FR2573870A1 - METHOD OF USING A MICROSCOPE-TYPE INSTRUMENT - Google Patents

Abstract

ACCORDING TO THIS PROCEDURE FOR THE USE OF AN INSTRUMENT 10 INCLUDING MEANS 16 FOR MICROSCOPIC ANALYSIS OF PARTICLES IN A FLUID FLOW THROUGH A CHAMBER 12, 20, 22, 24 INCLUDING AN IMAGE FORMATION ZONE 14 ON WHICH ARE DIRECTED THE MEANS OF ANALYSIS 16, AND MEANS 26, 28 SERVING TO LAY OUT THE SAMPLE OF FLUID AND AN WRAPPING FLUID IN THE FORM OF A SENSITIVELY FLAT FLOW, A FLAT LAMINAR FLOW IN THE IMAGE FORMING AREA 14, THE MEANS OF ANALYSIS 16 ARE DIRECTED IN THIS ZONE FOLLOWING A DIRECTION PARALLEL TO THE THICKNESS OF THIS ZONE, WE CHOOSE THE WORKING DISTANCE AND THE DEPTH OF FIELD OF THE LENS OF THE MEANS OF ANALYSIS 16 AND THE FLOW IS MAINTAINED OF THE TOTAL FLUID SO THAT THE PARTICLES OF THE FLUID SAMPLE CIRCULATE WITHIN THE DEPTH OF FIELD OF THE MEANS OF ANALYSIS 16. APPLICATION ESPECIALLY TO THE ANALYSIS OF BLOOD AND URINE PARTICLES.

Description

i

  The present invention relates to a method of

  of a microscope-type instrument and more particularly

  of a microscope-type instrument which includes a chamber

  flow for analyzing particles in a fluid sample passing through the chamber.

  Microscope-type instruments for analyzing

  particles, such as biological particles, for example

  are well known in the art. Typically,

  the development of these micro-level instruments is being

  cope on particles that are located on a coverslip or

  are suspended in a circulating fluid sample

  in a flow chamber. This last case is good

  known in the art. U.S. Patent No. 3,893,766 and the application

  US Patent No. RF 29,141 and US Patent No. 4,338,024

  describe a type of flow chamber, which can be used

  with a microscope-type instrument for the analysis of the particles passing through it. In all these documents, the flow chamber is characterized in that it comprises

  an inlet and an outlet equipped with a passage extending

  then admission to the exit. The passage has a zone

  image formation on which the instrument is directed.

  The flow chamber has a thickness which decreases substantially

  the extent of the passage from admission to the imaging area. In US Pat. No. 4,338,024,

  a wrapping fluid is also introduced into the chamber.

  flow to guide the fluid sample from inlet to outlet. In U.S. Patent No. 3,893,766, the wrapping fluid is conveyed by wrapping flow means, which includes a plurality of tubes extending in the direction of fluid flow.

  surrounding the tube carrying the sample.

  None of the documents cited, however, indicate or suggest

  manages the parameters necessary to make the mo-

  yens of microscopic analysis in relation to dimensions

of the flow chamber.

  In accordance with the present invention, a

  assigned use of a microscope-type instrument.

  This method of using a medium-sized instrument

  croscope comprising microscopic analysis means

  particles in a sample of fluid flowing from convert with a wrapping fluid into a flow chamber, which has an inlet and an outlet and a

  passage extending from admission to exit and

  seducing an imaging zone on which C is directed said microscopic analysis means, and means

  for distributing the fluid sample and the fluid of

  development from admission to the training area

  of images essentially according to a characteristic plane flow.

  by a width and a thickness, said fluid of enve-

  said fluid sample and fluid being driven

  then said admission to the exit, and said means of

  microscopic analysis comprising an optical lens serving

  to focus on the training area

  mages, is characterized in that it consists in choosing the flow rate of the enveloping fluid so as to produce a flat laminar flow at the image formation zone, directing said microscopic analysis means on

  said image forming zone in a parallel direction

  at said thickness, to choose the working distance of the-

  so-called lensoptic so that it is greater than the distance between the image plane and the outside of said chamber at said image forming zone, to choose the depth of field of said optical lens so that it is clearly less than the thickness of said chamber at said imaging area, and maintain the

  flow rate of said fluid sample and said casing fluid

  so that the particles of the fluid sample

  circulate in the space corresponding to the depth of field

  said optical lens at said training zone

{ion of images.

  Other features and benefits of this

  invention will emerge from the description given below which

  Referring to the accompanying drawings, in which: - Figure 1 is a cross-sectional view of an apparatus used for carrying out the method according to the present invention; FIG. 2 is a cross sectional view, at a

  greatly enlarged scale, of a part of the apparatus

  shown in Figure 1; FIG. 3 is a plan view of a device used

  for carrying out the process according to the present invention

vention; and

  FIG. 4 is a cross-sectional view of the ap-

same as Figure 3.

  Referring to FIG. 1, there is shown a cross-sectional view of an apparatus used for

  implementation of the method according to the present invention.

  The apparatus 10 includes a flow chamber 12 having an image formation zone 14, on which are located

  directed means of microscopic analysis 16. The means of

  Microscopic lysis 16 are arranged on one side of the chamber 12. A light source 18 provides illumination of the microscopic analysis means 16 and is located on the other side of the chamber.

  the chamber 12. The flow chamber 12 has an admitted

  20, an outlet 22 and a passage 24 extending from the

  Mission 20 to Exit 22. Passage 24 includes the area

  14. The fluid sample, which

  holds the particles of interest, such as

  full of blood or urine, is conveyed through the chamber

  12 when entering the inlet 20, and is

  continued through passage 24 to exit 22.

  Wrapping fluids are also sent to the chamber.

  flow 12 through the flow-through admissions.

  The fluid inlets 26 and 28 are located respectively on one side and the other side of the inlet 20 and in cmont of the latter. The distance between the inlet 20, where the fluid sample enters the passage 24, and the imaging zone 14 is designated by L. The passage

  24 is characterized by a thickness and a width that decreases

  shade as we move around

  passage 24 from admission 20 to the retreating zone

  21. from the narrowing zone 21 and

  at exit 22, the thickness of the flow chamber remains

  the constant, while the width increases. The sectional area

  cross-section of the flow chamber decreases since the admission

  20 to the narrowing zone.

  cross-sectional face increases. The means of analysis

  croscopically include a lens 30 which is shown on

Figure 2.

  Referring to FIG. 2, there is shown, in a cross-sectional view, a greatly enlarged scale, a part of the flow chamber 12 shown

  in Figure 1. The part of the flow chamber 12 represents

  FIG. 2 is the portion that is close to the imaging area 14. The optical lens 30 is focused on the imaging area 14. The optical lens 30 is characterized by having a In addition, the optical lens 30 has a depth of field FD. The thickness of the flow chamber 12 at

  the imaging area 14 is designated d. Finally, the

  fluid sample has a thickness t at the level of the

imaging area 14.

  According to the process according to the present invention, the

  fluid sample is introduced into the flow stream.

  enveloping fluid. The fluid (including the sample

  fluid and the enveloping fluid) passes through the

  narrowing 21 which has a section in cross-section

  rectangular shape whose width is equal to a multiple of its thickness. In the imaging zone 14, the fluid is maintained in the state of a flat flow. Typically the width is 0.813 mm and the thickness is 0.05 mm. The flow of the fluid is chosen so

  to obtain a laminar flow. As indicated in the

  No. 3,893,766, a laminar flow may now be

  naked by the means providing the enveloping flow which

  include a plurality of tubes directed to the center of the con-

  duit in the direction of the flow and surrounding the tube

  supply of the sample. The tubes act in such a way as to

  fish any turbulence so that the fluid entering

  the flow chamber is "collimated" and is not turbulent.

  When the fluid sample enters the chamber of

  flow, the fluid takes the form of a flow of fluid

  laminar. The laminar flow can be obtained without the

  of the tubes, if a low-viscosity fluid is introduced

  in a duct sufficiently long before it enters the flow chamber 12. Typically the speed

  of the enveloping fluid in the flow chamber at

  calf of the imaging zone 14 is within the range of

  me between 0.7 x 103 mm / s and 2.7 x 103 mm / s. Once the laminar flow is established, the flow of the wrapping fluid has a velocity profile. Preferably, the microscopic analysis means 16 are located at or above the place where the flow fluid has reached a substantially constant velocity profile, which presents

  the form of a parable. So the distance L is so ty-

spike equal to 12.7 mm.

  Once the distance between admission and

  image formation is determined, the external thickness

  re D at the imaging zone 14 of the chamber

  flow rate 12 is determined. WD working distance

  of the lens 30 must then be chosen greater than the dis-

  between the image plane and the outside of the listening room.

  12 at the imaging area 14.

  The working distance W of the lens 30 and the dis-

  At the outer image area D at the image formation area D at the image forming area being defined, the field depth FD of the lens 30 is chosen to be much smaller than the thickness D. The thickness T of the fluid sample at the imaging area 14 should be comparable or

  below the depth of the lens.

  T of the fluid sample at the zone of formation.

  14 is determined by the flow velocities

  of the fluid sample and the wrapping fluid.

  The flow rates of the fluid sample and the enveloping fluid

  may be adjusted by changing the thickness T of the fluid sample at the training area

  of images 14. Naturally, as indicated above, the

  bit of the enveloping fluid is limited, in order to obtain a

laminar flow.

  In the case where the thickness T of the fluid sample

  at the level of the imaging zone 14 is superior to

  to the depth of field FD of the lens 30, the method

  form of the present invention can be further implemented by changing the flow rate of the fluid sample and the wrapping fluid so that the particles, which are

  is interested in the flow of the fluid sample, cir-

  in the center of the stream of the fluid sample, where they are located within the depth of field of the

optical lens 30.

  Finally, to observe the particles in the sample

  fluid, one chooses as a light source 18 a

  Strobe sampling light. The duration of the

  the light source 18 must be sufficiently short

  ve to "freeze" the image of the fluid sample. naturally

  the duration of the impulse of the light source allows

  to "freeze" the image is determined by the flow of the sample.

  fluid. However, once the flow of the sample

  fluid flow is set, as indicated above, the duration

  The minimum of the impulse is then also determined.

  A specific embodiment will now be described.

  cific. A flow chamber 12 has dimensions of 0.4

  timestamp (width) and 0.005 centimeters (depth, dimension

  inside) at the imaging area. The length

  It is 3.81 centimeters. The distance L between the admission and the imaging zone 14 is chosen to be greater than five times the internal thickness (d) of the chamber at the level of the imaging zone 14. Preferably the distance

  L is 1.27 centimeters. At the level of the training area

  image 14, the outer thickness D of the chamber of

  flow 12 is equal to 0.7 centimeters or less. Preferably

  this thickness D is of the order of 0.157 centimeters. The instrument

  microscope is directed in a parallel direction.

  is related to the thickness D at the imaging training area.

  14. The working distance WD of the optical lens 30

  is chosen to be 0.137 centimeters, which is a higher value

  than half the thickness D at the level of the

  14. The depth of the lens of the lens op-

  FD is selected at a value between 0.6 and 4.5 microns, which is significantly less than the thickness D of the chamber 12 at the imaging zone 14. Typically such a lens optical 30 is of a type such

  manufactured by the so-called American Optical Manufacturing

  Ring Corporation and has a depth of field of + 1.1 micron.

  Finally, the flow rates of the enveloping fluid and the

  fluid sample are set so that the thickness T of the fluid sample at the imaging area 14 is less than or comparable to the depth of field

  F of the optical lens 30. The typical flow rates of the sample

  Fluid specimen and wrapping fluid are located in

  the range between 1/2 and 1/50. Preferably the

  Bits are equal to 0.0036 ml / s and 0.05 ml / s, respectively, for a total fluid flow rate of 0.054 ml / s. The pulse of the light source 18 has a duration of 2 microseconds,

  and is triggered 60 times per second.

  In the foregoing description of the present invention

  tion, a method for determining the parameters of

  optimum optical ranges for microscopic analysis means

  the development of which is carried out in the area of

  images of a flow chamber crossed by a

fluid sample.

Claims (12)

  1. Method of using a medium-type instrument   croscope (10) having microscopic analysis means (16) analyzing particles in a circulating fluid sample together with a wrapping fluid in a flow chamber (12), which has an inlet (20) and   an outlet (22) and a passage (24) extending from the   up to the exit and having a training area of i-   mages (14) on which said means for   Microscopic analysis (16), and means (26, 28) for distributing the fluid sample and the wrapping fluid from the inlet to the imaging area (14) substantially in accordance with a plane flow characterized by a width and a thickness, said enveloping fluid and   said fluid sample being driven from said ad-   mission (20) to the exit (22), and said means for analyzing   microscopic lysis (16) comprising an optical lens (30)   used to focus on the said area of   imation (14), characterized in that it consists of   - choose the flow rate of the envelope fluid   in order to produce a flat laminar flow at   calf of the imaging zone (14), - directing said microscopic analysis means (16)   on said image forming zone in a pa- goes to said thickness,   - choose the working distance from laditelentille op-   it is larger than the distance between the image plane and the outside of said chamber (12) at said imaging area (14),   - choose the depth of field of said optical lens   (30) so that it is well below the thickness   said chamber (12) at said formation zone images (14), and   maintain the flow rate of said fluid sample and   said fluid of envelopment so that the particles of said fluid sample circulate in the space corresponding to the depth of the field of said optical lens (30) at the level of   said image forming area (14).   2. Method according to claim 1, characterized in that it further includes the operating phases of: - admitting said fluid sample in the flow stream of said wrapping fluid, and passing said wrapping fluid and than   said fluid sample through a foreign device   beam (21) having a rectangular cross-sectional shape   gular and whose width is much greater than the thickness   sor. 3. Method according to claim 1, characterized in that said microscopic analysis means (16) are arranged   at a distance at which or beyond which the fluid of   development takes an essentially constant speed profile.   As. 4. Method according to claim 3, characterized in that   that said profile has the form of a parabola.   5. Method according to claim 1, characterized in that the thickness of said fluid sample, at the level of   said image forming area (14) is comparable to or less than   greater than the depth of said optical lens (30).   6. Method according to claim 1, characterized in that the outer thickness of said chamber (12) at the level of   of said imaging area (14) is equal to 0.7 timestamp or less.   7. Process according to claim 1, characterized in that   that the optical lens (30) has a depth of taken between 0.6 and 4.5 μm.   The method of claim 3, characterized in that the distance between the image forming zone (14) and said admission (20) is greater than five times the thickness interior of said chamber (12).   9. A method according to claim 1, characterized in that the ratio of the flow rate of said fluid sample to the flow rate of said enveloping fluid is in the range of between 1/2 and 1/50.   10. The method of claim 1, characterized in that it further includes the sample operating phase of the fluid sample at the training zone.   of images (14) for a period of time capable of freezing the image of the fluid sample.   11. The method of claim 9, characterized in that the flow rate of said fluid sample is equal to about 0.0036 ml / s.   12. The method as claimed in claim 10, characterized in that the sampling is carried out using pulses of a duration of two microseconds appearing at an equal rate. about 60 times a second.