JPH05846Y2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a flow cell measuring device, and more specifically, to a flow cell measuring device capable of simultaneously performing optical measurement and electronic particle capacity measurement. [Background of the invention] The origin of flow particle analyzers can be divided into two main lines of development, one of which is an optical line and the other an electronic line. Furthermore, various proposals have recently been made to combine optical and electronic methods. The basic book that covers the origin of the entire field was published in 1979 by Melamed, Mullaney, Mendelsohn, et al. FLOW CYTOMETRY
AND SORTING). In order to facilitate understanding of the features and advantages of the present invention and its background, a brief summary of the development of optical, electronic and combination methods is provided below. Optical Flow Particle Analyzer Optical flow cytometry has been developed over the last 25 years. These methods were originally used to count cells or particles by the change in absorption of the illuminating light as the cells passed through a glass capillary in the measurement channel of a photoelectric measurement device. Later improvements provided for the flow of particles, or thin sheaths of cells, through the observation area. In this case, a suspension of cells or particles under investigation is injected into a faster flow of fluid. This faster flow provides a sheath around the particles, creating a laminar flow. The addition of a laminar flow is important because it allows the use of larger diameter flows, thus minimizing clogging and directing the flow of the particle or cell sample precisely into the center of the analysis volume. This is because it can be placed. A book on thin-layer flow and the development of hydrodynamic focusing methods is ``Flow Cell Measurement and Analysis Methods.''
(FLOW CYTOMETRY AND SORTING). Patents relating to various aspects of this subject include U.S. Patent No. 3,299,354;
No. 3738759; No. 3740665; No. 3810010; No.
No. 3871770; No. 3984307; No. 4140966; No.
No. 4162292; and No. 4237416. In addition to absorption optical measurements, light scattering and fluorescence are both methods that are also used to analyze cells or particles. In this regard, reference is made to the extensive description of these methods and the bibliography contained in the above-mentioned book ``Flow Cytometry and Classification''. Patents related to light scattering methods include U.S. Patent Nos. 3614231; 3646352; 3669542;
No. 3705771; No. 3785735; No. 3786261; No.
No. 3873204; No. 3960449; No. 4053229; No.
No. 4173415; and No. 4188121. Patents related to fluorescence are U.S. Pat. No. 3,788,744;
No. 4225229; and No. 4243318. In 1965, Kamentsky et al. were the first group to perform optical measurements of two parameters in cells by flow methods. See "Spectrophotometer: New instrument for ultrarapid cell analysis" by Kamentsky et al., Science 150:630-631, 1965. In this study, UV absorption by the nucleic acids was measured, as well as light scattering analyzed. There are many known patents directed to multiple light measurements, for example U.S. Pat.
No. 3662176; No. 3850525; No. 4038556; No.
No. 3824402; and No. 3916197. The sensitivity of an optical measurement is a measure of how much fluorescence emitted by a particle is collected, or how much of the incident light that is scattered or absorbed is recollected. These parameters are related to the optical configuration of the instrument, including the numerical aperture (NA) of the lens system and whether the optical axis is parallel to, or transverse to, the flow of particles or cells. An optical device is described that measures fluorescence in a stream of particles parallel to the optical axis (axial flow). Some such devices conveniently use incident light illumination, where the same optical device is used to direct the light into the particle stream and collect the fluorescent light from the particles. A flow cytometer across the light is also described,
In this case the flow of particles or cells crosses the optical axis. For example, US Pat. No. 4,056,324; No. 4,225,229
No. 3720470; and “Science”
(SCIENCE) Volume 204, April 27, 1979, No.
See page 403. Although axial flow optical cytometers have shown some very good results for fluorescence measurements, there are some deficiencies. Light scattering parameters are not possible due to the difficulty of placing the detector after the cells being measured. Additionally, an objective lens with a significant depth of focus is required to focus through the trough of the flow over the emerging cells or particles. This arrangement significantly increases the vertical cell analysis volume due to the large depth of field, thus limiting the speed of cell analysis. If the vertical cell analysis volume is increased, more than one cell can be introduced into the analysis volume and detected as a single fluorescence pulse (matching problem). Finally, axial flow designs are not suitable for cell sorting per se because cells exiting the orifice are not captured in a laminar flow. Cross-flow optical cytometers can avoid some of the problems associated with axial flow. First of all, light scattering methods are possible since the detector can be placed around the incident light beam (ie the beam from the laser) intersecting the cross flow. With this arrangement, the intensity of light scattering can be measured perpendicular to the incident beam. However, these orthogonal methods
Measure the fluorescence at 90°. The amount of fluorescence measured from the cell at this angle is minimized due to internal absorption of the fluorescence before it exits at right angles to the incident beam.
This "dark field" illumination is used because it ensures a strong incident laser light that does not interfere with the fluorescence signal, which is measured at 90° to the incident light. Since these formats are used for airborne water flow, right-angle flow optical cytometers must use dry objectives that limit the numerical aperture to 0.7 or less. Electronic Particle Volume The basic idea of electronic counting of particles suspended in a conducting fluid passing through a microscopic aperture is disclosed in US Pat. No. 2,656,508 to Coulter. In this case, an electric field is applied to the opening or orifice to cause current to flow therethrough. This aperture current is interrupted as particles pass through the aperture, creating a measurable pulse that is used to count particles. 1958 “ Nature ” 182:234−
In ``Electronic Counting and Sizing of Bacteria,'' described in 235, Kubitschek attempted to relate pulse amplitude or area to particle size. A number of publications followed that attempted to explain and characterize this relationship. In a 1969 article, Grover et al. (1969, 9:13981414, Biophys J., ``Electrical sizing of particles in suspensions'') We explained the "edge effect" due to the compression of the electric field as we progress from the volume to the microscopic aperture. The article also describes hydrodynamic flow considerations and the relationship between particle volume and the observed change in current as the particle traverses the aperture. Particle trajectory sensitivity has been one problem in electronic cell volume analysis. The solution taken to these problems was to orient the particles along the axis of the aperture, make the aperture long with respect to its diameter, and electronically reject any pulse shape that is not Gaussian.
These methods include hydrodynamic focusing of particle streams.
Although hydrodynamic focusing is a reasonably effective solution to orbital sensitivity, the requirement to precisely center the particle injector with respect to the aperture necessitates a very complex transducer design. Other challenges encountered in the design of electronic particle detection transducers are (1) bubbles from degassing of the sample, electrode, or suspended fluid, and (2) foaming of the electrode product and its impact on biological cells due to changes in cell PH and toxicity. (3) so-called "reverse cursor" pulses due to particles that have already crossed the aperture circulation in the region of the aperture outlet, and (4) clogging of the aperture. Solutions to the bubble and electrode problems have focused on separating the electrodes into separate chambers. A reverse cursor pulse provides a second pulse to prevent recirculation.
orifices or by flooding the particle fluid into the exit chamber and by electronic identification of longer reverse cursor pulses. Aperture clogging posed a serious problem for these devices. The openings are typically 50-100 microns in diameter and are easily clogged. Backflow, fluid and sample filtration, and the miniature windshield wiper mechanism disclosed in US Pat. No. 3,259,891 have all been tried, with largely unsatisfactory results. The literature on electronic particle volumemeters describes various ways to obtain additional information about the particles using something other than direct current in the aperture. These include multifrequency alternating current devices, alternating current devices with electrodes inside the aperture, and combinations of alternating current and direct current that seek to change both resistance and capacitance as a particle traverses the aperture. Combined Optical and Electronic Particle Volumemeter It must be shown that more useful information about the properties of particles or cells can be obtained by the combined use of both electronic and optical methods than by the use of either method alone. 1970, "Academic press", New York, pages 131-159
Leif in "Automated Cell Identification and Classification Methods", described on page 1, proposed an instrument that performs multiple optical and electronic measurements. Later books included AMAC (Automated Multiparameter)
describes various types of such devices known as Analyzers for Cells. One type, known as AMAC 1, was published in 1973 Clin.Chem. 19:853.
-870, by Leif and Thomas in the article ``Electronic Cell Volume Analysis Using the AMAC Transducer.'' The instrument is an axial flow, transmission illuminator suitable for light scattering and electronic measurements. Electronic measurements were accomplished with this device, but optical measurements were not. In 1973, in the article ``A New Multiparameter Separator for Small Particles and Biological Cells,'' published in Rev.Sci.Instr. 44:1301-1310, Steinkamp et al. The transducer that performs the measurements has been described. However, the measurements were not taken simultaneously. Electronic measurements are taken first;
Fluorescence measurements were then taken downstream and after 135 microseconds at right angles to the cell flow. Kachel, US Pat. No. 4,198,160 also describes a transducer that makes both electronic cell volume and fluorescence measurements. Katzherr used incident light illumination for fluorescence and also used a hydrodynamic focusing injector to position the particles. This structure allows both parameters to be measured more closely relative to each other, but still not at exactly the same time. There are books describing an AMAC IV transducer, comparable to the Steinkamp device described above, with a water immersion objective that captures the fluorescence perpendicular to the laser excitation of the particle stream. Water immersion objectives have a larger numerical aperture than air objectives (1.0 vs. 0.7 or less);
Thus, the light gathering ability in fluorescence measurements is increased.
Although unique electronic and fluorescent measurements were performed, the combined measurements were not successful due to the time delay between the electronic and optical measurements. Prior art for combined electronic and optical measurements includes U.S. Pat.
Also includes issue 3770349. No. 3,710,933 to Fulwyler et al. describes an apparatus in which electronic and optical measurements are made sequentially and not at the same time. Patents No. 3675768 and No. 3675768 to Sanchez
No. 3770349 describes a device that is said to provide both electronic measurements and optical absorption measurements. All of the above combination methods used circular openings in the wall as transducers. However, although one experimental transducer utilizing a square aperture has been reported in the literature, there is no indication of how to make the square aperture. this is
Journal of Histochemistry and Cytochemistry, published in 1977.
Cytochemistry) Volume 25, No. 7, Page 827-No.
The AMAC transducer was described by R. A. Thomas et al. in the article "Combinatorial optical and electronic analysis of cells with an AMAC transducer" on page 835. The transducer used hydrodynamic focusing to direct the particle stream and orthogonal fluorescence detection to the laser excitation beam. Since the optical measurements were performed in a small cuvette with an electronic aperture, theoretically all measurements could have been taken at the same time. Separate electronic and fluorescence measurements have been reported, but no simultaneous measurements have been reported to date. Several common problems existed in prior art attempts at combined electronic and optical cellular calculators. If the measurements are not taken at exactly the same time, there are matching problems in accurately relating two sequential measurements to the same particle. Conflicting design considerations are involved when it is desired to perform electronic and optical measurements at exactly the same time. For electronic measurements, the aperture resistance must be large compared to the entrance and exit resistances. If this were not the case, the effective length (i.e. the distance over which the particles appreciably affect the current flow) would be long and the matching speed would be unacceptable. The particles must pass quickly from the region of low current flux to the region of high current flux and back again to the region of low current flux. moreover,
They must proceed on a trajectory that avoids the "edge effect." In optical measurements, the larger the numerical aperture (NA), the better the sensitivity of the measurement. That is, the resolution is N.
A. is directly proportional to the image brightness is proportional to (NA) ² ,
Depth of focus is inversely proportional to NA. However, a large NA means a large acceptance angle, which indicates that the object to be measured must be as close as possible to the condenser lens. In any axial flow combination transducer configuration, the electronic and optical design structures are mutually exclusive and compromises must be made to reduce the sensitivity of one or both measurements. For example, in the case of electronic measurements, the objective lens must be moved away from the aperture to minimize the exit volume, resulting in reduced NA and/or loss of measurement simultaneity. . In the cross-flow method, downstream optics are the only solution, resulting in loss of measurement simultaneity. Of all the prior art structures, the AMAC converter comes closest to solving the problem of combined electronic and optical measurements. In AMAC, the square electronic aperture was also an optical aperture. By using a square shape, the entrance/exit chamber had a large volume compared to the opening volume, thus accommodating electronic confinement. However, the angular shape sacrificed numerical aperture (NA) by limiting the acceptance angle to 90° for fluorescence measurements. The fluorescence was activated by a laser placed at right angles, which produced good light scattering properties at the expense of the light provided by the incident light illumination. As can be seen from the above brief discussion of some of the prior art, the field of flow cytometers continues to be very actively researched. There is a clear need for a flow cytometer that can perform electronic and optical measurements exactly simultaneously, without sacrificing any of the sensitivity possible with either method. Additionally, improvements in sensitivity are required for both electronic and optical measurement methods. OBJECTIVES OF THE INVENTION Accordingly, one objective of the present invention is to provide a method and apparatus that improves the sensitivity of flow cell measurements. Another object of the present invention is to provide a method and apparatus for flow cytometry that performs simultaneous electronic and optical measurements. Another object of the present invention is to provide a flow cytometry transducer in which the shape of the opening is such that it reduces electronic edge effects through the aperture while increasing electronic sensitivity. Another object of the present invention is to provide an electronic and optical flow cytometry transducer consisting of a plurality of cubic polygons forming a polygonal aperture. Another object of the present invention is to provide a flow cytometer that performs electron and light scattering measurements simultaneously. Another object of the present invention is to provide a process for simultaneously performing electronic and optical measurements, in which at least one of the plurality of elements forming the aperture of the cytometer is the first element in the optical device when performing optical measurements. The purpose of the present invention is to provide a cell measuring device. Another specific object of one embodiment of the present invention is to provide a flow cytometer with a triangular transducer aperture. Another aspect of the present invention is to provide a flow cytometer and method in which sonication is used as a means to clean a clogged orifice. Another object of the present invention is to provide a flow cytometer for simultaneous electronic and optical measurements, where incident light illumination and sensing are used for optical measurements. [Structure of the invention] Briefly stated, one embodiment of the invention:
A flow cytometer is provided having an aperture having a predetermined shape for the aperture and provided with an inlet chamber and an outlet chamber that reduce edge effects and increase the sensitivity of electronic particle volume measurements. According to one embodiment, the aperture is triangular and is made by assembling a plurality of truncated pyramids, the truncated surfaces forming the walls of the aperture. The shapes of the inlets and outlets created by the truncated pyramids are shaped to reduce edge effects from the flow of current through the apertures and to maintain a predetermined cross-sectional area with respect to the cross-sectional area of the apertures. Electrodes are provided for passing electrical current through the apertures for electronic particle or cell volume measurements. At least one of the elements forming the aperture wall is part of a lens system or is a transparent cover plate for an oil immersion objective used to simultaneously take optical measurements of particles or cells while they are in the aperture. be. These optical measurements can be performed using a fluorescence method, a light scattering method, or a combination of both. moreover,
According to one aspect of one embodiment of the present invention, a piezoelectric transducer is provided to ultrasonically clean any blockages in the aperture. [Embodiment] In its broadest aspect, the present invention is directed to an electronic or combined electronic/optical flow cytometer having an inlet chamber and an outlet chamber adjacent to an aperture and having a predetermined geometrical relationship to the aperture. The aim is to provide an aperture, or a sensing zone. The predetermined shape of the entrance/exit chamber according to the present invention reduces electronic "edge effects" and improves the sensitivity of electronic volumetric measurements of sample particles passing through the aperture. Prior art electronic particle volume counters provided an aperture or sensing zone in the form of a small round hole in the wall. FIG. 1 shows the pattern of current flux density flowing through such an aperture consisting of a round hole through the wall 1. As can be seen, the current flux density tends to concentrate at the edges of the round hole. These “edge effects”
This is a source of a lot of false information when trying to relate the magnitude of change in current to the size, or volume, of a particle. For example, slight differences in the trajectories of particles as they traverse the measurement zone result in differences in the amount of current blocked by particles of the same size. According to the invention, the opening is provided with an entrance chamber having a chamber wall provided immediately adjacent to the opening that has a predetermined geometrical relationship with the opening. The first aspect shown in FIGS. 2 and 3 assumes a chamber 2 with a uniform current flux density, as long as edge effects are reduced. This chamber 2 connects to an inlet chamber having an opening or wall 3 forming an angle θ with respect to the plane of the measuring zone 4 . Since the current follows the line of least resistance, the current flux through the area Z ₁ of chamber 2 passes through the volume relative to corner ₁ , the current flux through Z ₂ passes through the volume relative to corner ₂ , and so on until Z _o and _o It is possible to specify that as a first approximation method. That is, the degree expressed by the angle _o is
Z is inversely proportional to the current flux through _o . For a fixed quantity I _o (representing the percentage of the width of the measurement zone at the aperture entrance), the angle θ
The degree of angle _o corresponding to each changing value of can be calculated. In the prior art arrangement of holes in the wall, θ is equal to zero. If θ is equal to 90°, a measurement chamber without an entrance angle is described. From FIG. 3, for purposes of illustration, the aperture is 100 microns wide, and corner ₅₀ is defined as the angle made from the center of the aperture to its edge (making an angle θ with the wall of the inlet chamber). The other angles ₄₀ , ₃₀ , ₂₀ , and ₁₀ are the angles made between the center of the aperture and 40, 30, 20, and 10 microns from the center, respectively. The distance L shown in FIG. 3 can be expressed in terms of θ by the following equation. (1) L=1/2 base tan θ=50 μm×tan θ Therefore, L can be obtained by changing θ. The value of L is determined as the angle θ increases or decreases. Therefore, the streamline density can be described at an aperture entrance at any distance from the center along the width of the aperture by finding for a given θ (or L used in the calculation). These arbitrary distances are
For example, it can be a multiple of I, which can be 10 μm. The value of _o is then given by the following formula: (2) _o = tan ^-1 nI/L - tan ^-1 (n-1) I/L Table I below shows the results of this method. The measurement zone or aperture entrance is divided into ten equal width segments, five on each side of the center of the aperture. The amount _1/10 represents a segment from the center that moves towards the edge by 10% of the width of the aperture, _1/20 from 10% - 20% of the width, _1/30 from 20-30% of the width, 1 / ₄₀ represents the segment from 30-40% of the width and _1/50 represents the edge of the opening from 40-50% of the width. [Table] [Table] As can be seen from Table 1, for an entrance angle of 1° (basically one hole in the wall), the change in value of _1/10 - _1/50 is extremely large, and the value change near the edge is extremely large. In other words, it becomes a particularly large value at _1/50 . As the entrance angle θ is increased, the dispersion (the difference in current flux density at the measurement entrance) decreases. At a θ of 50°, the value is almost constant over _1/10 - _1/50 . Figure 4 is a plot of these values. As can be seen from Figure 4, the curve is approximately
Focuses at 50° and maintains that state up to 90°. As can be seen from the curves in FIG. 4, a significant phenomenon of edge effects arises from entrance chamber angles θ of 5° or more. Thus, according to this aspect of the invention, the inlet chamber is fed into the sensing chamber;
The walls of the inlet chamber are arranged at an angle of 5° or more with respect to the plane of the opening to reduce edge effects. A similar concept applies to the exit chamber, the walls of which are arranged at an angle of 5 ° or more with respect to the plane of the opening. In addition to reducing edge effects in electronic particle volumetric cytometers, it is desirable to make the instrument as sensitive as possible. The current flux density of the aperture is inversely proportional to the cross-sectional area. Therefore, the current flux intercepted by the particle is maximized when the particle is confined to a small cross-sectional area. Therefore, as the particle progresses towards the measurement zone (i.e. where the current flux density is maximum), it rapidly progresses from an area of low current flux density to an area of high current flux density, i.e. an observable change in current. It is desirable to quickly progress from a zone with no current to a zone with maximum current change. According to the principles of the present invention, entrance and exit chambers are provided adjacent to both sides of the sensing opening, and the cross-sectional area of the entrance and exit chambers has a predetermined relationship to the cross-sectional area of the opening. First, “S”
A size parameter, referred to as a unit, is defined as the maximum linear dimension or width of the aperture along any plane passing through the axis of the aperture. This aspect of the invention allows the aperture to be of any cross-sectional shape, ie circular, angular, triangular, etc. The straight-line distance between the two "S" units is measured from either side of the opening to the doorway chamber, and it has been experimentally determined that the cross-sectional area of the doorway chamber at these points must be greater than 10 times the cross-sectional area of the opening. (See Figure 5). If the cross-sectional area of the entrance/exit chamber at these points is greater than 10 times the cross-sectional area of the aperture itself (preferably the cross-section is 20-30 times larger), a sharp, well-defined electron particle volume pulse from the electronic cytometer can be obtained. It is known that this occurs. In accordance with another aspect of the present invention, an improved method of fabricating an electronic/optical sensing device for a flow cytometer is discovered through the cooperation of a plurality of solid polygons. "Solid polygon" means a multi-sided three-dimensional polygon. In accordance with a broader aspect of the invention, a plurality of surfaces having surfaces operative to form an aperture or sensing zone and defining the shape of the entrance chamber so as to have a predetermined geometrical relationship with respect to the aperture or sensing zone. of these solid polygons are provided. It is within the scope of the present invention to provide such an assembly in which the aperture is triangular, square, pentagonal, etc. However, a triangular aperture is a preferred embodiment of the many advantages provided by the present invention. When performing optical measurements, at least one of the elements forming the wall of the aperture is part of an optical device. According to the invention, at least one element that is part of the optical device can be a transparent cover plate on which an oil immersion objective is placed, or a lens that itself forms the first optical element of the optical device. If desired, two or more of the elements forming the walls of the aperture, even all of them, may be provided as part of an optical device for introducing excitation light into or collecting light from the sensing body of the aperture. is within the scope of the present invention. FIG. 6 shows a front view of a portion of an actual device constructed in accordance with the principles of the present invention. FIG. 7 is a side view thereof, and FIGS. 8, 9 and 13 are various cross-sectional views thereof. In the drawing, the mounting plate 11
The two electrolyte containers 1 are connected via the clamp member 12.
3 and 14 are installed. Electrolyte container 13
communicates through conduit 16 with a suitable source of electrolyte. The electrolyte container 14 is coupled through its conduit 17 to a storage field for used electrolyte, which storage field may include a vacuum source or the like to create a negative pressure. Alternatively, pressure may be coupled to the inlet vessel to create the necessary pressure differential between the inlet and outlet vessels. An electrode 18 is placed in the container 13 (which is the electrolyte supply side of the device). Electrode 18 can be a metal foil electrode or the like and is electrically connected to lead 19. In a similar manner, the container 14 has an electrode 2 therein.
1, which is electrically connected to the lead 22. A housing 23 is attached to the bottom of the container 13. Housing 23 has a passageway defined therein, indicated generally by reference numeral 24, leading to the interior of container 13. Similarly, a housing 26 is attached to the bottom of the container 14 and has a passageway therein indicated generally by the reference numeral 27;
This passage leads into the interior of the container 14. Housings 23 and 26 each include an O-ring assembly 28 and 29. O-ring assemblies 28 and 29 are made of ceramic.
Blocks 31 and 32 are entered and attached. A solid polygon 33 made of glass is glued to surfaces 31a and 32a of ceramic blocks 31 and 32. A "solid polygon" as used herein refers to a multi-sided three-dimensional shape. In a similar manner, another solid polygon 34 made of glass is glued to the opposite surfaces of ceramic blocks 31 and 32 (see cross-section in FIG. 7). As will be explained in more detail below, the glass solid polygons 33 and 34 cooperate to form an aperture in the sensing zone, indicated generally by the reference numeral 36 in FIG. Furthermore, as will be explained in more detail below, the glass solid polygons 33 and 34 are constructed to provide the opening with an entrance/exit chamber having a predetermined shape relative to the dimensions of the opening. Ceramic block 32 is provided with an inlet passage 37 that connects passage 24 to an inlet chamber formed by solid glass polygons 33 and 34.
In a similar manner, the outlet passage 38 spans the ceramic block 31 and the glass polygons 33 and 34.
The outlet chamber formed by the outlet chamber is connected to the passageway 27. In the particular arrangement shown and described, the two flat surfaces of the glass solid polygons 33 and 34 form the two walls of a triangular aperture in the sensing zone 36. If desired, the same third wall is used to create the third wall of the triangular opening and the third wall of the doorway chamber.
may include a solid glass polygon. However, according to the particular embodiment shown in the drawings, a different structure of glass solid polygons in the form of glass cover plate 39 is provided, and glass solid polygons 3
3 and 34 and ceramic block 31
and 32 serve to form the third wall of the triangular opening with the sensing band 36 and to form the third wall of the entrance/exit chamber. In this particular example, the glass
A fitting 41 is shown adapted to couple a micro-objective, such as an oil-immersion objective, for optically observing the sensing zone 36 through the cover plate 39. As shown in the drawing, ceramic block 3
2 includes an additional sample inlet passageway 42 that mounts a sample injector 44 through a suitable O-ring assembly 43. The sample injector 44 spans the passageway 37 adjacent to the beginning of the inlet chamber formed by the glass solid polygons 33 and 34. Sample injector 44 is adapted to couple to a sample source, such as a particle or biological cell, in a manner well known to those skilled in the art of flow cytometers. As shown in the figures, the sample injector spans the inlet chamber formed by the passageway 37 and the glass solid polygons 33, 34 at an angle with respect to the electrolyte flow path. For example, suitable provision for longitudinal adjustment of sample injector 44 is made by an O-ring assembly, passageway 37 and glass solid polygon 33,
The syringes are adapted to be placed at various points in the flow of electrolyte flowing through the inlet chamber formed by 34. According to a particular aspect of one embodiment of the invention, a sonicator in the form of a piezoelectric crystal 46 is mounted to mounting plate 11 and is provided with suitable leads 47 for connection to a power source. A spring assembly 48 may also be included as part of the sonicator. The manner in which the glass solid polygons 33, 34 cooperate to form the polygonal opening and the entrance/exit chamber is as follows:
This can be best seen from FIGS. 10, 11 and 12. FIG. 10 shows glass solid polygons 33 and 34 which are truncated pyramids according to this particular embodiment. As shown in the figure, the glass solid polygon 33 has four sides 33a, 33b, 3
There are 3c and 33d. Similarly, the glass solid polygon 34 (which can be the same as 33) has four sides 34a, 34b, 34c and 34.
There is d. Each glass solid polygon 33 and 34
has truncated portions generally designated 33e and 34e. As shown in FIG. 11, the two glass solid polygons 33 and 34 have faces 33d and 34d.
are connected to each other by gluing the polygons, and both polygons are aligned. When this is achieved, the truncated portions 33e and 34e form two walls or sides of the triangular opening (as shown in this particular example). In the arrangement of FIG. 11, the glass cover plate 39 seen in FIG. 6 is attached to the bottom of the assembly shown in FIG.
form the walls or sides of Figures 10 and 11
As will be clear from the figures and FIG. 12, the truncated portions 33e and 34e form both the cross-sectional area of the sensing aperture and its depth, so that they fix the volume of the sensing aperture. As is clear from the figures, adjacent surfaces 33c and 34a form the volume and cross-sectional area of the inlet or outlet chamber, and corresponding adjacent surfaces 33a and 34c form the other inlet or outlet chamber. Returning to FIGS. 6-9, the illustrated apparatus generally functions as follows. The flow of electrolyte is
The sensing zone 36 (triangular aperture in this particular embodiment) is made from the electrolyte container 13 back to the electrolyte container 14 . This electrolyte flow is created by a device such as a vacuum source connected to conduit 17 of vessel 14. The AC/DC power supply is connected between leads 19 and 22, so that the current flow is between electrodes 18 and 2.
1 through various passageways and sensing zones or openings 36. A sample, such as particles or biological cells, is injected into the electrolyte stream by sample injector 44 .
As the samples are injected into the flow of electrolyte through the aperture or sensing zone 36, as they pass through the aperture they generate a pulse representative of particle volume measurement. The electrical resistance between electrodes 18 and 21 is influenced in a known manner. One aspect of the present invention allows simultaneous optical measurements of particles or biological cells as they traverse the aperture or sensing zone 36, as described in more detail below with respect to FIG. That is, the glass cover plate 39 itself constitutes one of the walls of the aperture and is part of a monolithic optical device onto which the particles or cells are focused while the aperture is present. In this way, electronic and optical measurements are performed on exactly the same particles at exactly the same time and no correlation problems arise. The angles and dimensions of the various surfaces of the glass solid polygons 33 and 34 affect both the fluid flow characteristics and the electric field characteristics through the sensing zone or aperture 36. As known in the art, to minimize turbulence that disturbs the position of particles in the sensing zone.
It is important to have a laminar flow of the electrolyte containing the sample through the sensing zone or aperture. Part 1 of this invention
The triangular laminar flow sheath provided by the triangular aperture and triangular entrance/exit zone created by the two embodiments provides exceptional positional stability to the injection stream of particles injected from the sample injector 44. It turned out to be provided. The angle of the entrance and exit chamber walls affects both the fluid flow and electric field characteristics of the device. The geometry of the inlet and outlet chambers can be calculated to create a continuous fluid boundary layer from the inlet through the sensing zone to the outlet chamber to minimize turbulence. Additionally, this geometry allows for the transition from areas of low current flux (i.e., the entrance chamber) to areas of high current flux (i.e., the sensing zone or aperture).
to and further back to the region of low current flux (i.e. the exit chamber), making a smooth transition. This smooth transition reduces the "edge effect" induced by abrupt transitions from large to small volumes (ie, prior art hole-in-the-wall methods). By limiting edge effects, the particle encounters a region of uniform current flux as it travels through the aperture or sensing zone. This leads to improved sensitivity of electronic measurements. With the wider aspect of the invention, the walls of the doorway chamber are made such that they make an angle of more than 5° with respect to the plane of the opening so as to reduce edge effects. Further, in accordance with the wider aspect of the invention, the walls of the doorway chamber are two "S" units apart from the sides of the sensing aperture, and the cross-sectional area of the doorway chamber is at least ten times larger than the cross-sectional area of the aperture. These shape considerations dictate constraints on the shape of the solid polygons used to form the apertures and entrance/exit chambers in accordance with that aspect of the invention. In accordance with embodiments of the invention in which solid polygons forming openings and entrance/exit chambers are used, it has been found that a truncated pyramid as described above is an advantageous shape for a solid polygon; It is not limited to that. Using truncated pyramids, triangular openings and entrance chambers can be made by assembling three identical truncated pyramids, or square openings and entrance chambers can be made by assembling four identical truncated pyramids. made with Figure 4 shows the ratio of the cross-sectional area of the inlet chamber to the cross-sectional area of the aperture for triangular and square orifices assembled by joining together a plurality of truncated pyramids, at a distance of ``S'' from the aperture, i.e., the entrance to the sensing zone. It is a plot that relates to something that has been created.
The angular reference of the curve in FIG. 4 indicates the angle made by the flat wall of the entrance chamber with respect to the axis of the opening. Using curves such as those shown in FIG. 4, the angle of the truncated pyramid used in the assembly of the transducer can be determined according to the principles of the invention to achieve the desired relationship between the cross-section of the entrance chamber and the cross-section of the aperture. It can be calculated by
As seen in the case of a triangular aperture with an entrance angle of 30°, at a distance of two "S" units from the measuring zone, the particles influence the current by about 3% of its maximum value. This is the difference between the inlet cross-sectional area and the measuring zone cross-sectional area.
It is guided by the ratio of 1/1. Therefore, a particle in the measurement zone will suffer a 3% matching error by the next particle two "S" units away from the measurement zone. In the particular embodiment of the invention shown in FIG. 6, the two truncated pyramids forming the two walls of the doorway chamber are oriented at 50° with respect to the plane of the opening;
This led to negligible edge effects in electronic measurements. Furthermore, the ratio of the cross-sectional areas of the entrance and exit chambers was about 40:1 for the two "S" units from the opening side. This was found to produce a sharp, well-defined electron particle volume pulse. As will be clear from the drawings, according to a particular embodiment of the invention, the flow of the electrolyte containing the sample from the inlet chamber through the aperture or sensing zone and out of the outlet chamber is an arcuate flow; is at the top or "knee" of the arch. This is important from a light sensing point of view. As is clear from the drawing, light sensing takes place through a glass plate 39 forming one of the walls of the sensing zone. Because the particle path is arcuate, particles in the sensing zone remain in focus as they traverse the optical path through the aperture or sensing zone. Particles that do not reach the sensing zone or leave the sensing zone are not in focus and therefore do not affect the optical measurement of particles that are in the sensing zone. According to one feature of one embodiment of the present invention, the sample injector 44 is not coaxially centered as in the prior art. Rather, the sample injector is placed at an angle to the triangular flow sheath of electrolyte. As the particle injector is moved from edge to edge of the triangular flow sheath, the position of the particles in the sheath, or in the sensing zone or aperture, is changed. In this way, precise positioning of the sample flow within the electrolyte is achieved so as to further minimize field edge effects experienced by the particles. Providing a triangular sensing zone or aperture has the advantage of providing exceptional stability to the triangular flow sheath, as well as other advantages. In electronic detection of particle properties, the volume of the sensitive band or aperture (ie, the product of cross-sectional area and length) is one of the factors determining measurement sensitivity. For small particles, the cross section needs to be small to maximize sensitivity. However, the smaller the cross-section, the greater the tendency for clogging. Therefore, it is advantageous to reduce the cross-section and make the dimension across the cross-section as large as possible to reduce clogging. For an equilateral triangle, the longest dimension is 1.35 times as large as a circle with the same cross-sectional area. In accordance with a particular embodiment of the invention, the triangular orifice or aperture is 100 μm on a side and has a length.
It was something like 100μm. The triangular aperture shape also provides important advantages with respect to optical measurements of particles in the aperture. The sensitivity of any optical measurement is related to how much of the total light emitted by the particle can be controlled, or how much of the incident light that is scattered or absorbed is recollected. Using a triangular aperture sensing zone, 120° per plane of light collection can be achieved in the case of an equilateral triangle with the axis of the triangular cross section. In the perpendicular axis, 180° is obtained, depending on the shape of the entrance hole. One way to create a practical optical boundary is by considering the state of the art of available micro-objectives. The numerical aperture (N.
A.) is 1.25−1.4. NA is given by the following formula: NA=n <sub>0</sub> Sinθ where n <sub>0</sub> is the refractive index of the object space and θ is half the acceptance angle of the light. Since these lenses operate maximally in object space with n <sub>0</sub> = 1.515, the range of θ is determined for these lenses, i.e. NA
=1.25=1.515Sinθ. Sinθ <sub>L</sub> = 1.25/1.515 = 0.825 Sinθ <sub>U</sub> = 1.4/1.515 = 0.92 NA = 1.25, θ <sub>L</sub> = 55° NA = 1.4, θ <sub>U</sub> = 67° Therefore, the actual optics of the best commercially available micro-optical device Shape limit is half of the light acceptance angle
It is between 55° and 67°. This exactly matches the range of acceptance angles provided by one side of the triangular opening. According to one particular embodiment of the invention, shown in FIG. 13, in which the optical arrangement is shown in diagrammatic form, an oil immersion objective 51 is provided next to the glass cover plate 39 and the sensing zone or aperture 36 is focused on. a suitable light source 52 that introduces excitation light into the sensing zone via a device such as a beam splitter 53;
is equipped. If a fluorescence measurement is to be performed on a sample located in the sensing zone 36, the light source 5
2 can be, for example, a mercury vapor lamp or a laser emitting in the ultraviolet spectrum. In any case, the excitation light is introduced into the sensing zone or aperture 36 through the oil immersion objective 51. If the samples flowing through the aperture or sensing zone 36 are properly prepared, ie, colored in the case of biological cells, they will emit a characteristic fluorescence. This light is collected by an oil immersion objective 51 and sent via a beam splitter 53 to a photodetector 54, such as a photomultiplier tube or the like. In the particular embodiment described, the aperture is triangular, so that
The light passes through the oil immersion objective 5 over an acceptance angle of 120°.
1, giving an NA of 1.3. As an alternative to providing the glass cover plate 39 as part of the optical arrangement, the element forming the aperture or that wall of the sensing zone can be the lens itself. solid polygons 33 and 34 and cover plate 3
In the explanation of No. 9, they were said to be made of "glass." No particular limitation is intended by the use of such terminology; the important point is that the elements forming the walls of the sensing zone may be of optical quality such that they may form part of an apparatus for making optical measurements with respect to the particles present in the sensing aperture. It is important to have the following. In a particular embodiment, solid polygon 3
3 and 34 are precisely lapped at the desired angle to form the desired truncated pyramid.
It is made from an optical flat body of BK7 glass. Whenever choosing materials for solid polygons 33 and 34, care must be taken to use materials for blocks 31 and 32 (FIG. 1) that have comparable coefficients of thermal expansion. In the description of FIG. 7, piezoelectric crystal 40 was mentioned. Providing such an ultrasonic treatment device has proven to be a very effective means of cleaning out any blockage that may occur in the sensing aperture 36. The momentary application of electrical power to the piezoelectric crystal causes sonic vibrations to flow through the electrolyte in the cytometer and has been found to be extremely effective in clearing blockages in the sensing aperture 36. 7th
As an alternative to providing a piezoelectric crystal secured to the cytometer mounting plate as shown in the figures, a piezoelectric crystal or other element producing ultrasonication can be coupled to the sample injector 44. This serves to create a localized sonication of the electrolyte near the sensing aperture 36. Suitable electronic circuits for processing, recording and displaying data from electronic and optical measurements are described in the literature and are well known to those skilled in the art of flow cytometry. FIG. 20 shows in block diagram form one example of an electronic arrangement suitable for this purpose. The electronic channel includes a pulse storage and shaping circuit 106.
, a subsequent amplification/filter circuit 107 , and a multi-parameter analyzer 109 for transmitting the electronic measurement signal.
an analog-to-digital converter 108 coupled to the analog-to-digital converter 108; The optical channel includes a suitable photodetector 111 coupled to a multiparameter analyzer 109 via an amplifier/filter circuit 112 and an analog/digital converter 113. If desired, real time display circuitry 114 may be included to display relatively unprocessed signals from the electronic and optical channels. Figure 20 shows only one optical channel entering the multi-parameter analyzer. Of course, if more than one photodetector is used in various embodiments of the present invention, a corresponding plurality of optical channels are provided. As is conventional, multi-parameter analyzer 109 inputs to a suitable computer 116 which can perform various data manipulations and correlations. computer 1
Connected to 16 are a data storage device 117 and a graphics device 118 for creating graphs and the like. Figure 14 shows two of the triangular apertures or sensing zones.
more than one side can be used to perform optical measurements;
Figure 3 represents one of many possible alternative embodiments of the present invention. In FIG. 14, a triangular aperture or sensing zone 56 is shown as being constituted by the oblique planes of three solid polygons 57, 58 and 59. solid polygons 57, 58 and 59
can be made of glass and have an oblique surface 57 forming the walls of the triangular aperture or sensing zone 56.
a, 58a and 59a. The solid polygon itself covers each side 57a, 58a of the aperture.
and 59a may be polished to act as a lens. Such additional lens elements may be provided in the optical arrangement on three sides of aperture 56 if desired, but are not shown in FIG. 14 for simplicity. Regarding the sides of the aperture formed by the surface 57a, the light source 61 and the beam
In order to excite the particles (indicated by dashed lines 60) by the splitter 62, a device may be provided for introducing excitation light into the aperture. A device such as a photodetector 63 is provided to detect light emitted from the particles 60 through the wall 57a of the aperture 56. In a similar manner, devices in the form of a light source 64 and a beam splitter 66 are provided for introducing excitation light into the aperture through the aperture wall 58a. Similarly,
A photodetector 67 is provided to detect light emitted from the particles 60 through the aperture wall 58a. In a similar fashion, a light source 68 and a beam splitter are provided which introduce excitation light into the aperture through the aperture wall 59a. A device such as a photodetector 71 is provided to detect light emitted by the particles through the wall 59a. The light sources 61, 64 and 68 can all be the same, ie UV sources, such as for fluorescence measurements of the particles 60, but they can also be different. If desired, only one light source is provided, in which case analyzers are provided on two or all three sides of the aperture 56. The important point is that by collecting light from all three sides of the aperture 56, the effective N.A. is three times the NA for one side.
A. In other words, 3.9 is obtained, and the sensitivity of optical measurements is greatly improved. Various different schemes are possible that use only one light source and one photodetector and simultaneously increase the effective NA. Thus, in FIG.
a, and direct the excitation light from the particle 60 to the surface 5.
Only one light source 64 and one photodetector 67 can be provided for collection at 8a. To increase the effective NA, surfaces 57a and 59a may be mirrored or other surfaces of polygons 57 and 59 may be mirrored so that all light emitted from particle 60 is detected by photodetector 67. The light can then be reflected back onto the surface 58a of the aperture 56. Alternatively or additionally, the reflective surface can serve to reflect a portion of the excitation light onto the particles 60. FIG. 15 shows another possible embodiment of the invention in which light scatterometry is combined with fluorometry. In FIG. 15, an oblique surface 72 forming the two walls of a triangular aperture or sensing zone 74 is shown.
solid polygons 72 and 7 with a and 73a
3 is provided. In the particular embodiment shown in FIG. 15, a glass cover plate 76 is shown constituting the third wall 76a of the opening 74, but it will be understood that another solid polygon may be used instead of the glass cover plate 76. can be used. For fluorescence measurements, the light source 77 and beam splitter 78 are
Light from light source 77 is combined via suitable optics (not shown) through wall 76a to focus on particles (indicated by dashed line 79) in aperture or sensing zone 74. If the particles are suitably prepared, such as by coloring, the fluorescent light emitted by the particles is focused onto a photodetector 81 via a suitable optical device (not shown). So far, the optical device operates as shown in and described in FIG. 13. However, in addition to this, provision can be made to simultaneously carry out light scattering measurements on the particles or samples 79. In this case, a suitable light such as a red laser beam is applied to the aperture 7.
The light source 82 and the beam coupled to the wall 76a of
A splitter 83 can be provided. Multiple detectors, such as those shown by photodetectors 84, 86, and 87, are provided with various angles θ <sub>1</sub> , θ around the elements forming transducers for detecting, for example, forward light scattering, 90° light scattering, and backscatter, respectively. <sub>2</sub> and θ <sub>3</sub> . Conveniently, in forward light scattering measurements,
The apex of the triangular aperture (reference number 75) serves as a field stop. By incorporating the arrangement shown in FIG. 15 into the apparatus of FIG.
It can be seen that the and light scattering measurements can all be made simultaneously on the same particle or cell sample in the sensing zone or aperture. The important advantages of the triangular orifice or sensing zone according to the principles of the present invention are applicable to combined electronic and optical cytometers as described above, as well as to purely electronic flow cytometers. 1st
Figures 6 through 19 relate to embodiments of the present invention suitable for use in existing electronic flow cytometers. In this embodiment, there is a source of electrolyte 89 within supply container housing 88 . An inlet housing 91 is provided and the supply container housing 8
8 and is suitably fluid-tightly sealed thereto by a device such as an O-ring 92. The inlet housing 91 has a passageway 9
A centrally located inlet vessel 93 is provided which communicates with the electrolyte 89 via 4. A sample injector 96 is placed in the center of the inlet vessel 93. The three truncated pyramids 97, 98 and 99 are attached to each other in such a way that the truncated portions of each form a triangular shaped opening or sensing zone 101. As with other embodiments of the invention, the angular walls of the truncated pyramids 97, 98 and 99 form suitably shaped entrance and exit zones with respect to the opening 101. The upper part of the inlet housing 91 is provided with angular surfaces 91a, 91b, etc., which form truncated pyramids 97, 98 and 9.
9, these truncated pyramids are glued to surfaces 91a, 91b, etc.
An outlet housing 102 is provided with a centrally located outlet receptacle 103. Outlet housing 1
The bottom part of 02 has surfaces 102a, 102b, etc., which match the mating surfaces of the truncated pyramids 97, 98 and 99, and the mating surfaces are glued. As seen in the drawing, the sample injector 9
6 is placed so that the sample is injected into the center of the inlet chamber formed by the walls of the truncated pyramids 97, 98 and 99, and across the center of the opening or sensing zone 101. In operation, a suitable device, such as a vacuum source, is coupled to the outlet housing 102 in a manner well known to those skilled in the art of flow cell measurement, and the supply vessel 88 is moved from the supply vessel 88 to the inlet vessel 93 through the opening or sensing zone 101 to the outlet vessel. A flow of electrolytic solution 89 coming out of 103 is created. Appropriate equipment, such as electrodes and a power source, are provided to create an electric current through the electrolyte. The sample injected from the sample injector 96 enters the electrolyte flow sheath and passes into the opening or central portion of the sensing zone 101 . This alters the current through aperture 101 and produces a pulse that is detected by appropriate electronic circuitry well known to those skilled in the art of flow cytometers. The advantages of triangular apertures are most applicable to electronic measurements of the apparatus shown in FIGS. 16-19. The triangular flow sheath of the electrolyte is formed through the opening 101.
It exhibits exceptional stability in its laminar flow properties through. The sample flow therefore passes through the opening 101 in a very thin layer without any turbulence. Angling the truncated pyramid forming orifice 101 to be compatible with the principles of the present invention results in a transition from a region of low current flux in the inlet chamber to a region of high current flux in the opening and then a region of low current flux in the exit chamber. A smooth transition occurs back to . This smooth transition reduces edge effects. Further, as previously discussed, by creating an entrance/exit chamber with a predetermined cross-sectional area relative to the cross-sectional area of the aperture according to the principles of the present invention, the "effective" electron length of the sensing band is reduced, resulting in a sharp, well-defined pulse. This creates an extremely sharp pulse of electrons caused by the particle or sample passing through the aperture or sensing zone. Turning now to Figures 21 through 33, there are examples of many different tissues prepared for DNA or electronic cell volume analysis using one embodiment of the flow cytometry transducer of the present invention. It is shown. In particular, this data was obtained using the flow cytometer shown in FIG. FIG. 21 is a combined electron particle volume and fluorescence (530 nm) histogram for a Duke Scientific 10 micron diameter fluorescent sphere. Figures 22 through 26 show DNA histograms (i.e., fluorescence measurements) for small mice, common mice, and humans. Thus,
Figure 22 is a flow cell measurement DNA histogram of cell nuclei from the liver of a Wistar mouse.
Figures 3 and 24 are similar DNA histograms of cell nuclei from normal CF1 miniature mouse kidney and liver, Figure 25 is a flow cytometric DNA histogram of cell nuclei from normal human breast tissue, and Figure 25 is a flow cytometric DNA histogram of cell nuclei from normal human breast tissue. Figure 26 is a flow cytometric DNA histogram of cell nuclei from a patient with breast cancer. All tissue samples used for the data in Figures 22 to 26 were made using DAPI, or DNA
It was created using a cell nucleus isolation medium containing a specific dye. The cell nucleus isolation medium used was Jerry T. Thornthwaite.
Thornthwaite) in April 1981, entitled ``Cell Nucleus Isolation Media and Methods for Isolating Cell Nuclei.''
This is disclosed in a patent application filed on May 24th.
As can be seen from Figures 22-26, the coefficient of variation is in the range of 1-2% for the DNA histograms, demonstrating the effectiveness of the optical aspects of the transducer of the present invention. FIG. 27 is an electronic cell volume histogram of Wistar mouse liver cells enzymatically degraded with collagenase and trypsin and measured by the transducer of the present invention. Examples of combinatorial electronic cell nuclear volume (differentiated from cell volume) and DNA fluorescence are shown in Figure 28.
This is shown in FIGS. 29 and 31. Again, these samples are from the 22nd to the 2nd
It was prepared using the same cell nucleus separation medium as the sample in Figure 6. In Figures 30a and 30b, 256 channel single parameter data of CF1 miniature mouse liver cell nuclei by electronic nuclear volume and DNA fluorescence is shown. These data demonstrate simultaneous analysis of diverse samples using fluorescence and electronic impedance measurements using the transducer of the present invention. FIGS. 31 and 32 further illustrate the electronic cell nuclear volume and DNA
Showing the simultaneity of fluorescence measurements. Figures 31 and 32
The figure reproduces dual-channel oscilloscope tracings of electronic and optical measurements on the liver of a small mouse. Again, small mouse tissue samples were made using the same nuclear isolation media and dyes as in the previous example, and the optical measurements were fluorometry. As can be seen from these figures, the transducer of the present invention indeed achieves electronic volumetric and optical measurements simultaneously. Although various aspects of the invention have been described and illustrated with respect to embodiments, it is not meant to limit the invention to these specific embodiments. Various modifications may be made within the scope of those skilled in the art without departing from the true spirit and scope of the invention.

[Brief explanation of the drawing]

FIG. 1 is a cross-sectional view of a flow cytometer aperture according to the prior art showing edge effects on current flux density; FIG. 2 is a schematic diagram of a flow cytometer aperture with an inlet chamber with angled walls; Figure 3 is similar to Figure 2, showing the various angular relationships for the aperture of the flow cytometer, and Figure 4 shows how the current flux of the edge effect changes as the angle of the entrance chamber wall changes. Figure 5 is a graph showing the ratio of the entrance chamber cross section to the opening cross section for various shapes of the entrance chamber and for various distances to the entrance chamber.
The figure is a front view of one embodiment of the flow cell measuring device according to the present invention, FIG. 7 is a side view of the cell measuring device shown in FIG. 6, and FIG. 8 is a cross-sectional view of a part of the cell measuring device shown in FIG. 6. , FIG. 9 is a cross-sectional view taken along line 9--9 of FIG. 8, FIG. 10 is a plan view of the truncated pyramid components of the cytometer of FIG. 6, and FIG. 12 is a bottom view of the assembly shown in FIG. 11, and FIG. 13 is a partial cross-sectional view of a part of the cytometer shown in FIG. Diagram of the optical sensing array of
FIG. 4 is a diagram representing an optical sensing array of a flow cytometer according to one embodiment of the present invention, FIG. 15 is a diagram similar to FIG. 14 showing another embodiment of the optical sensing array, and FIG. 17 is a cross-sectional view of a portion of the transducer of FIG. 16; FIG. 18 is along the axis of the inlet chamber of the transducer of FIG. 16; FIG. Figures 19 and 20 show the triangular inlet chamber and opening of the transducer of Figure 16.
Figure 21 is a block diagram of a suitable electronic circuit used with the cell measuring device of the present invention, Figure 21 is a combined electronic particle-fluorescence histogram of Duke Scientific's 10 micron diameter fluorescent sphere, and Figure 22 is Wistar. Flow cytometry of cell nuclei from the liver of a mouse DNA histogram. Figure 23 shows flow cytometry of cell nuclei from the kidney of a normal CF1 small mouse.
DNA histogram, Figure 24 shows the flow of cell nuclei from the liver of a normal CF1 small mouse.
Histogram, Figure 25 is a nuclear flow cell measurement DNA histogram from normal human breast tissue.
Figure 26 is a flow cytometric DNA histogram of cell nuclei from a patient with thoracic cancer, Figure 27 is an electronic cell volume plot of enzymatically digested Wistar mouse liver, and Figure 28 is a cell nucleus from a patient with thoracic cancer. Figure 29 is a combined plot of electronic nuclear volume and DNA fluorescence for cell nuclei from the liver of a Wista mouse; Figures 30a and 30b are
Figure 31 shows the electronic cell nucleus volume and fluorescence brightness histogram of liver cell nuclei of CF1 small mice.
Combined plots of electronic nuclear volume and DNA fluorescence of CF1 small mouse liver cell nuclei, Figures 32 and 33 show actual oscilloscope plots for simultaneous electronic nuclear volume and fluorescence pulse measurements of small mouse liver cell nuclei. This is a reproduction diagram of racing. Explanation of symbols, 36... Sensing zone, 33, 34...
Solid polygon, 44... Sample injector, 46...
Ultrasonicator, 13, 14... Electrolyte container, 1
8, 21...electrode, 39...glass cover plate.

Claims (1)

[Claims for Utility Model Registration] (1) A flow cytometer that simultaneously performs electronic and optical measurements of individual particles or cells: a sensing zone 36 having an opening with an axis; adjacent planes of the plurality of polygons 33, 34 form a polygonal opening in the sensing zone 36, and other surfaces of the polygons 33, 34 form an entrance/exit chamber; consists of a plurality of fixedly connected polygons 33, 34, and at least one polygon 33, 34 forms an optical element of an optical detection system, which optical detection system Devices 41, 51, 53, 72, 7 for introducing excitation light into the aperture through the optical element
3, 76 and a device 41, 51, 54, 63, 6 for collecting light from the sample particles in the aperture.
7, 71, 109; an inlet passageway arrangement 37 for producing a flow of electrolyte for the provision of a laminar flow of said particles and an outlet passageway arrangement 38 for conducting said laminar flow of said particles from said sensing zone 36; a particle injector 44 that injects the particles into the electrolyte before reaching the sensing zone 36; one or more electrodes coupled to the inlet passageway; a device coupled to the outlet device for stabilizing the flow of electrical current between the electrodes and through the aperture; a monitoring device for monitoring the flow of electronic current through the aperture; The passage devices 37, 38 are
tubular arcuate fluid passageways, each located at a predetermined angle to said central axis of the aperture, said aperture being located at the apex of the arch at which the central axis of the aperture is aligned with the optical axis of the detection system. When the particle crosses the aperture in a direction parallel to the central axis of the aperture, the particle will be in focus only if it is at the apex of the arch, and the particle will be in focus if it approaches the aperture or moves away from the aperture. A flow cytometer characterized in that the optical detection system is located on the outside near the arch apex to avoid overlapping. (2) In the flow cell measuring device according to claim 1, the particle injection device 44 is located at a predetermined angle with respect to the flow of the electrolyte, and the electrode A flow cytometer configured to include an adjustment device for adjusting said particle injection device 44 to selectively inject particles into different regions of the flow of a space charge layer. (3) The flow cell measuring device according to any one of claims 1 and 2, further comprising a sonic vibration generator 40 that generates sonic vibrations through the electrolyte near the sensing zone 36 in order to eliminate clogging. Including, flow cytometer. (4) The flow cell measuring device according to any one of claims 1 to 3, wherein the opening has a triangular cross section perpendicular to the central axis of the opening. (5) In the flow cell measuring device according to any one of claims 1 to 4, the inlet chamber is formed between an end of the opening and an end of the inlet passage device 37, and the outlet chamber is A flow cytometer formed between the other end of the opening and the end of the outlet passageway device 38, such that the inlet and outlet chambers are part of an arcuate liquid passageway. (6) In the habituation cell measuring device according to claim 5, at least one of the inlet chamber and the outlet chamber forms an angle of at least 5° with respect to a surface end of the opening perpendicular to the central axis of the opening. at least one of the inlet and outlet chambers has a wall disposed with a distance from the aperture equal to twice the width of the aperture along a plane passing through the central axis of the aperture; With a cross-sectional area 10 times larger,
Flow cytometer. (7) The flow cell measuring device according to claim 1, wherein the opening is formed of a polyhedral surface. (8) The flow cell measuring device according to claim 4, wherein the wall of the triangular opening is formed by the truncated surfaces of two truncated pyramids and an optically flat plate. . (9) In the flow cytometer according to any one of claims 1, 4, 7, and 8, the device for collecting light collects light scattered by particles. at least one positioned at a predetermined angle with respect to the excitation light for detection.
A flow cytometer comprising three optical detectors 63, 86, 87. (10) In the flow cytometer according to any one of claims 1, 4, 7, and 8, the device for collecting light detects fluorescence emitted by particles. Flow cytometer comprising at least one optical detector 54,63 for. (11) In the flow cytometer according to any one of claims 4 and 7, at least one wall or surface of the opening is a mirror surface, so that the sample can be passed through the other wall or surface of the opening. A flow cytometer that reflects light emitted by particles.