Method and apparatus for label-free identification of cells and cell subtypes

Abstract

An apparatus and method for label-free identification of cells and cell subtypes is disclosed. Particles are suspended in a fluid. The particles and fluid pass through a flow channel. A light source illuminates the particles flowing past the light source with light. As the particles scatter the light, detectors measure the characteristics of how the particles scatter the light. Enough measurements are made sufficient to distinguish types and / or sub-types of the particles without using attachment molecules.

Description

METHOD AND APPARATUS FOR LABEL-FREE IDENTIFICATION OF CELLS ANDCELL SUBTYPES

[0001] The present invention pertains to a method and apparatus for label-free identification of cells and cell subtypes.BACKGROUND

[0002] Blood contains a large variety of cell types. The large majority of blood cells are red blood cells (RBCs), but there are many other types of cells. The other types include many types that play a role in immune response and defense against disease. These cell types are referred to as “suspended” because they circulate in the blood and do not (perhaps until activated) adhere to fixed tissues.

[0003] It is valuable to be able to distinguish these many cell types from one another. Distinguishing cell types allows for counting of the numbers of these types (referred to as cytometry) and for separating cells of different types into separate volumes of fluid (referred to as cell sorting). In particular, sorting comprises two activities, namely, distinguishing or identifying cells by type, and separating cells of distinct types once they have been distinguished by type. For any one cell, these activities are sequential in the sense that a given cell is first identified by type and then separated. This sequential operation allows for cells to be sorted as they flow through a sorting device, wherein each cell as it is flowing through the device is first identified by type and then separated.

[0004] The types of cells can be thought of as hierarchical whereby gross or high-level types can be refined by description of subtypes. Conventional cell sorters can be thought of as sorting cells by their high-level types. To do this level of sorting, conventional cell sorters make use of a few measurements of cells based typically on optical scatter and fluorescence. For example, measurements of optical attenuation (or zero-degree scatter), optical scatter at a few chosen angles, say 15 degrees, 90 degrees, and intrinsic fluorescence at two fluorescent wavelengths, amounting to 6 measurements, might be used to identify 2 to 6 high-level types.

[0005] However, it is often valuable to be able to sort based on more refined subtypes of cells. For this purpose, the few optical scatter or intrinsic measurements described above are not sufficient. In this case, what has been done prior to this invention is to use antibodies coupled to exogenous fluorescent markers. These antibodies couple to protein structures on the surface of cells, which protein structures are distinct according to the subtype of the cell. This technique is called Fluorescently Activated Cell Sorting (FACS). The advantage of FACS isthat very fine distinctions between closely related subtypes can be identified because the antibodies are very specific. The great disadvantage of FACS is that the cells are inevitably altered by the attachment of the antibody. This alteration prohibits the reintroduction of FACS-identified cells into a human recipient.SUMMARY

[0006] The present invention pertains to an apparatus and method for label-free identification of cells and cell subtypes. Particles are suspended in a fluid. The particles and fluid pass through a flow channel. A light source illuminates the particles flowing past the light source with light. As the particles scatter the light, detectors measure the characteristics of how the particles scatter the light. Enough measurements are made sufficient to distinguish types and / or sub-types of the particles without using attachment molecules.

[0007] These and other objects and advantages of the various embodiments of the invention will be recognized by those of ordinary skill in the art after reading the following detailed description of the embodiments that are illustrated in the various drawing figures.BRIEF DESCRIPTION OF DRAWINGS

[0008] The accompanying drawings, which are incorporated in and form a part of this specification and in which like numerals depict like elements, illustrate embodiments of the present disclosure and, together with the detailed description, serve to explain the principles of the disclosure.

[0009] Figure 1 shows one embodiment of an optical apparatus for label-free identification of cells and cell subtypes.

[0010] Figure 2 shows an embodiment comprising an actuating mechanism which allows particles of distinct subtypes to be collected in distinct containers.

[0011] Figure 3 is an example wherein the radiation from the source is arranged to illuminate the particles from substantially one direction.

[0012] Figure 4 shows an example wherein the single-element or multi-element detectors are placed at positions such that those positions correspond to specific angles relative to the incident direction.

[0013] Figure 5 shows an example wherein a colored filter can be placed between the illuminated particle and one or more of the detector elements such that the one or more detector elements detect optical radiation at a subset of wavelengths of radiation.

[0014] Figure 6 shows an embodiment wherein the light source may comprise a laser.

[0015] Figure 7 shows one embodiment wherein more than one source may illuminate the particle from more than one incident direction.

[0016] Figure 8 shows an embodiment wherein the flow channel comprises one or more surfaces where each point on each surface causes radiation to refract by a refraction angle as the radiation passes through the that point on that surface where the refraction angle at that point on that surface corresponds to the direction of propagation of the radiation as it arrives at that surface

[0017] Figure 9 shows an embodiment wherein one or more surfaces of the flow channel cause radiation to fall on detectors which are not necessarily continuous

[0018] Figure 10 shows an embodiment wherein at least one of the surfaces of the flow channel through which the incident radiation passes is flat and perpendicular to the direction of the incident radiation

[0019] Figure 11 shows an embodiment whereby at least one of the surfaces of the flow channel are curved.

[0020] Figure 12 shows an embodiment wherein the emitted radiation substantially passes through curved surfaces.

[0021] Figure 13a shows an embodiment wherein the location of the subregion may be chosen so that cells pass through the center of the beam of incident radiation.

[0022] Figure 13b shows another example for a curved surface that is circular in crosssection.

[0023] Figure 14 shows an embodiment wherein at least a portion of either incident or emitted radiation undergoes total internal reflection from at least a portion of the surfaces of the flow channel

[0024] Figure 15 shows an embodiment wherein the measurement of the intensity of emitted radiation is analyzed to determine parameters related to shifts in the position of the particles in the flow channel

[0025] Figure 16 shows measurements of intensity at chosen specific angles of emitted radiation are preferentially incorporated into an analysis method.

[0026] Figure 17 shows an embodiment whereby the more than one source may each emit radiation distinctly over ranges of wavelengths.

[0027] Figure 18 shows that more than one source may emit over substantially different bands that are shifted from one another.

[0028] Figure 19 shows that at least one source may provide the slight shift in emission via frequency or wavenumber or wavelength modulation of the at least one source.

[0029] Figure 20 shows a substantially large number of detector elements is provided, and in addition an attenuation mask is provided

[0030] Figure 21 shows an embodiment wherein more than one attenuation mask is provided.

[0031] Figure 22 shows an embodiment wherein a light modulator may be utilized to modulate the phase and / or amplitude of the light source before interacting with the particles.

[0032] Figure 23 shows an embodiment wherein a light modulator may be utilized to modulate the phase or amplitude of the radiation from the particles

[0033] Figure 24 shows an embodiment of modulating the light source before interacting with the particles in phase and / or amplitude, as well as modulating the phase and / or amplitude of the radiation from the particles.

[0034] Figure 25 shows hydrodynamic focusing using sheath flow comprises multiple flow ports to support irregular flow channels.

[0035] Figure 26 shows how multiple configurations of the invention may be utilized in a connected configuration.

[0036] Figure 27 shows an example whereby the propagating light source is replaced with a near-field optical probe.

[0037] Figure 28 shows a cell (or particle) that is surrounded by a medium that is conductive of electrical current.

[0038] Figure 29a shows an embodiment wherein the proximate electrode is approximately one or more cell diameters away from the cell and approximately one or more cell diameters in transverse dimension.

[0039] Figure 29b shows an embodiment wherein the smaller electrode is closer to the cell than approximately the transverse dimension of the electrode.

[0040] Figure 30 shows at least two electrodes that are placed proximate to the cell and with transverse dimensions smaller that the cell.

[0041] Figure 31 shows two electrodes that are placed substantially on the same side of the cell.

[0042] Figure 32 shows an embodiment wherein the cell is annotated with surface features.DETAILED DESCRIPTION

[0043] Reference will now be made in detail to the various embodiments of the present disclosure, examples of which are illustrated in the accompanying drawings. While described in conjunction with these embodiments, it will be understood that they are not intended to limit the disclosure to these embodiments. On the contrary, the disclosure is intended to cover alternatives, modifications and equivalents, which may be included within the spirit and scope of the disclosure as defined by the appended claims. Furthermore, in the following detailed description of the present disclosure, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. However, it will be understood that the present disclosure may be practiced without these specific details. In other instances, well-known methods, procedures, components, and circuits have not been described in detail so as not to unnecessarily obscure aspects of the present disclosure.

[0044] Embodiments of the present invention pertain to identifying finely distinguished cell types without the use of attachment molecules, particularly antibodies. This is referred to as a label-free method. The present invention provides an apparatus and method for label-free identifications and thus label-free cell sorting.

[0045] The various embodiments of the present invention relate to the use of more channels of information than are used in conventional cell sorting, each channel representing a measurement made on the particle or cell, and where the greater number of such channels, as compared to conventional cell sorting, give sufficient additional information to distinguish sub-types of particles or cells. The additional channels are subdivided into channels derived from optical measurements (optical channels) of the particle or cell, and channels derived from measurements that are not optical in their origin (non-optical channels). Given that conventional cell sorting is done with optical information, the present invention considers optical channels that are in addition to those conventionally used. The non-optical channels are substantially all in addition to conventional cell sorting. Below are descriptions of the additional optical channels with particular attention to how such channels differ from conventional cell sorting. The non-optical channels are described separately.

[0046] The basic concept of additional optical channels of information is to use light to illuminate particles or cells and to distinguish the subtle differences between how different types of particles or cell subtypes scatter light. The differences in scattering are due to smalldifferences in internal and external geometric structure of the particles or cell subtypes. The differences can be in both the amount of scattered light and direction of the scattered light relative to the direction and characteristics of the particle-illuminating light. The particles or cells can be configured to be illuminated with light and the scattered light can be detected in a variety of different ways to help differentiate particle subtypes.

[0047] Figure 1 shows and embodiment of a flow channel through which particles 101 suspended in fluid may flow, a light source 102 of optical radiation in the visible or near- visible range which source is arranged to illuminate particles as the particles flow through the flow channel 103, one or more single-element or multi-element detectors 104-107 of optical radiation, where each detector element of each single-element or multi-element detector produces a signal that is a measurement of the intensity of the optical radiation that is emitted from the particle being illuminated, a data-acquisition system 108 such as one or more analog to digital converters (ADCs) that measure the signals from each of the detector elements along with a computer system 109 to operate the ADCs and to collect and store such measurements at sequential instances of time, and an analysis method that analyzes the collected measurements from one or more of the instances of time and determines the subtype of the illuminated particle. A number of analysis methods are well known in the art such as Principal Component Analysis and Logistic Regression. In addition, we have used Deep Neural Networks to analyze such measurements. We use visible (380 - 750 nm) near-infrared (750 - 1300 nm), or near ultra-violet (200 - 380 nm) optical radiation because the particles are most commonly suspended in an aqueous fluid and light propagates more readily in aqueous solutions at visible and near visible (e.g., near infrared) wavelengths. For nearultraviolet (nUV) light, the absorption of nUV by water is mitigated by using optical cuvettes or flow channels that are very thin, for example with thickness between 10 and 250 microns. Optical radiation emitted from the particle being illuminated is commonly scattered light and that scattered light can be emitted in different directions from the particle based on characteristics of the illuminating light (e.g., wavelength, polarization, incident direction), the size of the particle, the shape of the particles, and the internal and external structure of the particle. In comparison, emitted light generated from fluorescence interaction is typically emitted in all directions equally. In some embodiments, the particles comprise cells.

[0048] In addition to detecting the differences in particle subtypes, there may be a practical need to sort particles based on subtype for subsequent processing. Figure 2 shows anembodiment comprising an actuating mechanism which allows particles of distinct subtypes to be collected in distinct containers. Such an actuating mechanism could be based on piezoelectric actuation to add mechanical pressure to the fluid flow to redirect particles, applying various electric fields to the particle to move that particle within a flow, applying focused laser pulses to create vapor bubbles to direct or move particles, or other methods. Collecting subtypes in distinct containers allows for further downstream processing of the particle subtypes of interest, including genetic modification of desired subtypes in the case of cells, and / or reintroduction into a human recipient.

[0049] In further embodiments, the number of cells flowing through the flow channel is counted by recording each time the scattered radiation rises above the background signal that is present when no cells are present in the flow channel. Each subtype may be counted by recording each time an algorithm, as described above for determining subtypes, produces an output for that subtype above the background signal produced when no cell of that subtype is present in the flow channel. The number of cells, either in total or of a particular subtype recorded over a length of time may be related to the number of cells of that type per unit volume of fluid by dividing the number by the flow rate of the fluid through the flow channel, which ratio is a concentration of that subtype. Determining the concentration of subtypes can be used in optimizing further downstream processing of the particles.

[0050] In some embodiments, as shown in Figure 3, the radiation from the source is arranged to illuminate the particles from substantially one direction where the range of angles at which in radiation from the source illuminates the particle is between 0 degrees and 20 degrees, and preferentially between 0 degrees and 5 degrees, thus defining the incident direction. This is an important characteristic of the illumination as the direction of scattered light is dependent on incident direction. Limited angular range can be achieved by the use of lasers, or by the use of lenses in between a radiation source such as an LED and the particles.

[0051] Figure 4 shows an example wherein the single-element or multi-element detectors are placed at positions such that those positions correspond to specific angles relative to the incident direction. Such specific angles can be described by two values, for example, latitude and longitude on a sphere where the north pole corresponds to the incident direction. This is important because different subtypes of particles will scatter light at different angles depending on the internal and external structural differences between subtypes.

[0052] Figure 5 shows an example wherein a colored filter can be placed between the illuminated particle and one or more of the detector elements such that the one or more detector elements detect optical radiation at a subset of wavelengths of radiation. In further embodiments, the subset of wavelengths corresponds to wavelengths of fluorescent emission from the particle. These colored filters may block or allow passage of different wavelengths of light to detector elements, including the wavelength(s) of the illuminating light. If the illuminating light is in the visible or near-visible wavelength range, colored filters that block the illuminating light wavelength range may be detecting fluorescent emission from the particle. Alternatively, color filters that allow passage of the illuminating light wavelength can be used to detect scatter from both the particles and from the overall system while potentially rejecting any fluorescent light or background light.

[0053] The source that illuminates the particles may provide radiation at a subset of wavelengths in the visible or near-visible range. In further embodiments the source may provide radiation over a narrow range of wavelengths. A narrow range of wavelengths can contain light energy with a range of wavelengths of less than 200 nm, or less than 100 nm, or preferentially less than 20 nm or less than 2 nm. In some embodiments the source may comprise a laser as shown in Figure 6. In alternative embodiments, the source may comprise a light-emitting diode (LED), a hot-filament bulb, an arc lamp or any other source of visible or near visible radiation along with an optical filter to allow only a narrow range of wavelengths to fall on the particles. Choice of illumination wavelength and specific implementation (laser or LED or other illumination source) will depend on a number of factors, including illumination level (photon flux) requirements, overall system cost, and particle scattering properties at specific wavelengths.

[0054] Figure 7 shows one embodiment whereby more than one source may illuminate the particle from more than one incident direction. In these embodiments, the detector elements collect radiation from more than one specific angle, each specific angle corresponding to one of the incident directions. In further embodiments, each source illuminates the particle during distinct periods of time, whereby each detector element measures emitted radiation during each period at the specific angle relative to each incident direction. This potentially allows for additional particle subtype discrimination as the scattered light (direction and intensity) isdependent on the physical properties of the particles (e.g., size, shape, internal geometry) as well as the properties of the incident light (e.g., wavelength, polarization, and incident angle).

[0055] In some embodiments, the flow channel comprises one or more surfaces where each point on each surface causes radiation to refract by a refraction angle as the radiation passes through the that point on that surface where the refraction angle at that point on that surface corresponds to the direction of propagation of the radiation as it arrives at that surface, relative to the direction normal or perpendicular to that surface at that point, and to the refractive indexes of the materials on either side of the surface, as shown in Figure 8, according the Snell’s law. This is because the fluid flow may be encased in a flow channel embedded within a device, such as a microchip or flow cell. One or more surfaces of the flow channel cause radiation to fall on detectors which are not necessarily continuous, where continuous implies a sequence of detector elements directly adjacent to one another, as shown in Figure 9. Therefore, detectors may be placed specific angles around the particle rather than detecting scatter at all angles from the particle, potentially saving on cost and complexity of the system.

[0056] In Figure 10, at least one of the surfaces of the flow channel through which the incident radiation passes are flat and perpendicular to the direction of the incident radiation, that is, perpendicular to the incident direction. This geometry can simplify the design of the optical system and provide for a more consistent interaction between the illumination source and the particle.

[0057] Figure 11 shows an embodiment whereby at least one of the surfaces of the flow channel are curved. The curvature of the curved surface is chosen to optimize the propagation direction of the emitted radiation where the chosen curvature mathematically determines the surface in such a way that the direction of the normal to the surface at each point results in the direction of the emitted radiation arriving at a point to be refracted according to Snell’s law to a desired direction. The desired direction may allow for the emitted radiation leaving the surface to be deposited on a chosen detector element. In further embodiments, the curvature comprises a section of an ellipse, or a circle, or some other mathematically defined curve. This geometry can simplify the design of the optical system and provide for a more consistent interaction between the particle and the detector(s).

[0058] In some embodiments, surfaces of the flow channel are arranged such that incident radiation passes through planar surfaces, the orientations of which planar surfaces are related to the incident direction. For example, the orientation of the planar surface may be chosen to be perpendicular to the direction of the incident radiation to prevent refraction of the radiation to a different direction according to Snell’s law. For another example, the planar surface may be not perpendicular to the direction of the incident radiation so that any reflection of incident radiation by the planar surface backward along the incident direction does not enter a laser generating the incident radiation and cause that laser to function poorly. In addition, in these embodiments, the emitted radiation substantially passes through curved surfaces, the shapes of which curved surfaces are related to the ranges of specific angles of the emitted radiation, as shown in Figure 12. For example, the emitted radiation, which exits the region of the cell at a range of angles, can meet the curved surface at a point where the surface at that point is perpendicular to that angle of emitted radiation arriving at that point. This geometry can simplify the design of the flow channel and the optical system and provide for a more consistent interaction between the illumination source, the particle, and the detector(s).

[0059] Hydrodynamic focusing may be incorporated which causes the particles to flow through a subregion of the cross-sectional area of the flowing fluid. In further embodiments, the location of the subregion relative to the cross-sectional area of the flowing fluid is chosen to optimize the direction of propagation of radiation relative to the surfaces of the flow channel. For example, the location of the subregion may be chosen so that cells pass through the center of the beam of incident radiation as shown in Figure 13a. For another example, for a curved surface that is circular in cross-section, the cells may pass through the center of curvature of the circular cross-section, as shown in Figure 13b. In all these cases, because the particle is in a consistent location, this would simplify the design of the optical system and provide for a more consistent interaction between the illumination source and the particle. Hydrodynamic focusing may be achieved using a sheath flow of an additional fluid in the flow channel.

[0060] Figure 14 shows an embodiment whereby at least a portion of either incident or emitted radiation undergoes total internal reflection from at least a portion of the surfaces of the flow channel. This internally reflected light, if detected separately with a dedicateddetector, may also allow additional differentiation between particle subtypes by measuring emitted radiation that would otherwise be lost.

[0061] Figure 15 shows an embodiment whereby the measurement of the intensity of emitted radiation is analyzed to determine parameters related to shifts in the position of the particles in the flow channel, for example, the displacement of the cell with respect to the center of the incident radiation beam. In further embodiments, those parameters relate directly to the position of the particle in the flow channel, for example, the distance of the cell from the top and bottom walls or right and left walls of the channel. In alternative embodiments, those parameters relate to adjustments made in the analysis of the detected radiation, which adjustments allow for the proper interpretation of the measurements in the face of shifts of the position of the particles. In further embodiments, those parameters are determined in part utilizing measurements of intensity of forward specific angles of emitted radiation where the forward specific angles imply the direction of the emitted radiation is close to the direction of the incident radiation.

[0062] Figure 16 shows measurements of intensity at chosen specific angles of emitted radiation are preferentially incorporated into an analysis method where, for example, the chosen specific angles produce signals that change relatively strongly when one subtype of cell is being measured vs. when a different subtype is being measured. In further embodiments the chosen specific angles are related to specific characteristics of one or more subtypes of particles. In further embodiments, the specific characteristics correspond to specific subtypes of cells. In some embodiments, the chosen specific angles do not include substantially forward specific angles. Analysis methods can provide information on the utility of specific angles of emitted radiation to the determination of specific subtypes of particles. For example, using sensitivity analysis, the specific angles that show the greatest effect on the differentiation of subtypes of cells can be determined. In further embodiments, the positions of detectors are chosen to preferentially measure intensity at specific angles of substantially high utility.

[0063] Figure 17 shows an embodiment whereby the more than one source may each emit radiation distinctly over ranges of wavelengths. In further embodiments, those distinct emissions may comprise substantially different bands of wavelengths. This potentially allows for additional particle subtype discrimination as the scattered light (direction andintensity) is dependent on the physical properties of the particles (e.g., size, shape, internal geometry) as well as the properties of the incident light (e.g., wavelength, polarization, incident angle).

[0064] Figure 18 shows that more than one source may emit over substantially different bands that are shifted from one another. In this embodiment, the shift in the emission of the more than one source produces changes in the emitted light vs specific angle. Such changes may be smaller than the change in measured intensity from one detector element to a directly adjacent detector element, thus providing sampling of the distribution of intensity versus specific angle that is higher than the change in specific angle from element to element. This potentially allows for additional particle subtype discrimination as the scattered light (direction and intensity) is dependent on the physical properties of the particles (e.g., size, shape, internal geometry) as well as the properties of the incident light (e.g., wavelength, polarization, incident angle).

[0065] Figure 19 shows that at least one source may provide the slight shift in emission via frequency or wavenumber or wavelength modulation of the at least one source. In a further embodiment, the slight shift may comprise emission at a sequence of wavelengths, wavenumbers, or frequencies. Such a sequence may constitute a chirp of the emission. This potentially allows for additional particle subtype discrimination as the scattered light (direction and intensity) is dependent on the physical properties of the particles (e.g., size, shape, internal geometry) as well as the properties of the incident light (e.g., wavelength, polarization, incident angle).

[0066] In some embodiments, the measurement of intensity may occur at multiple times where such multiple times are during the time duration defined by the passage of one particle through the incident radiation. For any one such particle, measurement at such multiple times may constitute data that are more informative of the subtype of such a particle than are the data from any one of such multiple times. In further embodiments, the number of such multiple times is chosen to provide sufficient data to distinguish such a subtype while at the same time not overburdening the system with an overly large volume of data. In an alternate further embodiment, the number of such multiple times is chosen to balance, according to some definition of balance, the sufficiency of data to distinguish particle subtypes against the volume of data.

[0067] Figure 20 shows a substantially large number of detector elements is provided, and in addition an attenuation mask is provided. In further embodiments, such a combination of the substantially large number of detectors and the attenuation mask is chosen to emphasize emitted radiation of high utility in distinguishing subtypes of particles and to attenuate emitted radiation of low utility in distinguishing such subtypes. In further embodiments, the attenuation mask comprises substantially full transmission of emitted radiation for chosen specific angles together with substantially complete attenuation of emitted radiation for other specific angles.

[0068] In Figure 21, more than one attenuation mask is provided each such mask corresponding to a distinct subset of detectors. In further embodiments, each of the more than one attenuation masks is chosen to provide data of high utility in identifying a corresponding chosen subtype of particles.

[0069] In Figure 22, a light modulator may be utilized to modulate the phase and / or amplitude of the light source before interacting with the particles, substantially increasing the radiation of the incident particles.

[0070] In Figure 23, a light modulator may be utilized to modulate the phase or amplitude of the radiation from the particles, substantially decreasing the non-particle background signal.

[0071] Figure 24 shows an embodiment of modulating the light source before interacting with the particles in phase and / or amplitude, as well as modulating the phase and / or amplitude of the radiation from the particles, the light source may utilize methods to decrease the noise of the incident radiation below the shot noise limit by manipulating the quantum state of the incident radiation.

[0072] Figure 25 shows hydrodynamic focusing using sheath flow comprises multiple flow ports to support irregular flow channels, such as a rectangle. In some embodiments, multiple independent configurations of embodiments of the invention may be utilized to measuremultiple independent streams of particles. This method of multiplexing would increase the processing throughput of the system.

[0073] Figure 26 shows how multiple configurations of the invention may be utilized in a connected configuration, measuring the same stream of particles with sequential configurations of embodiments of the invention.

[0074] Figure 27 shows an example whereby the propagating light source is replaced with a near-field optical probe. The near-field probe does not produce an outgoing wave that propagates to the detector or detectors. However, when a cell or other small particle is very close to the end of the probe, it may convert some light to an outgoing propagating wave that can reach detectors. In addition, the characteristics of the outgoing wave are dependent on fine (sub-wavelength size) features at the surface of the cell that is very near to the end of the near-field probe. This is advantageous in that the subtypes of cells are usually defined in terms of surface features, for example, clusters of differentiation, or CDs. In alternate embodiments, the wide variety of detection schemes described in previous embodiments can be used together with the near-field source.

[0075] In further embodiments, multiple near-field optical probes may be placed sequentially such that a given cell or particle is near to each probe in turn. In this case, the detectors would measure scattered radiation corresponding to potentially multiple different regions of the surface of the particle or cell. In further embodiments, the cell is sequentially rotated and placed near to each probe, in which case the scattered radiation corresponds to different regions along a circumference of the cell.

[0076] Non-optical channels are now described in detail. Typically, a non-optical channel comprises any measurement of one or a combination of properties relating to a cell that are not optical properties.

[0077] Figure 28 shows a cell (or particle) that is surrounded by a medium that is conductive of electrical current. At least one electrode is placed proximate to the cell, and at least one additional electrode is placed in contact with the medium. The electrical impedance between the two electrodes is measured, either by impressing a current between the two electrodes and measuring the voltage difference between the electrodes, or by applying a voltage between the electrodes and measuring the current that flows between them. If the proximate electrodeis approximately one or more cell diameters away from the cell and approximately one or more cell diameters in transverse dimension (Figure 29a), then the measurement involves current flowing through the whole volume of the cell and in a portion of the medium surrounding the cell, in which case the impedance will represent an aggregate impedance of the combination of the cell impedance and the impedance of the medium. If the transverse dimension of the proximate electrode is smaller than the transverse dimension of the other electrode, then current must bunch up as it approaches the smaller electrode, and as a result, the measured impedance will be dominated by the impedance of the material near the smaller electrode. If the smaller electrode is closer to the cell than approximately the transverse dimension of the electrode (Figure 29b), then the impedance will be dominated by the impedance of the materials of the cell. Such materials are at least the cell membrane, the cell membrane surface proteins, the cell cytoplasm and the cell organelles. The diameters of cells range from less than five microns to greater than 20 microns. Therefore, the proximate electrode may have transverse dimension between 0.1 micron and 50 microns. Preferentially the transverse dimension may be between 1 micron and 5 microns. The distance of the proximate electrode to the cell may be between 0.1 micron and 50 microns, and preferentially between 1 micron and 5 microns.

[0078] Figure 30 shows at least two electrodes that are placed proximate to the cell and with transverse dimensions smaller that the cell. In this case, the impedance is dominated by the combination of the impedance of the materials of the cell in the regions near to the electrodes.

[0079] Figure 31 shows two electrodes that are placed substantially on the same side of the cell. In this case, the impedance is dominated by the impedance of the materials of the cell that are proximate to the pair of electrodes.

[0080] In an alternate embodiment, more than two electrodes are place in an array and the cell is placed sequentially such that different regions of the cell are proximate to different pairs of electrodes. In Figure 32, the cell is annotated with surface features: Feature A and Feature B. The cell first appears over the first pair of electrodes when surface feature A happens to be closest to the electrodes. The first pair of electrodes measures impedance dominated by the proximate material of the cell, which in this case includes feature A. Then, later, the cell appears over the second pair of electrodes. Feature B happens to be near the second pair of electrodes. The second pair of electrodes measures impedance dominated bythe proximate material of the cell, which in this case includes feature B. One may suppose that feature A and feature B are characteristic of a first subtype of cells. If a second cell came by the electrodes and only feature B was detected, then we have evidence that the second cell is not of the first subtype but rather of a second subtype.

[0081] The concept of the previous paragraph can be extended, in additional embodiments, to incorporate more than two pairs of electrodes. The cell may be manipulated to present different portions of its surface to different pairs of electrodes. (This can be done by methods well known in the art such as fluid-flow based methods.) In this way a collection of measured impedance values is obtained which contains information about the arrangement of different materials of the cell including surface features in different regions of the cell, thus constituting information about the structure of the cell. To the extent that the electrodes and their separations are very small, for example between 0.5 and 2 microns and many pairs are available, the information about the structure of the cell can be very fine-grained, thus allowing the distinguishing of many different subtypes that only differ from each other in a few respects. A single sensor or pair of sensors is used and the cell is sequentially repositioned to present different regions of its surface to the single sensor or pair of sensors. In further embodiments more than a single sensor or pair of sensors is used and the cell is sequentially repositioned to present different regions of its surface to the sensors. In this way one may obtain measurements in total numbering approximately the number of sensors or pairs of sensors multiplied by the number of repositionings, therefore constituting a rich data set that may be able to distinguish many subtypes of cells.

[0082] In an additional embodiment, the cell is sequentially rotated and placed proximate to each successive pair of electrodes. (Again, methods for rotating the cell are well known in the art.) In this case, impedance values obtained constitute information about the fine-grained structure of the cell sampled around a circumference of the cell. In a further embodiment, mathematical techniques, such as impedance tomography, can be used to reconstruct the structural distribution of materials of the cell from such circumferential information. A single sensor or pair of sensors is used and the cell is sequentially repositioned to present different regions of its surface to the single sensor or pair of sensors. In further embodiments more than a single sensor or pair of sensors is used and the cell is sequentially repositioned to present different regions of its surface to the sensors. In this way one may obtain measurements in total numbering approximately the number of sensors or pairs of sensorsmultiplied by the number of repositionings, therefore constituting a rich data set that may be able to distinguish many subtypes of cells. The impedance can be measured at DC or at one or more finite frequencies. Such frequencies can range from 1 Hz to 10 GHz, and preferentially from 1 kHz to 1 GHz.

[0083] In some embodiments, ultrasonic transducers are placed proximate to the cell. Ultrasound energy is generated which passes through the cell and is detected by receiving transducers. The detected signals constitute information about the arrangement within the cell of the mechanical properties of the materials of the cell. Such ultrasound energy can be at frequencies between 30 MHz and 3 GHz, and preferentially between 1 GHz and 3 GHz, which frequencies yield appropriate wavelengths of ultrasound so as to be sensitive to object the size of a cell. The concepts described above can be applied to any sensing mechanism that can be miniaturized to be less than the size of a cell. Examples are, small electrode impedance as described above, near-field optical probes as described above, and ultrasound transducers as described above as well as other sensors known in the art. In a variety of embodiments, an array of sensors that can be miniaturized to be smaller than approximately the size of a cell measures properties of the cell dominated by different regions of the surface of the cell by sequentially presenting the cell in different orientations.

[0084] Although the subject matter has been described in language specific to structural features and / or methodological acts, it is to be understood that the subject matter defined in this disclosure is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts described above are disclosed as example forms of implementing this disclosure.

[0085] Embodiments according to the invention are thus described. While the present invention has been described in particular embodiments, the invention should not be construed as limited by such embodiments, but rather construed according to the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method to identify particle subtypes, comprising: suspending particles in a fluid; passing the particles through a flow channel; illuminating the particles flowing through the flow channel with light; measuring characteristics of how the particles scatter the light, wherein enough measurements are made sufficient to distinguish sub-types of the particles without using attachment molecules.

2. The method to identify particle subtypes of Claim 1, wherein the particles comprise cells, the sub-types comprise cell sub-types, and the molecules comprise antibodies.

3. The method to identify particle subtypes of Claim 1 comprising label-free cell identifications and label-free cell sorting.

4. The method to identify particle subtypes of Claim 3 further comprising reintroducing sorted cells into a human recipient.

5. The method to identify particle subtypes of Claim 1 wherein the characteristics of how the particles scatter the light comprise the amount of scattered light and direction of the scattered light relative to the direction and characteristics of the light illuminating the particles.

6. The method to identify particle subtypes of Claim 1 further comprising taking at least six measurements at different angles of light scattered from a particle as it flows through the flow channel.

7. The method to identify particle subtypes of Claim 6 wherein the measurements are taken by at least six detectors physically fixed at different locations relative to a light source illuminating the particles flowing through the flow channel.

8. The method to identify particle subtypes of Claim 7, wherein the detectors are placed at positions corresponding to specific angles relative to the incident direction.

9. The method to identify particle subtypes of Claim 6, wherein the measurements are taken by a camera.

10. The method to identify particle subtypes of Claim 1 further comprising analyzing light emitted from the particle in different directions based on characteristics of the illuminating light, the size of the particle, the shape of the particle, and the internal and external structure of the particle to determine the subtype of the particle.

11. The method to identify particle subtypes of Claim 1 further comprising illuminating the particles with light from at least two separate light sources.

12. The method to identify particle subtypes of Claim 1 further comprising recording a number of a particular subtype particle that flows through the flow channel to determine a concentration of that particular subtype particle.

13. The method to identify particle subtypes of Claim 1, wherein a test sample comprises at least four different cell types.

14. The method to identify particle subtypes of Claim 1 further comprising genetically modifying an identified subtype of cell which can then be inserted into a human.

15. The method to identify particle subtypes of Claim 1 further comprising hydrodynamic focusing to cause the particles to flow through a subregion of a cross-sectional area of the flow channel.

16. The method to identify particle subtypes of Claim 1 further comprising at least one surface of the flow channel through which incident radiation passes are flat and perpendicular to a direction of the incident radiation.

17. The method to identify particle subtypes of Claim 1 further comprising at least one surface of the flow channel is curved.

18. The method to identify particle subtypes of Claim 1 further comprising measuring the characteristics of the particle with a non-optical channel.

19. An apparatus for identifying cell subtypes, comprising: a flow channel through which a test sample comprising particles passes; an optical radiation source for illuminating the particles; a detector system for measuring light reflected by the particles at different scatter angles, wherein enough scatter light measurements at the different scatter angles are taken to differentiate between the different cell subtypes in the test sample in a label free process.

20. The apparatus for identifying cell subtypes of Claim 19, wherein the detector system measures at least six different scatter angles per particle.

21. The apparatus for identifying cell subtypes of Claim 20, wherein the detector system comprises at least six different detector elements situated at different locations which produces signals that are measurements of intensities of optical radiation that are emitted from the particles being illuminated.

22. The apparatus for identifying cell subtypes of Claim 19 further comprising a data acquisition system that measures signals from the detector system.

23. The apparatus for identifying cell subtypes of Claim 19 further comprising a computer system to collect and store the measurements at sequential instances of time and analyzed collected measurements to determine the subtype of an illuminated particle.

24. The apparatus for identifying cell subtypes of Claim 19 further comprising a sorting mechanism that enables particles of a selected subtype to be collected after it is identified.

25. The apparatus for identifying cell subtypes of Claim 19 further comprising a counter for determining a concentration of a particular particle subtype associated with the test sample.

26. The apparatus for identifying cell subtypes of Claim, wherein the detector system comprises detector elements placed at locations corresponding to specific angles relative to an incident direction.

27. The apparatus for identifying cell subtypes of Claim 19 further comprising a colored filter that is placed between an illuminated particle and at least one part of the detector system.

28. The apparatus for identifying cell subtypes of Claim 19 further comprising a second optical radiation source for illuminating the particles.

29. The apparatus for identifying cell subtypes of Claim 19, wherein at least six different scatter angles are measured to identify a particle.

30. The apparatus for identifying cell subtypes of Claim 19, wherein the detector system comprises at least six separate detectors.

31. An apparatus for identification of cell types, comprising: a conductive medium that is conductive to an electrical current, wherein a cell resides within the medium; a first electrode placed in contact with the medium; a second electrode placed in contact with the medium, wherein an electrical impedance between the first electrode and the second electrode is measured to identify the cell.

32. The apparatus of Claim 31, wherein the first electrode and the second electrode are placed proximate to the cell and comprise transverse dimensions smaller than the cell.

33. The apparatus of Claim 31, wherein the first electrode and the second electrode are placed on a same side of the cell.

4. The apparatus of Claim 31, wherein the first electrode and the second electrode are placed in an array and the cell is placed sequentially wherein different regions of the cell are proximate to different pairs of electrodes.

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