprojections is reconstructed to form a 3D image of the cell.
• FIG. 1 schematically shows an example of a system for 3D imaging of cells in an optical tomography system employing ultraviolet radiation.
• FIG. 2 schematically shows an alternate example of a system for 3D imaging of cells in an optical tomography system with ultraviolet radiation using a UV camera and optional adaptive optics.
• FIG. 3 schematically shows an embodiment of a temperature-controlled housing for use in an optical tomography system.
• FIG. 4 schematically shows a side view of an example of a microfluidics cartridge as used in a raceway configuration for imaging cells.
• FIG. 5 schematically shows a top view of an example of a microfluidics cartridge as used in a racetrack configuration for imaging cells.
• FIG. 6 schematically shows an optical tomography process including separate imaging stages along the same pathway.
• Capillary tube has its generally accepted meaning and is intended to include transparent microcapillary tubes and equivalent items with an inside diameter generally of 500 microns or less.
• Depth of field is the length along the optical axis within which the focal plane may be shifted before an unacceptable image blur for a specified feature is produced.
• Object means an individual cell, item, thing or other entity.
• Pulseudoprojection includes a single image representing a sampled volume of extent larger than the native depth of field of the optics. The concept of a pseudoprojection is taught in Fauver 744.
• specimen means a complete product obtained from a single test or procedure from an individual patient (e.g., sputum submitted for analysis, a biopsy, or a nasal swab).
• a specimen may be composed of one or more objects. The result of the specimen diagnosis becomes part of the case diagnosis.
• Sample means a finished cellular preparation that is ready for analysis, including all or part of an aliquot or specimen.
• a tube 22 such as a capillary tube, microcapillary tube or equivalent, is positioned to be viewed by a microscope 16 including a microscope objective 18 and a tube lens element 52.
• a rotation mechanism for example, a rotary motor 20 is attached to the tube 22.
• An axial translation mechanism for example motor 34, is coupled to the microscope objective.
• a radiation source 29 is positioned to illuminate a part of the tube 22 including a biological object 1 held therein. The radiation source 29 generates radiation having a spectral bandwidth limited to wavelengths between 150 nm and 390 nm.
• the radiation source 29 comprises multiple sources 30, 31 transmitting at least two selected wavelengths that are detected concurrently by a first light detector 10 and a second light detector 14.
• Optional filters 12A, 12B are selected to block fluorescence having a wavelength longer than the UV limited spectral bandwidth, such as native tryptophan fluorescence, and/or increase separation of differing ultraviolet radiation signals.
• the radiation source may advantageously be incorporated into a computer-controlled light source and condenser lens assembly 56.
• the computer-controlled light source and condenser lens assembly 56 may further include condenser lens optics 24, 26 a light diffuser 28 and the radiation source 29.
• the tube 22 is placed in a viewing area between two optically flat surfaces such as a standard microscope slide 23A and a standard microscope coverslip 23B.
• the interstices between the tube 22 and the microscope slide 23A and coverslip 23B are filled with optical gel 32 or an equivalent material such as inorganic and organic oils, having an index of refraction that also substantially matches those of the tube 22, and the microscope slide and coverslip.
• the tube 22 itself may advantageously be coated with an oil of similar optical properties.
• the outer diameter of the tube 22 may be, for example about 250 microns.
• a typical refraction index, n, matched to capillary tubing used in an optical tomography system is about 1.48, for example, at 590 nm, but the dispersion curve moves sharply upward in the UV.
• Estimated refractive index of fused silica capillary tube is 1.51 at 250nm, and transmittance of DUV by UV grade fused silica is about 90%.
• the biological object 1 may advantageously be selected from the group consisting of a cell, a live cell, a fixed cell, an unfixed cell, a frozen cell, a thawed cell, a desiccated cell, a cloned cell, a mobile cell, an immobilized cell, an encapsulated cell, a cell nucleus, cell parts, an organelle, a sub-cellular component, chromosomes, and equivalent materials.
• the optical tomographic imaging system 11 may advantageously employ illumination radiation having a frequency that stimulates native fluorescence from the biological object, where the light detectors and image processor further include modules for measuring the stimulated fluorescence.
• the biological object is contained in aqueous environment 2.
• the aqueous environment 2 comprises physiological buffered saline or other solutions as described below.
• a beamsplitter 15 is positioned to split radiation transmitted through the biological object into at least two selected wavelengths.
• the beamsplitter may advantageously be selected from the group consisting of a polarizing beam splitter, a Wollaston prism, a birefringent element, a half-silvered mirror, a 50/50 intensity beamsplitter, a dielectric optically coated mirror, a pellicle film, a dichroic beamsplitter, mirror, prism, diffractive optical element, grating, and equivalents.
• the first light detector 10 is positioned to sense radiation transmitted through the biological object 1 , the microscope objective 18, the beamsplitter 15 and a first set of the optional filters 12A.
• the second light detector 14 is positioned to sense radiation transmitted through the biological object 1 , the microscope objective 18, the beamsplitter 15 and a second set of the optional filters 12B.
• the first and second light detectors 10, 14 may each particularly include a pixel array detector sensitive to ultraviolet light, where each pixel array detector is selected to detect a different one of the two selected wavelengths.
• a computer 41 includes an image processor 40 coupled to receive data from the first and second light detectors 10, 14.
• a reconstruction module 42 is coupled to the image processor 40, where the reconstruction module processes the data to form a 3D image of the cell using reconstruction algorithm techniques such as taught in Fauver 744 for example.
• the image processor 40 transmits processed image data to the 3D image reconstruction module 42 which may advantageously be coupled to an optical display 44 for operator viewing.
• User interface 46 can be provided for operator control and information purposes.
• the user interface 46 may be a GUI interface or the like coupled to the computer 41.
• the axial translation mechanism 34 comprises a piezoelectric transducer or equivalent device.
• a controller 35 linked to control the piezoelectric transducer may advantageously be a computer, computer module or the like, where the piezoelectric transducer is controlled to axially move the objective lens 18.
• the optical tomographic imaging system 11 is configured through use of filters and radiation sources to image cells using wavelengths limited to between 240 nm and 300 nm.
• Radiation detected by the first detector 10 may have wavelengths primarily in a first range between 260 nm and 265 nm.
• Radiation detected by the second detector 14 may have wavelengths primarily in a second range between 280 nm and 285 nm.
• the first range operates to enhance natural radiation absorbance by DNA and RNA.
• the second range operates to enhance natural radiation absorbance by protein.
• the first and second wavelength ranges may be provided using a pair of radiation sources, each source transmitting one of the two selected of wavelength ranges.
• One of the detectors may be tuned to detect absorbance around 270 nm near hydrophilic surfaces such as DNA and protein.
• the radiation may be measured in time series using time to separate signals.
• the radiation source may be pulsed in a time series to cause pulsed excitation of the cells in order to increase signal to noise, separating signals.
• a radiation source at 260 nm may be pulsed at a time, T 0 , followed by a 280 nm pulse at T 1 , followed in turn by one or more laser pulses at n subsequent time increments, T n , where n is any number denoting a subsequent point in time.
• the native tryptophan fluorescence can be measured to obtain a secondary measure of protein and its confirmation and constituents, such as amino acids.
• a third beam splitter would be required unless time series illumination is used.
• beamsplitter 15 would split all DUV light (240-300 nm) to the DUV light detector 14 while the lower frequency fluorescence signal would be detected by a fluorescence light detector 10 (>300 nm).
• Operation of DUV light sources 30, 31 can be in time-series so radiation absorbance primarily by nucleotides (260-265 nm) can be captured at time T 0 while radiation absorbance primarily by amino acids (280-285 nm) can be captured at time Ti using the same detector 14.
• Discussion of filters 12A, 12B is warranted in this example as the set before the fluorescence detector will be the standard long-pass fluorescence emission filters while the set before the DUV detector will be DUV band pass filters or short-pass fluorescence blocking filters.
• laser light is incident at an oblique angle relative to the objective lens optical axis, blocking the unscattered light and allowing dark-field measurement of the scattering profile at higher scattering angles.
• laser scattering at visible wavelengths may be found in U.S. Patent No. 6,741 ,730, issued 5/25/2004 to Rahn, entitled “Method and Apparatus for Three- Dimensional Imaging in the Fourier Domain," which is incorporated herein by reference.
• laser illumination parallel to the optical axis is used.
• a disk of absorbing material is located in the back focal plane of the objective. The diameter of the absorber is only large enough to block unscattered and very low- angle scattered light. The resulting annular aperture permits a dark-field measurement of the scattering profile at higher scattering angles.
• live stain either absorbance or fluorescence
• standard bright-field transmission mode removing diffraction analysis
• antibody/probe and nanoparticle is used in dark-field illumination mode for molecular specific labeling of proteins and/or DNA in the living cell.
• the image reconstruction module 42 determines a size of a voxel in the reconstructed 3D image.
• the reconstruction module 42 may further include a module constructed in accordance with known software engineering techniques for measuring a concentration of molecules absorbing the radiation by measuring the absorbance per voxel.
• the optical tomographic imaging system 11 lends itself nicely to DUV absorbance imaging.
• the condenser optics 56 may include, for example, a DUV condenser lens (for example, model UV-Kond, from Zeiss, Germany) and objective lens 18 may comprise a lens such as available from Zeiss, 100x, 1.25 NA, Ultrafluar, or a custom 265 nm objective lens, as available from Optics Technologies, Inc., Rochester, NY.
• filters 12A, 12B may include a band pass filter with a bandpass from 250 nm to 290 nm as available from Chroma Technology Corp.
• Useful CCD cameras include CCD cameras from Sony Corporation of Japan, the PhotonMax model camera from Princeton Instruments, Trenton, NJ, or devices from Sarnoff Imaging, Princeton, NJ.
• Live cell imaging often requires the specimen stage and glycerol, oil, or water- immersion objective lens to be temperature controlled.
• the materials must be UV transparent for the short transmission distances (path lengths) required for imaging isolated cells in a microcapillary tube of 50 microns in diameter.
• the cell medium should be a physiological buffer solution that may have higher refractive index to help match to the cell plasma membrane.
• Additives to the aqueous solution may include, but are not limited to, polyethylene glycol (PEG), glycerol, modified or derivative PEGs, and agarose gel.
• the cell medium cannot be well matched to the glass used for the microcapillary tube, then increasing the inner diameter may help reduce the degree of refraction at the inner tube wall.
• the refractive index should be able to be matched well with the outer tube wall since no biocompatibility needs to be addressed. However, materials that do not fluoresce within the wavelength range of signal 250 nm - 290 nm should be considered when the rotational joint is being selected.
• FIG. 2 an alternate example of system for 3D imaging of cells in an optical tomography system with ultraviolet radiation using a UV camera and optional adaptive optics is schematically shown.
• the requirement for live cell imaging imposes a restriction on the types of aqueous and physiological buffer solutions and thus on the range of refractive index that can be used around the cell.
• This embedding medium surrounding the cell and within the tube is expected to have sufficient refractive index mismatch with standard dry or oil immersion microscope objectives to cause aberrations in the resulting images. Compensation for this index mismatch can be designed for a specified imaging depth or distance from objective lens to cell that contains physiological buffer.
• adaptive optics The optical component used for such dynamic aberration compensation is often a spatial light modulator or a deformable membrane mirror.
• An adaptive reflection mirror is the preferred component in a DUV microscope due to the non-optimal transmission properties of DUV light through sophisticated optical components.
• a system for 3D imaging of cells 200 includes several components that are the same as or similar to those described above with respect to FIG. 1.
• a tube 22 is positioned relative to a microscope objective 18 for viewing an object of interest 1.
• a microscope 16 includes an objective lens 18 and a tube lens element 52.
• the microscope objective 18 is aligned along an optical axis 202.
• only a single ultraviolet (UV) camera 48 is used for acquiring images of the object of interest.
• the UV camera 48 is also aligned along the optical axis 202.
• a fluorescence-blocking filter 50 Interposed between the UV camera 48 and the tube lens element 52 is a fluorescence-blocking filter 50.
• the fluorescence blocking filter 50 is selected to block longer wavelength fluorescence and/or increase separation of differing ultraviolet radiation signals.
• the aqueous environment 2 and object of interest 1 may cause a sufficiently large refractive index mismatch between microscope objective 18 and tube 22 and optical gel 32 or equivalent to necessitate the use of an adaptive mirror 54 with associated adaptive optics (AO) controller 201 to reduce depth-dependent image aberrations.
• This adaptive optics component can be an optional element located between the radiation source 29, optical elements 27 and condenser lens 24. Whether unpowered or energized at a constant wavefront compensation (2D) profile, the adaptive mirror 54 becomes a static 90-degree turn in the optical system that may compensate for a single depth level.
• images from the UV camera 48 are transmitted to the image processor 40.
• the image processor transmits processed image data to the 3D image reconstruction module 42 which may advantageously be coupled to the optical display 44 for operator viewing if desired.
• User interface 46 is provided for operator control and information purposes.
• the user interface 46 may be a GUI interface or the like.
• a temperature-controlled housing 300 contains an object of interest, such as a biological cell 1 , or other biological material, is contained in a tube, capillary tube, or microcapillary tube 22, that is positioned relative to a microscope objective 18.
• the microcapillary tube 22 is rotatable by a rotary motor 20 to allow controlled rotational motion 21 of the cells 1 within the microcapillary tube 22.
• the cell 1 and gel 32 can be advanced within the capillary tube 22 along the horizontal axis by positive pressure applied, for example, by a syringe 80.
• Another motor 34 controls vertical axial movement of the microscope objective 18, and tube lens 52.
• the microcapillary tube 22 is encased within optical gel or refractive index matching medium 32 and is part of and atop of the sample-condenser light assembly 56.
• a power amplifier 60 provides energy for the temperature controller 64 that responds to at least one sensor 74 and that may be further regulated with computer and electronic input 78 to maintain the desired temperature within specified ranges, such as 5 to 39 degrees C.
• a warm-blooded animal cell such as a human requires tight temperature control, i.e. 36 degrees C with range of +/- 0.5 degrees C. Regulation of temperature as well as microfluidic conditions facilitates keeping cells alive (i.e.
• thermoelectric heaters/coolers 70 may be positioned in throughout the system and may be positioned both above and below the microcapillary tube 22 provide thermal energy for fine temperature control. Additional locations for Peltier heaters/coolers 70 may be advantageous in specific embodiments.
• Alternatives to thermoelectric heater/coolers and fans are the options of temperature controlled water circulator or equivalents around a chamber that encloses the microscope. In some embodiments temperatures of about 35 degrees C to about 36 degrees C are used, in others higher or lower temperatures may facilitate study of specific biological processes or for use of specific reagents in living cells.
• Biological objects 1 such as living cells, are injected into the microcapillary tube 22 via the syringe device 80 where pressurized capillary flow 84 moves the biological objects 1 to a viewing window beneath the objective lens 18 of the microscope 16.
• At least one radiation source 29 e.g. DUV and visible light
• the radiation wavelengths of about 260 nm to about 280 nm are used.
• the radiation passes through the light diffuser 28 and condenser lens assembly 24, 26, as part of the sample-condenser light assembly 56.
• the integrated sensors 74, temperature controller 64 and fan 68 maintain the temperature to maintain and increase cell viability.
• the system allows numerous variations to study living cells under defined and controlled conditions.
• the optical tomography system described above and elsewhere uses temperature control and microfluidics to maintain suitable conditions such that any living biological material may be examined including, but not limited to, cells from humans, as well as cells from any other species.
• the cells, or other biological material flow through one or more tubes (e.g. microcapillary tubes) to facilitate imaging.
• the microcapillary tube 22 comprises a straight tube of more than one channel. It is recognized that the optical tomography system may be used to harvest cells or sub-cellular material in certain embodiments.
• the system senses radiation including imaging signals emanating from macromolecular complexes, nucleoprotein, DNA, RNA, or protein, comprised in living cells, or in some cases non-living cells, or fragments thereof.
• Cells comprising component DNA, RNA, and protein complexes may be treated with chemicals, biological agents, including, but not limited to biologically active molecules, nanoparticles, modified nanoparticles, microspheres, protein protocells, antibodies, biomarkers, cytokines, other nucleotides, other proteins, or alternately mechanically manipulated by micromanipulation or other treatments (e.g. transfection reagents, viruses, liposomes, and like agents) to alter or facilitate molecular uptake or affect other cellular processes during the imaging process.
• biological agents including, but not limited to biologically active molecules, nanoparticles, modified nanoparticles, microspheres, protein protocells, antibodies, biomarkers, cytokines, other nucleotides, other proteins, or alternately mechanically manipulated by micro
• Biological or chemical agents may be labeled or modified with chromophores and fluorophores.
• Embodiments also use nanoparticles that are modified by labeling with gold, colloidal gold, iron, and iron oxide, and like molecules that have absorption, fluorescence, and scattering properties acting as optical contrast mechanisms in the 3D image or diffraction pattern.
• Use of nanoparticles and microspheres in addition to chromophores and fluorophores allows enhanced 3D contrast.
• cells could be treated with agents that affect the cell cycle, cellular differentiation, infectivity, reduce or increase pathogenicity, or the cells can be further manipulated to alter sub-cellular compartmentalization.
• the expression and display of cell surface biomarkers or chromatin or other cellular nucleoprotein or macromolecular complexes could be examined during all or some of these treatments.
• the living cells or other biological material are illuminated with multiple wavelengths of radiation.
• a plurality of pseudoprojection images of the cell, or other biological material that are formed from the computer processing of input images may be processed using ratio imaging techniques.
• the ratio imaging includes images formed from radiation wavelengths of about 260 nm to about 280 nm.
• a rotary motor 20 includes a shaft 121 coupled to turn a belt 188, where a second end of the belt 188 is coupled to rotate a microcapillary tube 22.
• the microfluidics cartridge 400 operates with positive pressure and negative pressure 120 to move the cells in a raceway with a secondary channel 504 to supply nutrients and oxygen, remove metabolic waste, and allow drugs to interact with cells in physiological buffer (as best shown in FIG. 5).
• a bearing or friction fit 92 allows the microcapillary tube 22 to rotate while an object, such as a cell, passes through the tube.
• a microscope 16 including condenser illumination assembly 56 is positioned proximate the cartridge to view the object along the optical axis of the microscope 16.
• FIG. 5 a top view of an example of a microfluidics cartridge as used in a racetrack configuration for imaging cells is schematically shown.
• the microfluidics cartridge 400 is coupled in a fluidic racetrack configuration 500.
• the racetrack configuration includes an imaging area 116 along the optical axis of the objective lens including an optical window. Also included is an entrance valve 96, an exit valve 124 and a first channel 502.
• the first channel 502 is in fluid communication with a secondary channel 504.
• the channels may be joined, for example, with a semi-permeable membrane 104.
• the entire racetrack is maintained in a temperature controlled environment such described herein with respect to FIG. 3 using Peltier heater/cooler elements or equivalents.
• the racetrack and channels comprise conduit.
• Fresh nutrients, oxygen, buffer (pH, osmolarity, etc), optional drugs and the like as needed to maintain cell viability may be introduced through the secondary channel 504 as indicated by flow arrows 108.
• the semi-permeable membrane 104 may be replaced by a joined channel with non- turbulent parallel flows allowing diffusion of small molecules and solutions while maintaining cells within their original streamlines of microfluidic flow. Shear stress within physiological range is possible with slow flow rates while channel geometry, fluid viscosity, temperature, and cell type also play a role.
• a trough 100 serves as a housing for the rotation motor and belt used to rotate the microcapillary tube 22 while cells travel through the tube.
• Positive and negative pressure 120 is applied to control pressurized flow 84 throughout the racetrack.
• a an exit valve 124 can be used to direct selected cell 1 by flowing fluid into a discard channel or for harvesting the live cell.
• the specimen being examined may be a biopsy from a fine needle aspirate (FNA).
• FNA fine needle aspirate
• the resulting sample of live cells may be split into several different racetracks with separate entrance valves (not shown).
• Each sub-sample being examined may be exposed to different drugs (such as drug A, drug B, drug combination A+B, and control - no drug), and the response may be monitored as real-time feedback for the purpose of personalized drug response for the patient.
• the racetrack configuration is useful as a research/drug discovery instrument.
• live cells may be circulated in the racetrack while imaging in 3D.
• Each live cell in the sample may be exposed to a chemical and environmental protocol and small changes in cellular response may be indicative of a desired cell type. Variations is apoptosis, mitosis, necrosis, secretion, and other programmed cell responses to stimuli can be measured at high sensitivity in realtime.
• the live cells When the live cells exhibit desired characteristics, the cells may be harvested.
• labeled nanoparticles like antibody/DNA labeling of gold or nanospheres can be used with live cells to label specific proteins, chromatin, and DNA.
• gold nanoparticles or colloidal gold have both absorption and scattering contrast and are biocompatible with living cells.
• Fluorescently-labeled nanospheres and microspheres can have absorption, fluorescence, and scattering as optical contrast mechanisms in the 3D image or diffraction pattern.
• Using nanoparticles in addition to chromophores and fluorophores will allow a third contrast enhancement, which is scattering.
• a means for imaging the scatter signal as high contrast on a "black" background or field is to illuminate with light that is incident at an angle of incidence beyond that of the imaging objective lens, so only the signal scatter is collected.
• the image is analogous to that of fluorescence imaging where the illumination photons are rejected from the final image.
• Live cell imaging in 2D using dark-field microscopy is being conducted at Duke University, see, for example, Curry, A., Hwang, W. L., and Wax, A. (2006), "Epi- illumination through the microscope objective applied to dark-field imaging and microspectroscopy of nanoparticle interaction with cells in culture,” Optics Express 14(14): 6535-6542.
• Diffraction pattern measurement is a non-imaging technique that is complementary to the above imaging techniques which measure the spatial pattern in 3D of DNA, chromatin, proteins, and their specific labeling enhancements.
• Disease specific signatures of diffraction may be found at specific spatial frequencies, which are measured at specific scattering angles from the cell. Since the zero order light from the laser beam is orders of magnitude greater than the weakly scattered light from live cells, the technique of oblique illumination of the cell is proposed to greatly reduce this zero order light from reaching the optical detector or camera. This technique is similar to dark-field microscopy using nanoparticles as discussed above.
• FIG. 6 an optical tomography process including separate imaging stages along the same pathway is shown. Separate imaging stages may be processed along the same pathway, such as a single microcapillary tube.
• visible light diffraction analysis and cell counting 602 may be done at a first stage 611 , followed by visible light imaging 604 at a second stage 612.
• 280 nm absorption imaging 606 may be conducted at a third stage 613, followed by 260 nm absorption imaging 608 at a fourth stage 614.
• the cell should be aligned within the limited field of view at each stage as the cell continuously moves down a single rotating capillary tube.
• the 280 nm absorption imaging includes illuminating the object 1 with DUV light at a first wavelength in the range of about 275 nm to 285 nm.
• the 260 nm absorption imaging includes illuminating the object 1 DUV light at a second wavelength in the range of about 255 nm to 265 nm.
• a single imaging stage that combines one or more image contrast mechanisms, such as absorption at wavelengths of 260 nm and 280 nm, measuring DUV absorption and native fluorescence, or measuring absorption at more than two visible wavelengths for one or more live stains.
• the components for combining optical imaging techniques can use multiple optical components for beam splitting and combining (dichroic or polarization beamsplitters) and possibly multiple cameras.
• a single camera and detection pathway can be used if the multiple excitation light sources are pulsed in time series or filter wheels or actual sources are physically moved or shuttered in time series.
• the single stage for imaging and measurement allow for stopped flow axial transport of the cells for precise alignment with the field of view.
• dark-field imaging of live-cell stain with nanoparticle scatterers may advantageously be combined with oblique illumination of the cell with a laser for diffraction pattern analysis.
• This technique may be run at higher speeds and may be an initial stage before the slower and subsequent 3D imaging stage if initial results warrant a detailed 3D image of a particular cell.
• the system provides an optical tomography process including separate imaging stages along the same pathway.
• a plurality of biological objects is transported along a pathway 25 to the first stage 611.
• At least one object of the plurality of objects is illuminated with visible light at the first stage to produce a diffraction pattern and the diffraction pattern is sensed by a light sensor.
• the diffraction pattern is analyzed to produce a diffraction analysis.
• the at least one object 1 is illuminated with visible light and the visible light emanating from the at least one object is sensed to produce a first plurality of pseudoprojection images.
• the at least one object 1 is illuminated with DUV light at a first wavelength and the DUV light at a first wavelength emanating from the at least one object is sensed to produce a second plurality of pseudoprojection images.
• the at least one object is illuminated with DUV light at a second wavelength that is sensed to produce a third plurality of pseudoprojection images.
• a plurality of objects may be sorted or otherwise classified using a sorter 610.
• the sorter 610 may be any of many types of conventional classifiers, usually embodied in software residing in a computer such as a statistical sorter, adaptive classifier, neural network or equivalents.

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