Claims:
1. A flow cytometer comprising:
a sheath flow type flow-cell comprising a capillary assembly and a plurality of connectors to facilitate the capillary assembly to be joined to other fluidic pathway components;
a light source such as a laser or the like whose light is focused on the flow inside the said flow-cell;
a forward scattered light detecting system for detecting light scattered from particles/cells in the same direction as the incident light;
a second light detecting system for detecting light scattered at a right angle with respect to the direction of the said incident light;
a fluorescent light detecting system for detecting fluorescent light emitted at right angle with respect to the direction of the said incident light.

2. The flow cytometer according to claim 1, wherein the incident light passes through a first set of lenses to focus the laser light onto the flow-cell.

3. The flow cytometer according to claim 1, wherein forward light scattering system comprises a second set of lens for collecting light emerging from the flow-cell, and at least one light sensor in the form of a photodetector or charge-coupled device (CCD) for detecting scattered light coming from the second set of lens.

4. The flow cytometer according to claim 1, wherein the second light scattering system comprises a third set of lens for collecting light emerging from the flow-cell, and at least one light sensor in the form of a photodetector, charge-coupled device (CCD) or photomultiplier (PMT) for detecting scattered light coming from the third set of lens.

5. The flow cytometer according to claim 1, wherein the second light scattering system comprises a third set of lens for collecting light emerging from the flow-cell, one or more dichroic mirrors or filters and at least one light sensor in the form of a photodetector, charge-coupled device (CCD) or photomultiplier (PMT) for detecting fluorescent light coming from the third set of lens.

6. The flow cytometer according to claim 2 or 3, where the first and second set of lenses are on opposites of the flow-cell.

7. The flow cytometer according to claim 2 or 3, where the first set of lenses are on opposites of the flow-cell focus the laser beam in an elliptical shape with the short axis of the ellipse along the flow direction and the long axis of the ellipse perpendicular to the flow direction.

8. The flow cytometer according to claim 7, where the forward scattered light from particles/cells passing through the second set of lenses is collected by the sensor over angles from 0.5 degrees to 10 degrees from the axis.

9. A sheath flow type flow-cell device for flow-cytometer, comprising a capillary assembly and a plurality of connectors to facilitate the capillary assembly to be joined to other fluidic pathway components;
wherein the capillary assembly comprises a first capillary of circular cross section and a second capillary of square cross section; the capillary assembly being so designed that the outer diameter of the first circular capillary matches the inner width of the square capillary; and
characterized in that the plurality of connectors comprises a cross adapter having four-way-channel wherein a sample liquid having particle to be analyzed enters the first capillary through the top channel of the cross adapter and a sheath liquid enters the cross adapter through one of the side channels, the first capillary extending longitudinally from first channel of the cross adapter to the bottom channel just to enter the second capillary to be positioned tightly and concentrically over the second capillary thus providing only the corners of the second capillary for sheath liquid to flow downstream and communicate with the first circular capillary at its end enabling hydrodynamic focusing of the sample fluid;
and a tee-adapter having three-way-channel where the bottom end of the second capillary is connected via a top channel thereby assembling the connectors and capillaries co-axially resulting the flow speed of the core stream of particles obtained increasing up to 0.5 m/s and the transit time of particles being about 0.1 msec.

10. A sheath flow type flow-cell device for flow-cytometer, comprising a capillary assembly and a plurality of connectors to facilitate the capillary assembly to be joined to other fluidic pathway components;
wherein the capillary assembly comprises a first capillary of circular cross section and a second capillary of square cross section; the circular capillary being tapered circular cross section at the end; the capillary assembly being so designed that the outer diameter of the first circular capillary matches the inner width of the square capillary; and
characterized in that the plurality of connectors comprises a cross adapter having four-way-channel wherein a sample liquid having particle to be analyzed enters the first capillary through the top channel of the cross adapter and a sheath liquid enters the cross adapter through one of the side channels, the first capillary extending longitudinally from first channel of the cross adapter to the bottom channel just to enter the second capillary to be positioned tightly and concentrically over the second capillary thus providing only the corners of the second capillary for sheath liquid to flow downstream and communicate with the first circular capillary at its end enabling hydrodynamic focusing of the sample fluid;
and a tee-adapter having three-way-channel where the bottom end of the second capillary is connected via a top channel thereby assembling the connectors and capillaries co-axially.

11. The device as claimed in claim 9 or 10, wherein the first capillary is mounted inside the cross adapter from the top channel via a plastic tubing, preferably Teflon tubing, fitted tightly inside the cross-adapter fixture by a nut and the corresponding ferrule.

12. The device as claimed in claim 9 or 10, wherein the tee and cross-adapter are constructed in plastic or metal, preferably, micro-tight ethylene Tetraflurorethylene (ETFE) or PEEK or stainless steel.

13. The device as claimed in claim 12, wherein the ETFE tee-adapters and cross-adapters comprise plastic or metal flangeless nuts and ferrules, preferably Perfluoroalkoxialkane (PFA) flangeless nuts and ETFE ferrules to hold the tubing tightly inside the nut.

14. The device as claimed in claim 9 or 10, comprises silicone tubing to provide cushioning effect to the capillaries.

15. The device as claimed in claim 9 or 10, wherein the capillaries are constructed of glass and/ or quartz.

16. The device as claimed in claim 11, wherein the diameter of the Teflon tubing for the sheath is kept larger than that used for the sample flow injection.

17. The device as claimed in claim 9 or 10, wherein one of the side channels of the cross adapters introduces a burp line which is a tubing to discard bubbles created inside.

18. The device as claimed in claim 9 or 10, wherein the square capillary is jacketed with a silicone sleeve inside the plastic or metal nut and ferrule, preferably ETFE nut and ferrule, to avoid any breakage upon vibrations.

19. The device as claimed in claim 9 or 10, wherein the flow-cell can be mounted upside down to avoid bubbles from hindering the flow of the sample.

20. The device as claimed in claim 9 or 10, does not require any additional internal part for the centering of the round capillary inside the square capillary.
, Description:
TECHNICAL FIELD OF THE INVENTION
Embodiments of the invention described herein pertain to design and characterization of a sheath flow-cell applicable to a flow cytometer and flow cytometer including the sheath flow-cell.

BACKGROUND OF THE INVENTION
Flow cytometry is a powerful tool for measuring microscopic or sub-microscopic particles or biological cells [Harald B Steen. Flow cytometer for measurement of the light scattering of viral and other submicroscopic particles. Cytometry Part A: the journal of the International Society for Analytical Cytology, 57(2):94–99, 2004], [Howard M Shapiro. Practical flow cytometry. John Wiley & Sons, 2005]. It facilitates the identification of the different types of cells and particles within a heterogeneous population. This technique is based on the light-scattering and fluorescence emitting properties of cells and particles traversing through the region of illumination and the analysis of the scattering light can distinguish particles/cells based on their size, shape, granularity and fluorescence. This principle is also used in 5-part haematology analyzers for obtaining a count of the five different white blood cells in blood. A suspension of cells/particles is made to flow through a narrow channel so as to maintain laminarity with the aid of additional fluid inside a cuvette-based chamber [PJ Crosland-Taylor. A device for counting small particles suspended in a fluid through a tube. Nature, 171(4340):37–38, 1953]. A typical flow cytometer or a 5-part haematology analyzer is comprised broadly of three interdependent functional units, namely, the optical source with one or more lasers and a sensing system including the flow chamber and optical assembly, the second being a hydraulic system that delivers sheath fluid and sample, and controls the passage of cells through the interrogation or sensing region, and finally the data collection and analysis system that interprets the signals from the sensing region. In order to ensure smooth and ordered passage of particles through the flow cell, most instruments use a sheathing technique to confine cells to the center of the sample flow stream; this centered flow reduces clumping of the cells and allows the scattered light from each individual particle to be recorded separately. The pressurized sample fluid containing cells enter the flow cell through a small aperture ensheathed by another fluid [Didier Lefevre, Henri Champseix, and Serge Champseix. Apparatus for counting and determining at least one leucocytic sub-population, August 11 1992. US Patent 5,138,181]. The sheath fluid offers a hydrodynamic focusing effect and directs the cells in a single line with appropriate spacing between cells. Precise and accurate positioning of the sample fluid containing cells within the sheath fluid in a flow cytometer is the prime criterion ensuring efficient operation of the instrument. The flow rates of the sample and sheath fluids within the flow cell are so adjusted that the cells flow one behind another through the interrogation region. Various cell sorters have been reported that work on this principle of hydrodynamic focusing and have a cuvette-based chamber for cell separation and sorting [Paul Robinson. Mack Fulwyler in his own words. Cytometry Part A: The Journal of the International Society for Analytical Cytology, 67(2):61–67, 2005]. Some inventions include fixed cuvette chambers with replaceable nozzles to allow removing of substrate held at a registered location on a cuvette-based flow-cell [Pierce O Norton, David R Vrane, and Shervin Javadi. Fixed mounted sorting cuvette with user replaceable nozzle, April 10 2007. US Patent 7,201,875].

Many instruments use a cuvette-based flow-cell machined in such a way that the sheath and the sample fluid enter a square quartz cuvette, the sheath fluid entering from the side and the sample fluid from the center, enabling hydrodynamic focusing of the sample stream by the sheathing stream [Richard Channing Moore and Anthony Ferrante. High viscosity sheath reagent for flow cytometry, July 05 2001. WO200148455]. The final channel dimensions are a few hundred micrometers while the final sample core width is focused to a few tens of micrometers, typically around 20 to 30µm, inside the final channel [Katharina Ruzicka, Mario Veitl, Renate Thalhammer-Scherrer, and Ilse Schwarzinger. The new hematology analyzer Sysmex XE-2100: performance evaluation of a novel white blood cell differential technology. Archives of pathology & laboratory medicine, 125(3):391–396, 2001]. Apart from being expensive by virtue of the sophisticated cuvette cell and the machined fixtures being employed, there are certain limitations to commercial flow-cell designs. In those devices, a thick quartz cuvette sits inside a stainless-steel fixture, which is not only difficult to machine but also subjects the quartz cuvette to additional torque, causing uneven abrasions or damage to the quartz cuvette, as seen in previous reports of construction of Ortho Cytofluorographs in the 1970s [Kimia Sobhani, David A Michels, and Norman J Dovichi. Sheath-flow cuvette for high-sensitivity laser-induced fluorescence detection in capillary electrophoresis. Applied spectroscopy, 61(7):777– 779, 2007]. Previous inventions have employed round capillaries in place of square cuvettes for collection and excitation of light. However, it has been observed that square geometry of the capillaries or cuvettes provides an even and flat window for the light illumination, preventing any unnecessary refraction or scatter from the curved interfaces of a round geometry. Also, in many of the capillary-based flow-cells [D Peters, E Branscomb, P Dean, T Merrill, D Pinkel, M Van Dilla, and JW Gray. The LLNL highspeed sorter: Design features, operational characteristics, and biological utility. Cytometry: The Journal of the International Society for Analytical Cytology, 6(4):290–301, 1985], the inventors in some designs, integrate the quartz cuvette in their assembly for the final focusing, again increasing the overall cost and maintenance of the device. Some inventors have reported designs of microfluidic flow-cells to focus the cells in an efficient way [Mahesh Kumar, Supriya Yadav, Ashish Kumar, Niti Nipun Sharma, Jamil Akhtar, and Kulwant Singh. MEMS impedance flow cytometry designs for effective manipulation of micro entities in health care applications. Biosensors and Bioelectronics, 142:111526, 2019; Daniel Spencer, Gregor Elliott, and Hywel Morgan. A sheath-less combined optical and impedance micro-cytometer. Lab on a Chip, 14(16):3064–3073, 2014; Gwo-Bin Lee, Chen-I Hung, Bin-Jo Ke, Guan-Ruey Huang, Bao-Herng Hwei, and Hui-Fang Lai. Hydrodynamic focusing for a micromachined flow cytometer. J. Fluids Eng., 123(3):672–679, 2001]. Such designs use far less consumables for the same throughput of cells, although the cost of fabrication of microfluidic flow circuits and the accompanying instrumentation is significantly high. Further, these flow-cells are specifically designed for microfluidic devices and cannot be integrated on benchtop cytometers. Also, many microfluidic flow-cells are made of polydimethyl siloxane (PDMS), which is a polymer used in conjunction with a glass covering, engraved with channels for the fluid flow. Although being bio-friendly, it generates autofluorescence upon being subjected to laser, leading to poor performance of the device. The microfluidic chamber/cell apparatus in many of the reported works requires a microscope for collection of the fluorescence signals followed by image analysis, thereby making the overall device expensive and infeasible for a resource-constraint setting.

Given the high cost of thick quartz flow-cells that are used in conventional cytometers, CN108444897 involves a sheath flow chamber formed by a nested structure of glass tubes with different tube diameters. Specifically, they propose square inner capillary tube for the inner sample flow, which is surrounded by another square capillary of a larger cross-section that carries the sheath liquid while a circular ring or a spacer is placed in between to separate and align the two capillary tubes. In CN111337416, multiple inner glass tubes are placed in a single rectangular sheath flow chamber, again using a ring/spacer to separate the inner sample tube from the outer sheath flow tube. However, the complex design requires placing of the spacer and its attachment to the walls of the capillary is difficult and cumbersome. Further, most commercial flow-cells, which are square in cross-section, have sides between 200-400 µm so as to robustly and reproducibly focus the sample into a narrow stream. The aforementioned methodology is difficult to fabricate since it would require fitting a sleeve and an inner capillary (for sample) of even smaller cross-section inside a capillary of inner sides of 200-400 µm.

In WO201275358, the sheath liquid flows through an array of tubes with one of more of the inner tubes carrying the sample. The outer support tube is then thermally tapered in the downstream position so as to focus the sample stream into a narrow region. Here, the complexity of the design and its fabrication arises from the need to arrange the multiple tubes inside an outer support tube, bind the multiple tubes to each other and the support tube followed by thermal tapering of the support tube.

In US7835000 and US8767208, a small sample capillary tube is inserted into a 250 µm by 250 µm square flow channel of a thick quartz flow-cell. The proposed flow-cell is demonstrated for low flow rates where the transit time of the particles through the detection region is of the order of 100 microseconds or more, resulting in particle speeds of a few centimetres per second. Further, the flow-cell was used to collect side scatter from the detection region. The results imply that the embodiment is not suitable for fast flows where the transit time of particles is less than 100 microseconds or for detecting forward scatter of particles, which require sample flow to be focused within a narrow region of 50 µm, preferably within 30 µm.

Therefore, there remains a need for an economical and accurate light scattering flow-cell system which eliminates many of the expensive components used in commercial instruments especially for resource-constrained settings.

SUMMARY OF THE INVENTION
The following disclosure presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later.

The object of present invention is to overcome the problems of prior arts.

Another object of present invention is to attend to the need for an economical and accurate light scattering flow-cell system which eliminates many of the expensive components used in commercial instruments especially for resource-constrained settings.

Another object of present invention is to employ a leak-free sheath flow enclosing a sample flow containing particles through an air tight polymer or metal fixture.

Yet another object of present invention is to introduce a small (mini-fluidic), simple, light, low-cost, reliable, reproducible and durable design of the capillary flow-cell.

Another object of present invention is to provide a bio-compatible supply system where sheath liquid contacts only non-metal parts thus avoiding metal corrosion and contamination of the sheathing liquid.

Another object is to introduce a flow cytometer with easily available and inexpensive optical and electronic components capable of performing the blood cell counting.

Further object of the present invention is to provide a generic design enabling scope for modifications in the design as in introducing bubble-free apparatus and 3-D printed designs.

According to the first aspect of the present invention, there is provided a sheath flow type flow-cell device for flow-cytometer, comprising a capillary assembly and a plurality of connectors to facilitate the capillary assembly to be joined to other fluidic pathway components;
wherein the capillary assembly comprises a first capillary of circular cross section and a second capillary of square cross section; the capillary assembly being so designed that the outer diameter of the first circular capillary matches the inner width of the square capillary; and
characterized in that the plurality of connectors comprises a cross adapter having four-way-channel wherein a sample liquid having particles to be analyzed enters the first capillary through the top channel of the cross adapter and a sheath liquid enters the cross adapter through one of the side channels, the first capillary extending longitudinally from first channel of the cross adapter to the bottom channel just to enter the second capillary to be positioned tightly and concentrically over the second capillary thus providing only the corners of the second capillary for sheath liquid to flow downstream and communicate with the first circular capillary at its end enabling hydrodynamic focusing of the sample fluid;
and a tee-adapter having three-way-channel where the bottom end of the second capillary is connected via a top channel thereby assembling the connectors and capillaries co-axially.

According to another aspect of present invention there is provided a sheath flow type flow-cell device for flow-cytometer, comprising a capillary assembly and a plurality of connectors to facilitate the capillary assembly to be joined to other fluidic pathway components;
wherein the capillary assembly comprises a first capillary of circular cross section and a second capillary of square cross section; the circular capillary being tapered with a circular cross section at the end; the capillary assembly being so designed that the outer diameter of the first circular capillary matches the inner width of the square capillary; and
characterized in that the plurality of connectors comprises a cross adapter having four-way-channel wherein a sample liquid having particle to be analyzed enters the first capillary through the top channel of the cross adapter and a sheath liquid enters the cross adapter through one or both of the side channels, the first capillary extending longitudinally from first channel of the cross adapter to the bottom channel just to enter the second capillary to be positioned tightly and concentrically over the second capillary thus providing only the corners of the second capillary for sheath liquid to flow downstream and communicate with the first circular capillary at its end enabling hydrodynamic focusing of the sample fluid;
and a tee-adapter having three-way-channel where the bottom end of the second capillary is connected via a top channel thereby assembling the connectors and capillaries co-axially.

While assembling the capillaries co-axially within the tee and cross-adapter, the round capillary is made to just enter the square capillary such that the exposed corners allow the sheath fluid to come and communicate with the round capillary at its end. This geometry ensures that the pressurized sheath fluid coming from the corners of square capillary provides hydrodynamic sheathing to the sample fluid emitting from the circular capillary sitting tightly inside the square capillary, with zero dead volume at the corners. This geometry focuses the sample fluid to a narrow stream containing particles/cells confined in a single line with adequate spacing in between. This kind of a flow makes it possible to analyze single cells allowing a detailed spatial measurement of the cell at every angle, in addition to preventing direct contact of the biological cells with the walls of the capillary which may otherwise cause damage and mechanical stresses to the cell body.

Other aspects, advantages, and salient features of the invention will become apparent to those skilled in the art from the following detailed description, which, taken in conjunction with the annexed drawings, discloses exemplary embodiments of the invention.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
The above and other aspects, features and advantages of the embodiments of the present disclosure will be more apparent in the following description taken in conjunction with the accompanying drawings, in which:

Fig. 1 illustrates the capillary based sheath flow embodiment (1 mm square capillary with tapered round capillary combination) with the inlet and outlet channels and the two ETFE adapters.

Fig. 2 shows the capillary based sheath flow embodiment (400 µm I.D. square capillary with 400 µm O.D. round capillary combination).

Fig. 3 shows the cross-sectional view of the inner geometry of the capillary assembly enabling hydrodynamic focusing of the sample fluid (a) and the isometric view of the combination of the concentric and co-axial capillaries (b).

Fig. 4 illustrates a schematic drawing of the capillary sheath flow cell based flow cytometer with proper fluidic, optical and electronic components aligned together.

Fig.5 shows the variation in sample core width with increase in the relative flow rate. The inner round capillary has inner and outer diameter of 300 µm and 400 µm, respectively, while the inner width of the square capillary is 400 µm. The sample core diameter reduces to 25 µm as a result of hydrodynamic focusing.

Fig.6 discloses the variation in sample core width with increase in the relative flow rate of the sheath and the sample flow as a result of hydrodynamic focusing for the 1 mm I.D. square capillary and a tapered inner round capillary set for two different sample flow rates,10 µl/min and 50 µl/min and comparison between the two.

Fig. 7 illustrates a comparison between the different sample flow rate variations with respect to the relative flow rate of the sheath and the sample (Qo/Qi) for the 1 mm square capillary and the 400 µm square capillary and plots representing variation of the width of the sample core (Rs/R) with respect to Qo/Qi obtained theoretically for both concentric cylindrical geometry and circular capillary within a square capillary configuration.

Fig. 8 illustrates the graph representing variation of the width of the sample core with respect to change in the relative flow rate with particles in the solution for the 1 mm square capillary to compare with the dye based experiments.

Fig. 9 illustrates a graph showing an overlap between the size distribution of 14-20 µm particles obtained via direct imaging and that obtained as a result of forward scattered intensity measurement.

Fig. 10 illustrates a graph showing an overlap between the size distribution of 10 µm particles obtained via direct imaging and that obtained as a result of forward scattered intensity measurement.

Persons skilled in the art will appreciate that elements in the figures are illustrated for simplicity and clarity and may not have been drawn to scale. For example, the dimensions of some of the elements in the figure may be exaggerated relative to other elements to help to improve understanding of various exemplary embodiments of the present disclosure. Throughout the drawings, it should be noted that like reference numbers are used to depict the same or similar elements, features, and structures.

DETAILED DESCRIPTION OF THE PRESENT INVENTION
The following description with reference to the accompanying drawings is provided to assist in a comprehensive understanding of various embodiments of the present disclosure as defined by the claims and their equivalents. It includes various specific details to assist in that understanding, but these are to be regarded as merely exemplary. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the various embodiments described herein can be made without departing from the scope and spirit of the present disclosure. In addition, descriptions of well-known functions and constructions may be omitted for clarity and conciseness.

The terms and words used in the following description and claims are not limited to the bibliographical meanings, but, are merely used by the inventor to enable a clear and consistent understanding of the present disclosure. Accordingly, it should be apparent to those skilled in the art that the following description of various embodiments of the present disclosure is provided for illustration purpose only and not for the purpose of limiting the present disclosure as defined by the appended claims and their equivalents.

It is to be understood that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a component surface" includes a reference to one or more of such surfaces.

All terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which various embodiments belong. Further, the meaning of terms or words used in the specification and the claims should not be limited to the literal or commonly employed sense but should be construed in accordance with the spirit of the disclosure to most properly describe the present disclosure.

The terminology used herein is for the purpose of describing particular various embodiments only and is not intended to be limiting of various embodiments. As used herein, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising" used herein specify the presence of stated features, integers, steps, operations, members, components, and/or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, operations, members, components, and/or groups thereof. Also, expressions such as "at least one of," when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

The present disclosure will now be described more fully with reference to the accompanying drawings, in which various embodiments of the present disclosure are shown. The present disclosure may, however, be embodied in many different forms and should not be construed as being limited to the various embodiments set forth herein, rather, these various embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the present disclosure. Furthermore, a detailed description of other parts will not be provided not to make the present disclosure unclear. Like reference numerals in the drawings refer to like elements throughout.

Provided herein is a sheath flow type flow-cell device for flow-cytometer, comprising a capillary assembly and a plurality of connectors to facilitate the capillary assembly to be joined to other fluidic pathway components;
wherein the capillary assembly comprises a first capillary of circular cross section and a second capillary of square cross section.; the capillary assembly being so designed that the outer diameter of the first circular capillary matches the inner width of the square capillary; and
characterized in that the plurality of connectors comprises a cross adapter having four-way-channel wherein a sample liquid having particle to be analyzed enters the first capillary through the top channel of the cross adapter and a sheath liquid enters the cross adapter through one or both of the side channels, the first capillary extending longitudinally from first channel of the cross adapter to the bottom channel just to enter the second capillary to be positioned tightly and concentrically over the second capillary thus providing only the corners of the second capillary for sheath liquid to flow downstream and communicate with the first circular capillary at its end enabling hydrodynamic focusing of the sample fluid;
and a tee-adapter having three-way-channel where the bottom end of the second capillary is connected via a top channel thereby assembling the connectors and capillaries co-axially.

Further provided herein is a sheath flow type flow-cell device for flow-cytometer, comprising a capillary assembly and a plurality of connectors to facilitate the capillary assembly to be joined to other fluidic pathway components;
wherein the capillary assembly comprises a first capillary of circular cross section and a second capillary of square cross section; the circular capillary being tapered circular cross section at the end; the capillary assembly being so designed that the outer diameter of the first circular capillary matches the inner width of the square capillary; and
characterized in that the plurality of connectors comprises a cross adapter having four-way-channel wherein a sample liquid having particle to be analyzed enters the first capillary through the top channel of the cross adapter and a sheath liquid enters the cross adapter through one of the side channels, the first capillary extending longitudinally from first channel of the cross adapter to the bottom channel just to enter the second capillary to be positioned tightly and concentrically over the second capillary thus providing only the corners of the second capillary for sheath liquid to flow downstream and communicate with the first circular capillary at its end enabling hydrodynamic focusing of the sample fluid;
and a tee-adapter having three-way-channel where the bottom end of the second capillary is connected via a top channel thereby assembling the connectors and capillaries co-axially.

While assembling the capillaries co-axially within the tee and cross-adapter, the round capillary is made to just enter the square capillary such that the exposed corners allow the sheath fluid to come and communicate with the round capillary at its end. This geometry ensures that the pressurized sheath fluid coming from the corners of square capillary provides hydrodynamic sheathing to the sample fluid emitting from the circular capillary sitting tightly inside the square capillary, with zero dead volume at the corners. This geometry focuses the sample fluid to a narrow stream containing particles/cells confined in a single line with adequate spacing in between. This kind of a flow makes it possible to analyze single cells allowing a detailed spatial measurement of the cell at every angle, in addition to preventing direct contact of the biological cells with the walls of the capillary which may otherwise cause damage and mechanical stresses to the cell body.

In an embodiment of present invention, the tee and cross-adapter are constructed by simple fluidic polymer components such as, but not limited to, micro-tight ethylene Tetraflurorethylene (ETFE). These adapters or connectors come with a true Zero Dead Volume (ZDV) internal configuration that reduces the formation of dead volume inside the channels or fluidic pathway enabling bubble-free movement of flow to the capillary assembly. The ETFE connectors or adapters are easy to assemble or disassemble and can be easily integrated with commercial capillary-based apparatus. All ETFE tee-adapters and cross-adapters come with such as, but not limited to, 1/4-28 Perfluoroalkoxialkane (PFA) flangeless nuts and ETFE ferrules to hold the tubing tightly inside the nut. These are inert bio-compatible polymeric adapters that ensure bio-chemical compatibility with the liquid flowing through. For applications involving high pressures of the order of about some thousands of psi, Polyether ether ketone (PEEK)or stainless-steel adapters can used with 1/16’’ OD PEEK or stainless-steel tubing. There are certain advantages in using these polymeric micro-tight adapters. In addition to ensuring laminar flow for hydrodynamic focusing, they are of customizable dimensions based on the nut and the ferrule size, they eliminate loosening of fittings upon twisting effects of the nuts, hold tight even through vibrations, can incorporate silicone tubing to provide cushioning effect to the capillaries. The cross adapter allows the sample and the sheath flow to enter while the tee adapter is used to hold the capillaries in place along with disposing off the waste. The flow-cell design may also encompass casings/fixtures made of other materials like UV-resins used for 3D printed designs, PEEK, stainless steel or Teflon.

In another embodiment of present invention, the capillaries are constructed of glass and/or quartz.

In one of the embodiments of present invention, the first capillary is mounted inside the cross adapter from the top channel via a Teflon tubing fitted tightly inside the cross-adapter fixture by a nut and the corresponding ferrule.

The sample fluid containing particles/cells is introduced through this pathway via the first circular capillary to communicate with the pressurized sleeving or sheath flow coming from the side(s) of the cross-adapter ends, via a Teflon tubing and the corresponding ferrule.

In a preferred embodiment, the diameter of the Teflon tubing for the sheath is kept larger than that used for the sample flow injection.

Another embodiment of present invention relates to one of the sides/channel pathways of the cross adapter can be made to introduce a burp line, a tubing to discard bubbles created inside. The burp line is closed during normal operation.

The top port of the cross-adapter carries the sample flow while the sheath liquid flows from the side ports of the adapter. The bottom port is used to introduce a square capillary (glass or preferably, quartz), via a nut and ferrule to hold it tightly.

In a preferred embodiment, the square capillary is jacketed with a silicone sleeve inside the ETFE nut and ferrule to avoid any breakage upon vibrations.

The downstream tubing from the tee adapter emits the waste solution into the discard or a reservoir that can be replenished.

The embodiment is mounted on an X-Z translation stage screwed securely to a metal C-frame with proper screws. The adapters come with clean grooves for mounting screws. The apparatus is placed with one of the flat surfaces of the square glass capillary facing the laser source. Light from the laser beam is focused to a narrow elliptical beam of a width of about 150 µm by a pair of orthogonally placed cylindrical lenses and is illuminated on to the square capillary carrying particles/cells. The particles scatter light in all directions and the small angle forward scatter is collected by an objective lens placed at a distance from the capillary flow cell assembly. The objective lens is aided with a beam stop of about a millimetre width which blocks the direct light from hitting the sensor for detection. A high speed camera (Photron FASTCAM camera), a CCD camera (PIKE, ALLIED Vision Technologies)as well as a colour CMOS camera (Infinity1 Lumenera Corp) have been used which capture the scattered light, hence measuring the count and size of particles traversing the width of laser beam.

EXAMPLES:
In order to test the feasibility of present invention, characterization experiments with a sheathing liquid and two different sample fluids, a dye and a suspension containing particles have been conducted. The sample stream was imaged to determine the extent of hydrodynamic focusing. Two separate designs were tested, one with the capillary set comprising of a round capillary of O.D. 400 µm inside a square borosilicate as well as a quartz capillary of I.D. 400 µm and 500 µm, respectively, and the other with a tapered round capillary of I.D. 800 µm inside a square capillary inside a square borosilicate capillary of I.D. 1 mm.

Referring to the drawings and specifically to FIG. 1, the capillary based sheath flow-cell assembly with all the associated parts intact has been depicted. This simple embodiment is a part of the prototype flow cytometer and is capable of performing the hydrodynamic focusing of particles using low-cost capillaries kept in a particular configuration aiding to the cuvette-based geometry necessary for the focusing of the sample fluid. The ETFE cross adapter 12 allowing for the passage of four channels and while the ETFE tee adapter 13 allowing for the passage of three channels have been shown separately, which represents the main embodiments holding the capillaries in a particular configuration. The sample flow 8 is made to enter the cross-adapter 12 from the top channel through a Teflon tube enclosing a circular capillary 6 of I.D. of 800µm with a taper towards the bottom (in one configuration) bringing the outer dimension of the capillary down to 190 µm. The capillary is centered and drawn inside the cross-adapter and made to emerge from the opposite channel end. The centering is made possible with the help of the 1/4 - 28 PFA flangeless nuts and ETFE ferrules 11 holding the Teflon tubing enclosing the round capillary. These nuts and ferrules ensure there is no leakage from the embodiment, in addition to holding the capillaries in place and providing jerk-free enclosure to the capillaries.

One of the side ports (channel 2) of the cross-adapter carries a highly pressurized sheath fluid/sleeving fluid. The sheath fluid is injected inside the inner chamber of the cross-adapter where it communicates with the sample fluid, however, there is no mixing of the two streams by virtue of the smaller dimensions of the adapter chamber inside. The other lateral channel of the cross-adapter is either restricted to introducing another sheath flow stream, or in modified designs, for burp line/air purge line for the removal of bubbles. The sheath and the sample liquids are pumped using syringe pumps (New Era Pumps, SyringePump.com). The bottom end channel of the cross-adapter holds the ETFE nut hosting the square capillary 5 (boro-silicate or quartz), which is jacketed by a polymeric sleeve (silicone tube) in the center. The square capillary 5, of an inner diameter of 1 mm, encloses the round capillary co-axially and concentrically in such a way that the configuration consists of a round capillary (borosilicate)fit inside a square capillary leaving the four corners of the square capillary exposed to the influx of the sheath stream throughout. Some portion of the round capillary protrudes to a level where it is visible inside the square capillary, although the length of the round capillary can be made shorter and the round capillary may be made to just enter the square capillary.

At the very bottom of the square capillary, the ETFE tee-adapter 9 is integrated with the square capillary via a specific sized nut and a ferrule in a similar manner. The tee-adapter is used in the downstream to eject the liquid from one lateral channel end 3 into a waste chamber while additionally providing leverage and mechanical strength to the design assembly thereby making the whole system sturdy and easy to handle. The other end of the tee-adapter 7 is typically blocked to prevent leakage and introduction of air/bubbles. The two adapters come with mounting through-holes 10,14 that are used to mount the assembly to a metal bracket or a translation stage mount.

Referring now to FIG. 2, there is shown a drawing of another similar embodiment made of the two adapters, enclosing a combination of two capillaries, one round capillary with an outer diameter of 400 µm 15 and an outer square capillary of inner diameter 400 µm 16 such that the round capillary sits tightly inside the round capillary so that there are only the corners left exposed for the sheathing liquid to enter. This design was constructed to check the accuracy of hydrodynamic focusing and compared with a larger diameter capillary combination as seen in the previous drawing.

Referring to FIG. 3(a), a cross-sectional view of the inner geometry of the capillary set shows a 800 µm I.D round capillary inside a square capillary with an inner width of 1 mm. The sectional view shows that the round capillary fits inside the square capillary such that the outer surface of the round capillary completely touches the inner surface of the square capillary. This combination ensures proper and robust alignment of the inner capillary. Any misalignment may lead to the deviation of the sample core stream away from the center of the square capillary. The sheath fluid enters the corners of the square capillary and joins the sample stream at the center coming from the top channel of the cross-adapter. The volumetric flow rate of the sheath stream is kept much higher than the volumetric flow rate of the sample fluid (typically of the order of 5-50 µl/min), the ratio of the sheath and the sample flow rates is typically maintained at about 200:1. The tip of the inner round capillary has been tapered to provide a smooth cuvette-based nozzle surface for the sheath flow stream to focus the inner sample fluid stream more efficiently. A typical nozzle structure for the channels providing hydrodynamic focusing of fluids has been reported in literature and the nozzle length is also kept as small as possible to provide a sharp convergent profile of the focused stream. The flow is laminar, with no diffusion or mixing taking place. This pressurized sheath flow focuses the sample fluid to a narrow core stream of width of a few microns. With our design, we have been able to focus to a width of about 25 µm, which matches that obtained using commercial flow-cells. Any deviation of the sample flow stream from the central region at the interrogation point may lead to wrongly detected particles, giving faulty signals, or no signals at all.

FIG. 3(b) shows an isometric view of a drawing of another combination of capillaries assembled to check the accuracy of our design. This configuration represents a 400 µm O.D round capillary inside a 400 µm I.D square capillary in a closely packed placement, such that here also only the corners of the square capillary are exposed allowing the sheath stream to flow and focus the sample stream. This combination was tested and the results showed that the sample width could be brought down smoothly to about 25 µm, close to what is obtained in commercial cuvette-based flow-cells.

In FIG. 4 is illustrated a simple schematic drawing of the laboratory set-up of the flow cytometer. The instrument is a simplified working model of the commercial cytometer that works on the principle of light scattering by small particles flowing through a flow chamber. In the present embodiment, the fluidic, the optical, the electronic and the sensing components are all integrated in proper alignment to achieve proper size measurement by light scattering. A 532 nm green laser (Laserglow TechnologiesTM) 21 is projected to illuminate a focused stream of particles traversing through the capillary based flow-cell 31. The laser beam spot is made to pass through a 2 mm pinhole/orifice 22 and is further focused into a small elliptical beam, with the short axis of the ellipse along the flow direction and the long axis of the ellipse perpendicular to the flow direction, by a set of orthogonally placed cylindrical lenses 23. The elliptical shape of the laser beam is necessary to incorporate any misaligned particles scattering laser light slightly off from the central maxima of the beam. The lenses and the orifice have been properly mounted in a compact metallic cage (Thorlabs, Inc.) that comes with easily adjustable cage mounts (Thorlabs, Inc.) 24. The illumination as well as the collection optics have been aligned with respect to the position of the interrogation region of the flow-cell and the laser. The ease in adjustment is ensured with using a XYZ translation stage with a custom-made C-bracket 26 that holds the flow-cell in perfect alignment and a similar custom made XY translation stage for the laser 32. The particles in the focused jet scatter laser light in all directions. Two directions are crucial in determining the size and shape of the particles or the scatterers. The forward scattered light is captured at small angles from the central axis which is ensured by a beam stop or an obscuration bar/blocker bar which prevents direct transmission of the axial beam permitting light scattered by the particles or cells at low angles (0.5-10 degrees [Howard M Shapiro. Practical flow cytometry. John Wiley & Sons, 2005], adjustable depending on the width of the laser beam and the speed of the particles, or vice versa) from the central axis. A 4X, 0.10 NA microscope objective (AmScopeTM) 27 is placed at its working distance from the source of forward scatter and is used to collect the forward scattered light and collimate it to strike the sensor 28. We have used a high-speed camera (Photron FASTCAM camera), a charge-coupled device (CCD) camera (PIKE, ALLIED Vision Technologies) as well as a color CMOS camera (Infinity1 Lumenera Corp) to capture and image the forward scattering. A photodetector with an amplifier may also be used to collect and amplify the forward scatter signals. The size of the scatterer is determined by the forward scatter signal, the more intense the signal is, the higher the peak is and the larger the particle is. The intensity of the scattered light also depends on the region of the laser beam illuminating the particle, the particle hitting the central maxima of the laser exhibits a highly intense scatter signal. This property is harnessed in enumerating and sizing particles. The forward scatter signals captured by the camera sensor can be analyzed using Image Analysis software to obtain the size distribution based on forward scatter intensity distribution. The side scattered light, also called orthogonal scattered signal, generally captured at larger angles, typically at 90 degrees from the axis [Howard M Shapiro. Practical flow cytometry. John Wiley & Sons, 2005] determines the shape and granularity of the particles/cells in focus. Since, the side scatter signal is low in intensity, it is captured using a photomultiplier tube (PMT) 29 which comes with an inbuilt series amplifier. For some applications involving measuring granularity or nucleic acid content or apoptosis, etc, fluorescence detection becomes important. A proper set of optical filters (bandpass, long pass, short pass filters) and dichroics, along with a PMT 30 are kept orthogonally on the other side of the flow-cell as shown in the figure to capture the fluorescence signals.

The side scatter signal and the fluorescence signals can be processed further by ADC (Analog to Digital converter) and data analysis to obtain the population of particles/cells exhibiting a particular property in study.

FIG. 5 presents the variation of the width of the sample stream measured at the focusing length (the length at which complete focusing has been achieved) with respect to increasing relative flow rate of the sheath and the sample fluid streams (sheath flow rate/sample flow rate). The experiments were performed using 400 µm OD round capillary inside a 400 µm ID square capillary, with a food coloring dye used as the sample fluid against distilled water as the sheath fluid. The flow rate of the sample fluid was fixed at 20 µl/min while the sheath flow rate was gradually increased from 100 µl/min to 1200 µl/min. The width of the sample core stream at the focusing length was measured by an image analysis software, ImageJ (FIJI) using photographs taken from a CMOS camera (Infinity1, Lumenera) kept at the detection region of the flow-cell with the light source being an LED lamp. The graph shows a smooth decreasing profile where sample core width decreases with an increase in the relative sheath and the sample flow rate. It was observed that the sample core width was reduced from 190 µm at a relative flow rate of 5:1 to 25 µm at a relative flow rate of 60:1 by hydrodynamic focusing. This simple and low-cost design was able to achieve effective focusing resulting in a narrow-focused sample core stream capable of allowing particles to flow in a single-line fashion so as to be detected efficiently by the narrow laser beam.

Referring now to FIG. 6, two plots has been shown, (a) graph representing variation in the sample core stream with increasing relative flow rate of the sheath and the sample measured at the focusing length at a sample flow rate of 10 µl/min and (b) graph representing variation in the sample core stream with increasing relative flow rate at a sample flow rate of 50 µl/min. The experiment was done using 1 mm ID square capillary with a tapered round capillary inside and a food coloring dye was used as the sample core fluid. The profile is a parabolic curve which shows decreasing width of the sample core with increasing relative flow rate, in both the cases. It is recommended to use lower flow rates for the sample fluid to maintain a uniform and narrow core width. The relatively low sample flow rate allows the cells to remain in the interrogation region slightly longer so that more accurate measurements of the particles are possible [10]. From the two graphs, the curve for 10 µl/min sample fluid flow rate is smoother and this combination of the flow rates of the sample and the sheath flows yields a final width of 27 µm for a sample flow rate of 10 µl/min and 34 µm for a sample flow rate of 50 µl/min. These graphs also include photographs of the square capillary region showing the focusing length and the point of measurement of the sample core width at different relative flow rates. These photographs were captured by a high-speed camera (Photron FASTCAM camera). It is evident that the capillary-based flow-cell is able to focus the sample fluid to the desired width acceptable for measurement of particles in a flow cytometer.

FIG. 7 compares graphs of different sample flow rates, namely,10, 20, 30 and 50 µl/min as a function of the sheath flow rates for 1 mm I.D square capillary with a tapered round capillary inside and a square capillary of I.D 400 µm holding a round capillary of O.D.400 µm. The sample core diameter for 1 mm square capillary measured at a specific focusing length came out to be 26, 33, 26 and 34 µm for the sample flow rate of 10, 20, 30 and 50 µl/min, respectively. The sample core diameter for 400 µm capillary was reduced to 25 µm.

FIG. 8 also includes variation of sample core width/diameter with respect to relative flow rate based on theoretical models on the relation between sample core width and the sheath flow rate for two different configurations.

The velocity distribution in a straight, square duct of that extends in the z-direction and with walls at, x = ±R and y = ±R, can be approximated by the expression [19],
(1)
where C1 is a function of the pressure gradient, liquid viscosity and inner width of the duct. The volume flow rate in a square duct, on the other hand, is given by,
, (2)
where (PL - P0)/L is the pressure gradient and µ is the viscosity.

The expression for C1 can be obtained by comparing the volumetric flow rate obtained from (1) with (2). On applying mass balance before and after the focusing of the sample fluid, we obtain a relation between the radius of the sample core (Rs) and the ration of sheath and the sample flow rate (Qo/Qi),
(3)
We also compared our measurements with the theoretical model based on the flow profile for two concentric cylinders [20],
(4)
Both equations (3) and (4) are plotted on the same graph (FIG.7) and the predicted profiles compare well with the measurements. These results validate the experimental results and confirm the correctness of the novel design.

Experiments were repeated with suspension of particles to test the feasibility of present invention for applications in flow cytometry. Experiments were conducted with an aqueous suspension of 14-20 µm polystyrene particles (Cospheric, Inc.) and used distilled water as the sheathing liquid. The trends were identical to those obtained with dye. FIG. 8 presents a plot showing decreasing sample (suspension of particles) core width with increase in the relative flow rate. The sample flow rate was fixed at 10 µl/min and increasing sheath flow rate from 500 µl/min to 2500 µl/min. We captured a movie, at a frame rate of 1000 fps, of the particles flowing using the high speed camera. The movie was processed in ImageJ and a single image was obtained by overlapping all frames and analyzed. The photographs in the graph show the overlay pictures of the movies representing the path of many particles traversing the capillary. Clearly, the width of the suspension stream reduces with increasing relative flow rate.

The final width of the sample core at the focusing length was found to be 28 µm, which is close to the value obtained in the experiments with the dye.

Forward scattering experiments with polystyrene model particles have also been performed and correlated the size distribution obtained via microscope imaging and that obtained from the forward scatter intensity measurement. According to the Mie Theory, the forward scattered signal from the particles/cells in focus is directly proportional to the cross-sectional area of the particle, regardless of its shape, and is independent of refractive index [Craig F. Bohren, Donald R. Huffman. Scattering and Absorption by Small Particles. John Wiley & Sons, 1983]. A 0.5% polystyrene model particle suspension was prepared with 0.1% Tween 20 as the surfactant to prevent agglomeration of the particles. A small volume of 14-20 µm polystyrene particle suspension was imaged under confocal microscope for obtaining the size distribution to be compared with that obtained via forward scatter intensity measurement of the same suspension of particles traversing through the flow-cell. Similar analysis was done for 10 µm polystyrene particle suspension. The flow-cell combination used for these experiments is a quartz square capillary with I.D. 500 µm enclosing a borosilicate round capillary with I.D. 300 µm. The particles are made to flow through the flow-cell at a flow rate of 5 µl/min, with the sheath flow rate up to 3500 µl/min, thereby driving the particles at a speed of about 0.5 m/s and transit times of about 0.1 msec. The forward scattered light is captured at a frame rate (fps) of 1000 in a high-speed camera and the scattered light intensity is obtained using ImageJ software.

FIG. 9 shows a graph presenting a direct correlation between the normalized Gaussian distribution of the 14-20 µm particles’ cross-sectional area, (d/dm)2 and the normalized Gaussian distribution of the forward scattered intensity, I/Im. Here, dm is the diameter of the particle with the highest occurrence in the mixture while Im is the intensity with highest occurrence in the scattered light intensity distribution. A microscope image of the 14-20 µm particles has been included in the inset of the figure for reference purpose. The overlap of the distribution of that obtained from image analysis and that from the forward scatter confirms the correctness of the measurement technique and the accuracy of the flow cell.

In order to test for smaller particles sizes, we performed experiments with polystyrene particles with mean size of 10 µm. In FIG. 10, a graph has been presented which shows an overlap between the normalized Gaussian distribution of the 10 µm particles’ cross-sectional area, (d/dm)2 and the normalized Gaussian distribution of the forward scattered intensity, I/Im. A microscope image of the 10 µm particles has been shown for reference purpose. In a similar manner, the results for the 10 µm show that the forward scatter intensity from the particles obtained via flow cytometry using our flow-cell is directly proportional to the cross-sectional area of the scatterers, thereby validating our claims on the performance of our flow-cell.

To conclude, it can be readily understood that the flow of particle suspension in the sample along with the sheathing liquid inside the capillary based flow cell, constructed according to the aforementioned method of the invention provides a compact, simple and low-cost apparatus that yields reproducible results comparable to those observed in commercial cuvette-based flow-cells. In addition to this observation, the experimental results on size characterization of polystyrene particles also show the substantiation that the current invention of the flow-cell along with the supporting optics and electronics can achieve a high throughput measurement of blood cells. The current invention can be further upgraded with minor modifications for commercial purposes.

The invention is low-cost and very simple to construct and handle, producing reliable focusing for flow cytometric applications. The aforementioned design is rather an example of one preferred embodiment of the invention. However, many other variations in our own design are possible without departing from the teachings of the invention, of which a few alternatives will now be described:
1. The flow-cell can be mounted upside down to avoid bubbles from hindering the flow of the sample.
2. The supply system can be replaced by stable miniature pumps or air compressors.
3. The optical and electronic components can be further simplified to make the instrument compact, point-of-care and bench-top.