Source: https://patents.google.com/patent/US7923252B2/en
Timestamp: 2018-12-10 11:42:01
Document Index: 516682766

Matched Legal Cases: ['Application No. 398582', 'Application No. 398582', 'Application No. 95', 'Application No. 95', 'Application No. 95', 'Application No. 95']

US7923252B2 - Droplet formation systems for flow cytometers - Google Patents
Droplet formation systems for flow cytometers Download PDF
US7923252B2
US7923252B2 US11069529 US6952905A US7923252B2 US 7923252 B2 US7923252 B2 US 7923252B2 US 11069529 US11069529 US 11069529 US 6952905 A US6952905 A US 6952905A US 7923252 B2 US7923252 B2 US 7923252B2
US11069529
US20050153458A1 (en )
A droplet forming flow cytometer system (1) allows high speed processing without the need for high oscillator drive powers through the inclusion of an oscillator or piezoelectric crystal (10) within the nozzle volume (3) and directly coupled to the sheath fluid. The nozzle container (27) continuously converges so as to amplify unidirectional oscillations (11) which are transmitted as pressure waves through the nozzle volume (3) to the nozzle exit so as to form droplets from the fluid jet. A variation in substance concentration is achieved through a movable substance introduction port (9) which is positioned within a convergence zone (32) to vary the relative concentration of substance to sheath fluid while still maintaining optimal laminar flow conditions. This variation may be automatically controlled through a sensor and controller configuration.
This application is a continuation of U.S. application Ser. No. 09/689,585 filed Oct. 12, 2000, now U.S. Pat. No. 6,861,265 to be issued on Mar. 1, 2005, which is a divisional of U.S. application Ser. No. 08/627,963, filed Apr. 16, 1996, now U.S. Pat. No. 6,133,044 issued on Oct. 17, 2000, which is a continuation of U.S. application Ser. No. 08/323,270 filed Oct. 14, 1994, now abandoned; each of these are hereby incorporated by reference herein.
To some degree the challenges for droplet formation may be the result of the fact that although drop formation has been modeled with significant theoretical detail, in practice it still remains a somewhat empirical subject. While on one level exhaustive mathematical predictions are possible, in practice these predictions can be greatly tempered—and are often revised—by the fact that materials limitations, inherent substance variations, and the like contribute heavily to the end result. A number of “advances” in this field have even proved to be either unnecessary or unworkable in practice.
An even more paradoxical situation exists with respect to the problem of maintaining laminar flow within the nozzle system of a droplet flow cytometer. Although those having ordinary skill in this field have known for years that maintaining laminar flow was desirable, until the present invention, practical systems utilizing replacement tips have not been optimally designed so as to achieve the goal of truly laminar flow. For instance, U.S. Pat. No. 4,361,400 as well as the 1992 publication by Springer Laboratory entitled “Flow Cytometry And Cell Sorting”, each show replaceable nozzle tip designs in which laminar flow is disrupted at the junction between the nozzle body and the nozzle tip. Again, such designs seem to present almost a paradox in that they obviously are not optimum from perspective of a goal which has long been known as those having ordinary skill in the art. The present invention not only recognizes this goal but also demonstrates that a solution has been readily available.
Accordingly, one of the objects of the invention is to provide for a low power system which allows high processing rates. In keeping with this object, one goal is to achieve direct coupling of the oscillations to the sheath fluid and thus minimize any losses associated with material interfaces. In keeping with this object, another goal is to provide for a system which actually amplifies the oscillations so as to produce acceptable fluid variations at the nozzle tip.
In order to form regular droplets, the preferred embodiment utilizes a piezoelectric crystal (10) to cause oscillations (11) within the sheath fluid. These oscillations are transmitted as pressure variations through to nozzle exit (6) and act to allow jet (12) to form regular droplets (13) through the action of surface tension. These processes are well understood and are further explained in a number of references including the 1992 reference entitled “Flow Cytometry and Cell Sorting” by A. Radbruch (© Springer-Verlag Berlin Heidelberg) and the 1985 reference entitled “Flow Cytometry: Instrumentation and Data Analysis” edited by Marvin A. Van Dilla, et al. (© Academic Press Inc. (London) Ltd.) each of which are incorporated by reference.
As shown in FIG. 1, one of the features of the preferred embodiment is the location of piezoelectric crystal (10) within nozzle volume 3. By this feature the oscillator acts to initiate oscillations (11) within the nozzle volume. The oscillator thus may be directly coupled to the sheath fluid. These oscillations are transmitted through the sheath fluid as it flows out nozzle exit (6) and forms droplets (13) below nozzle (6) in freefall area (7). Naturally, although shown to be directly below it is possible that the nozzle assembly could be oriented on its side or in some other relationships and so droplets (13) might form at some other location and yet still be characterized as “below” nozzle tip (6) since they will form in the direction that jet (12) is emitted from nozzle exit (6).
As may be easily understood from FIG. 1, this type of flow cytometer, a droplet flow cytometer, operates quite differently from a channel forming flow cytometer. In channel-type flow cytometers, oscillators and the theories involved are not relevant as no freefall or droplet formation is required. Further, while the nozzle exit orifice is approximately 50 to 150 microns in diameter in droplet forming flow cytometers, in channel-type flow cytometers, the orifice can be much larger —on the order of 1000 microns. This causes extremely different conditions and has resulted in the two fields being treated somewhat differently by those involved.
As shown in FIG. 1, piezoelectric crystal (10) is configured as a ring-shaped crystal which occupies most of the top end of nozzle container (2). This ring is mounted directly to nozzle container (2) in a manner so as to be situated within nozzle volume (3). It need not vibrate the nozzle container and, indeed is designed to avoid it. Its oscillations (11) may also be made to occur generally in a direction parallel to the central axis of nozzle container (2) as shown. Further, these oscillations (11) are essentially coupled to the sheath fluid, not to the nozzle container. Thus, rather than taking the directions suggested by some of the prior art involving moving the actual nozzle container, the present invention acts directly upon the sheath fluid to cause pressure variations within the sheath fluid. These pressure variations move down nozzle volume (3) and may actually be amplified by the shape of nozzle container (2) so as to cause surface tensions variations in jet (12) as it emerges from nozzle exit (6). These variations act to pinch off jet (12) and thus form droplets (13). Since the sheath fluid is not substantially compressible, these pressure variations may pass relatively unattenuated and in fact may be amplified through nozzle volume (3) to achieve the desired droplet formation effect. While others may have considered the desire to coupling directly to the sheath fluid, they failed to recognize ways to do this and did not recognize that they could have positioned the oscillator within the sheath fluid for most efficient coupling.
In addition to the aspect of maintaining laminar flow, the continuously converging nozzle container can provide amplification of the oscillations (11). Similar to horn and other designs, the continuous convergence combines with the principals of conservation of energy so that the amplitude of the oscillations actually increases as it passes from piezoelectric crystal (10) to nozzle exit (6). This amplification may be maximized not only by positioning the oscillator at or near the largest cross-sectional area but also by making oscillator surface (18) to have an area substantially as large as the largest cross-sectional area. In this regard by “substantially” it is meant that the oscillator should be as large as practically possible after consideration of the typical desire to introduce substance through the center axis of nozzle volume (3) as well as this invention's unique desire to maintain oscillator side (21) spaced apart from nozzle container (2). The amplification may also be enhanced by providing for continuous convergence from sheath fluid port (4) through to nozzle exit (6). As mentioned earlier, each of the foregoing aspects also contribute to the present invention's extraordinary reduction in input power requirements.
Unlike the designs shown in the prior art such as those shown in FIG. 5, nozzle tip (25) need not be sealed to nozzle body (24) on its inner surface. Instead, the nozzle body inner surface (26) joins smoothly with the nozzle tip inner surface (27) at tip joint (28). This smooth transition is to the degree necessary to maintain laminar flow in the particular application. It can be achieved through the inclusion of edge insert (29) within nozzle body (24) so as to allow nozzle tip (25) to be inserted into nozzle body (24). In this fashion seal (30) can be positioned so as to contact the outer surface (31) of nozzle tip (25) and thus avoid any adverse impacts on laminar flow within nozzle volume (3). By locating seal (30) off of inner surface (27) of nozzle tip (25), the seal can be kept away from areas which are important to laminar flow. As may be understood, a great variety of designs may be accomplished to achieve this goal. Importantly, it should be understood that inner surface (27) of nozzle tip (25) is defined merely with respect to its function, namely, the surface which contacts and directs the flow of sheath fluid of nozzle volume (3). Further, the definition of “smooth” is also relatively defined as those transitions which do not significantly interrupt laminar flow and thus do not degrade the performance of the flow cytometer. It should also be understood that the seal between any two components such as the seal between nozzle body (24) and nozzle tip (25) may be direct or indirect through the use of intervening materials or components.
Yet another independent aspect of the invention is the aspect of being able to adjust the location at which the substance is introduced. As mentioned earlier, those skilled in the art have long recognized the need to achieve variations in the entire process to accommodate variations in conditions practically experienced. As shown in FIG. 3, the present invention affords the ability to vary the rate at which substance is introduced without disrupting laminar flow and the like. This is achieved through positioning substance introduction port (9) within convergence zone (32) as may be easily understood and by varying the location of substance introduction port (9) within is convergence zone (32). As shown, substance introduction port (9) may move along the primary flow direction to maintain an optimal relationship to the flow of the sheath fluid. Through this technique, the relative concentrations of the substance introduced and the sheath fluid can be varied. This can act to avoid the resolution drop and the like which the prior art appeared to consider unavoidable as they adapted to changing conditions.
Further, since it may be desirable to maintain equal velocities at substance introduction port (9), and since substance tube (33) may be moved, it is possible to include a controller (34) which receives signals from some type of sensor (14) and which may act to control a movement mechanism (35) and thus automatically adjust the location of substance introduction port (9) within nozzle container (2). Further, controller (34) may act to additionally control the pressure of substance reservoir (8) and sheath reservoir (5) for automatic correlation of the various factors based upon location or other parameters sensed. Since the theoretical relationship between these factors is well known for optimal conditions and since the programming or wiring of such a design could be easily achieved by those skilled in the art, a variety of designs may be implemented to achieve this goal. Given the great variety of flow cytometer systems possible, it should be understood that a great variety of sensed values may be used ranging from concentration of the substance contained within substance reservoir (8), to the actual location of substance introduction port (9), to the pressure of the various sheath fluid or substance fluids, to some other property of the substance sensed by sensor (14). Each of these—or any combination of them and other factors—may be adjusted automatically to achieve desired relationships or to simply optimize results without regard to the actual predicted values. Naturally, in keeping with this broad concept it should be understood that sensor (14) may not be just one sensor but may in fact be a host of different sensors positioned at various locations depending upon the particular condition existing within the flow cytometer desired to be sensed. While, of course, the sensor (14) will only ascertain specific values, these values can indicate results which may be used to more appropriately adjust the location of the substance introduction port.
1. A method of creating a droplet from a jet of a flow cytometer comprising:
introducing a flow of sheath fluid into a nozzle, said sheath fluid having a primary flow direction through the nozzle;
converging said sheath fluid in a convergence zone in the nozzle;
introducing a flow of a substance at a location within said sheath fluid in said convergence zone;
adjusting the location along the primary flow direction at which said substance is introduced within said convergence zone;
allowing said sheath fluid to exit from said nozzle; and
forming at least one droplet from said sheath fluid after allowing said sheath fluid to exit from said nozzle.
2. A method of creating a droplet from a jet of a flow cytometer according to claim 1 wherein said adjusting the location at which said substance is introduced within said convergence zone comprises establishing a desired concentration of said substance relative to said sheath fluid.
3. A method of creating a droplet from a jet of a flow cytometer according to claim 1 or 2 wherein said adjusting the location at which said substance is introduced within said convergence zone comprises establishing laminar flow of said substance within said sheath fluid.
4. A method of creating a droplet from a jet of a flow cytometer according to claim 1 wherein said adjusting the location at which said substance is introduced within said convergence zone is automatic.
5. A method of creating a droplet from a jet of a flow cytometer according to claim 1 wherein said adjusting the location at which said substance is introduced within said convergence zone comprises:
sensing values representative of conditions within said flow cytometer; and
automatically moving said location based upon said sensed values.
6. A method of creating a droplet from a jet of a flow cytometer according to claim 5 wherein said conditions are at least one of the following: the pressure of said sheath fluid; the pressure of said substance; the location at which said droplet is formed; the rate at which droplet is determined to contain some of said substance; or a property of said substance.
7. A method of creating a droplet from a jet of a flow cytometer according to claim 1 wherein said substance is introduced through a substance tube and wherein said adjusting the location at which said substance is introduced within said convergence zone comprises replacing said substance tube.
US11069529 1994-10-14 2005-02-28 Droplet formation systems for flow cytometers Active 2019-08-28 US7923252B2 (en)
US08627963 US6133044A (en) 1994-10-14 1996-04-16 High speed flow cytometer droplet formation system and method
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US11069529 US7923252B2 (en) 1994-10-14 2005-02-28 Droplet formation systems for flow cytometers
US09689585 Continuation US6861265B1 (en) 1994-10-14 2000-10-12 Flow cytometer droplet formation system
US20050153458A1 true US20050153458A1 (en) 2005-07-14
US7923252B2 true US7923252B2 (en) 2011-04-12
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US09689585 Expired - Fee Related US6861265B1 (en) 1994-10-14 2000-10-12 Flow cytometer droplet formation system
US11069529 Active 2019-08-28 US7923252B2 (en) 1994-10-14 2005-02-28 Droplet formation systems for flow cytometers
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