GB2167880A

GB2167880A - Planar laminar sample flow through microscopic instrument

Info

Publication number: GB2167880A
Application number: GB08529039A
Authority: GB
Inventors: Fred H Deindoerfer
Original assignee: International Remote Imaging Systems Inc
Current assignee: Iris International Inc
Priority date: 1984-11-29
Filing date: 1985-11-26
Publication date: 1986-06-04
Also published as: JPS61132841A; GB8529039D0; DE3539922A1; AU4906085A; GB2167880B; AU563260B2; FR2573870A1; JPH0659782B2

Abstract

Particles in a fluid sample are passed into a flow chamber 12, which has an inlet 20 and an outlet 22 and a passageway 24 having a cross-sectional area which may decrease substantially as the passageway extends from the inlet to a constriction 21, the cross-sectional area thereafter increasing from the constriction 21 to an imaging area 14. The thickness of the passage 24 in a direction which is parallel to the direction in which a microscope 16 is directed, may decrease substantially from the inlet 20 to the constriction 21. From the constriction 21, however, the thickness remains a constant T. The fluid sample is introduced into the flow chamber 12 at a distance L sufficiently far away from the imaging area 14 to be flowing in a laminar stream having a constant velocity profile at the imaging area 14. The working distance Wd of the microscope lens 30 is greater than one-half the outer thickness D of the flow chamber 12 at the imaging area 14. The focal depth Fd of the lens 30 is much less than the thickness D. The flow rates of the fluid sample and the sheath fluid are such that the fluid sample has a thickness t at the imaging area 14 which is less than or comparable to the focal depth Fd of the lens 30. <IMAGE>

Description

SPECIFICATION A method of operating a microscopic instrument The present invention relates to a method of operating a microscopic instrument which has a flow chamber for analyzing particles in a fluid sample flowing in the chamber.

Microscopic instruments for analyzing particles, such as biological particles, are well known in the art. Typically, the microscopic instrument is focussed on the particles which are on a slide or are suspended in a fluid sample flowing in a flow chamber. The latter is well known in the art. US patent No 3,893,766 and RE 29,141 and US Patent No 4,338,024 disclose a type of flow chamber which can be used with a microscopic instrument for analysis of the particles flowing therein. In both of these references, the flow chamber has an inlet and an outlet with a passageway extending from the inlet to the outlet, the passageway having an imaging area where the microscopic instrument is directed, and a thickness which decreases substantially from the inlet to the imaging area.In US Patent no 4,338,024, a sheath fluid is also introduced into the fow chamber to guide the fluid sample from the inlet to the outlet. In US Patent No 3,893,766, the sheath fluid is conveyed by sheath flow means which comprises a plurality of tubes extending in the direction of the fluid flow and surrounding the sample tube.

None of the references, however, teaches or suggests the necessary parameters for operating the microscopic means in relationship to the dimensions of the flow chamber.

In an embodiment of the present invention, a microscopic instrument is operated which has a microscopic means for analyzing particles that are flowing in a flow chamber. The chamber has an inlet and an outlet and a passageway extending from the inlet to the outlet. The microscopic means is directed at an image area between the inlet and the outlet of the passageway. The passageway is characterised by a thickness which decreases initially from the inlet to the imaging area, reaching a constant thickness, and then remaining constant at the imaging area. A sheath fluid and the fluid sample are conveyed from the inlet to the outlet. The microscopic means further has an optical lens for focusing on the imaging area. In accordance with the present invention, the flow rate of the sheath fluid is selected to produce a planar laminar flow at the imaging area.The microscopic means is directed at the imaging area in the direction parallel to the thickness. The working distance of the optical lens of the microscopic means is chosen to be greater than one-half the thickness of the chamber at the imaging area.

The focal depth of the optical lens is chosen to be much less than the thickness of the chamber at the imaging area. The flow rate of the fluid sample is maintained such that the thickness of the fluid sample at the imaging area is less than the focal depth of the optical lens.

The invention will now be described by way of example with reference to the accompanying drawings, in which: Figure 1 is a cross-sectional view of an apparatus used in a method of embodying the present invention.

Figure 2 is an enlarged cross-sectional view of a portion of the apparatus shown in Fig. 1.

Figure 3 is a plan view of the apparatus of Fig. 1 of the present invention.

Figure 4 is a cross-sectional view of a constriction in the apparatus of Fig. 3.

Fig. 1 shows a cross-sectional view of an apparatus 10 useful in a method of embodying the present invention. The apparatus 10 comprises a flow chamber 12, having an imaging area 14 to which a microscopic means 16 is directed. The miroscopic means 16 is to one side of the chamber 12. A light source 18 provides the illumination for the microscopic means 16 and is to the other side of the chamber 12. The flow chamber 12 has an inlet 20, an outlet 22, and a passageway 24 from the inlet 20 to the outlet 22. The passageway 24 passes by the imaging area 14.

Fluid sample with particles of interest such as blood or urine is conveyed through the flow chamber 12 by entering the inlet 20, and is then conveyed through the passageway 24 to the outlet 22. Sheath fluids are also supplied to the flow chamber 12 through the fluid inlets 26 and 28. The fluid inlets 26 and 28 are to one side and the other side, respectively, of the inlet 20 and upstream of it. The distance from the inlet 20 where the fluid sample enters into the passageway 24 to the imaging area 14 is designated as L. The passageway 24 is characterised by a thickness and a width which decreases substantially from the inlet 20 to a constriction area 21. From the constriction 21 to the outlet 22, the thickness of the passageway 24 remains at a constant, while the width increases, as illustrated in Fig.

3. The cross-sectional area of the passageway 24 decreases from inlet 20 to the constriction 21. Thereafter, the cross-sectional area increases. The microscopic means has an optical lens 30 which is shown in Fig. 2.

Referring to Fig. 2, there is shown an enlarged cross-sectional view of a portion of the flow chamber 12 shown in Fig. 1. The portion of the flow chamber 12 shown in Fig. 2 is that portion near the imaging area 14. Optical lens 30 is focussed on the imaging area 14.

The optical lens 30 is characterised by having a working distance Wd. In addition, the optical lens 30 has a focal depth Fd. The overall or outer thickness of the flow chamber 12 at the imaging area 14 is D. Finally, the fluid sample has a thickness at the imaging area 14 of t.

In operation, the fluid sample is admitted into the flow stream of the sheath fluid. The fluid (comprising of the fluid sample and the sheath fluid) is passed through the restrictor 21 which has a rectangularly shaped crosssection, whose width w is many times its thickness T, as illustrated in Fig. 4. In the imaging area 14, the fluid is maintained in a planar flow. Typically, the width w is 0.813 mm and the thickness T is 0.50 mm. The flow rate of the fluid is chosen such that laminar flow results. As stated in US Patent No 3,893,766, laminar flow can be maintained by the sheath flow means which comprises a plurality of tubes extending through the portion central to the passageway in the direction of the flow and surrounding the sample tube.

The tubes function to prevent turbulence so that the fluid entering the flow chamber is "collimated" and is non-turbulent. As the fluid sample enters in the flow chamber, the fluid takes the form of a laminar fluid flow. Laminar flow can be achieved without the use of tubes if a slow velocity fluid is introduced into a substantially long conduit prior to entering the flow chamber 12. Typically, the velocity of the sheath fluid in the flow chamber in the imaging area 14 is in the range of 0.7X103 mm/sec to 2.7--103 mm/sec. Once laminar flow is established, the flow of the sheath fluid will have a velocity profile. Preferably, the microscopic means 16 is located at or after the sheath fluid has achieved a substantially constant velocity profile, which is in the nature of the shape of a parabola. Thus, the distance L is typically 12.7 mm.

Once the distance L from the inlet to the imaging area 14 is determined, the outer thickness D at the imaging area 14 of the flow chamber 12 is determined. The working distance Wd of the lens 30 must then be chosen to be greater than the distance from the image plane to the outside of the flow chamber 12 at the imaging area 14.

With the working distance Wd of the lens 30 defined, and with the outer distance D at the imaging area 14 defined, the focal depth Fd of the lens 30 is chosen to be much smaller than the outer thickness D.

The thickness t of the fluid sample at the imaging area 14 must be comparable to or less than the focal depth Fd of the lens 30.

The thickness t of the fluid sample at the imaging area 14 is determined by the flow rates of the fluid sample and of the sheath fluid. The flow rates of the fluid sample and the sheath fluid can be adjusted thereby varying the thickness t of the fluid sample at the imaging area 14. Of course as previously noted, the flow rate of the sheath fluid is constrained, in order to produce laminar flow.

In the event that the thickness t of the fluid sample at the imaging area 14 is greater than the focal depth Fd of the lens 30, the method of the present invention can still be practiced by varying the flow rates of the fluid sample and the sheath fluid such that the particles of interest in the fluid sample flow in the center of the fluid sample stream, where they are within the focal depth of the optical lens 30.

Finally, to view the particles in the fluid sample, the light source 18 is chosen to be a strobe light. The duration of the strobe of the light source 18 must be sufficiently short to "freeze" the image of the fluid sample. Of course, the duration of the strobe of the light source to "freeze" the image is determined by the rate of flow of the fluid sample. However, once the flow rate of the fluid sample is set, as set forth hereinabove, the minimum strobe duration is then also determined.

One specific embodiment will now be described. A flow chamber 12 has a passageway with the dimensions of 0.4 centimeters width at the imaging area by 0.005 centimeters depth, inner dimension, at the imaging area. The length is 3.81 centimeters. The distance L from the inlet 20 to the imaging area 14 is chosen to be greater than five times the inner thickness T of the chamber at the imaging area 14. Preferably, the distance L is 1.27 centimeters. At the imaging area 14, the outer thickness D of the flow chamber 12 is 0.7 centimeters or less. Preferably, the thickness D is on the order of 0.157 centimeters. The microscopic instrument is directed in a direction parallel to the thickness D at the imaging area 14. The working distance Wd of the optical lens 30 is chosen to be 0.137 centimeters which is greater than one-half the thickness D at the imaging area 14.The focal depth of the optical lens Fd is chosen to be between 0.6 and 4.5 micrometers, which is much less than the thickness D of the chamber 12 at the imaging area 14. Typically, such an optical lens 30 is one manufactured by American Optical Manufacturing Corporation and has a focal depth of t 1.1 micrometers.

Finally, the flow rates of the sheath fluid and the fluid sample are adjusted such that the thickness t of the fluid sample at the imaging area 14 is less than or comparable to the focal depth Fd of the optical lens 30. The typical flow rates ratio of the fluid sample and of the sheath fluid is in the range of 1 to 2 to 1 to 50. Preferably, the flow rates are 0.0036 ml/sec and 0.050 ml/sec, respectively for a total fluid flow rate of .054 ml/sec. The strobe of the light source 18 is at 2 microsecond duration, 60 times per second.

Thus there is disclosed herein a method for determining the optimal operational parameters for the microscopic means which is focused on the imaging area of a flow chamber having fluid sample flowing therethrough.

Claims

1. A method of operating a microscopic instrument having a microscopic means analyz ing particles in a fluid sample flowing with a sheath fluid in a flow chamber, said chamber having an inlet and an outlet and a passageway extending from the inlet to the outlet with the passageway having an imaging area which said microscopic means is directed, and means to distribute the fluid sample and the sheath fluid into substantially a planar flow from the inlet to the imaging area, said planar flow characterised by a width and a thickness, said sheath fluid and said fluid sample being conveyed from said inlet to the outlet, said microscopic means having an optical lens for focusing on said imaging area; said method comprising selecting the flow rate of the sheath fluid to produce planar laminar flow at the imaging area; directing said microscopic means at said imaging area in a direction parallel to said thickness; choosing the working distance of said optical lens to be greater than the distance from the image plane to the outside of said chamber at said imaging area; choosing the focal depth of said optical lens to be much less than the thickness of said chamber at said imaging area; and maintaining the flow rate of said fluid sample and said sheath fluid such that the particles of said fluid sample flow within the focal depth of said optical lens, at said imaging area.

2. The method of claim 1 further comprising the steps of: admitting said fluid sample into flow stream of said sheath fluid; and passing said sheath fluid with said fluid sam ple through a restrictor having a rectangular cross-sectional shape with the width substantially greater than the thickness.

3. The method of claim 1 wherein said microscopic means is positioned at a distance at or after the sheath fluid achieves a substantially constant velocity profile.

4. The method of claim 3 wherein said profile is in the shape of a parabola.

5. The method of claim 1, wherein the thickness of said fluid sample at said imaging area is comparable to or less than the focal depth of said optical lens.

6. The method of claim 1, wherein the outer thickness of said chamber at said imaging area is 0.7 centimeters or less.

7. The method of claim 1, wherein said optical lens has a focal depth of between 0.6 and 4.5 1m.

8. The method of claim 3, wherein the distance from the imaging area to said inlet is greater than five times the inner thickness of said chamber.

9. The method of claim 1, wherein the ratio of the flow rate of said fluid sample to the flow rate of said sheath is in the range of 1 to 2 to 1 to 50.

10. The method of claim 1, further comprising the step of strobing the fluid sample at the imaging area at a duration to freeze the image of the fluid sample.

11. The method of claim 9, wherein the flow rate of said fluid sample is approximately 0.0036 ml/sec.

12. The method of claim 10, wherein said strobe operates an approximately 60 times per second of two microsecond flash duration.