EP0172242A1 - Cytometres d'ecoulement - Google Patents

Cytometres d'ecoulement

Info

Publication number
EP0172242A1
EP0172242A1 EP85901277A EP85901277A EP0172242A1 EP 0172242 A1 EP0172242 A1 EP 0172242A1 EP 85901277 A EP85901277 A EP 85901277A EP 85901277 A EP85901277 A EP 85901277A EP 0172242 A1 EP0172242 A1 EP 0172242A1
Authority
EP
European Patent Office
Prior art keywords
light
flow cytometer
shell
analysis
zone
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP85901277A
Other languages
German (de)
English (en)
Inventor
Ralph-Michael Boehmer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Research Corp
Original Assignee
Research Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Research Corp filed Critical Research Corp
Publication of EP0172242A1 publication Critical patent/EP0172242A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G01N15/1436—Optical arrangements the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N2021/4704—Angular selective
    • G01N2021/4711—Multiangle measurement
    • G01N2021/4719—Multiangle measurement using a optical fibre array
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/08—Optical fibres; light guides

Definitions

  • the present invention relates to flow cyto- meters.
  • FCM Flow cytometers
  • a liquid flow system by which cells in suspension, which may be loaded with fluorescent dye, are transported in a vertical particle stream and passed singly, one after another, across a zone of analysis _ j . where they are exposed to an intense light beam. This zone may be located in open air or in a glass flow chamber; ii. A light source and focussing system- which directs a light beam (for example a laser beam) sharply focussed into the zone of analysis within the particle 0 stream such that only a single cell will be exposed to the beam; iii. An optical detection system, by which the scattered or fluorescent light pulses emitted by each cell at the moment when the cell passes across the beam, 5 is collected, selected according to wavelength and converted into electronic pulses; iv. An electronic analysis unit by which these pulses are processed and analyzed for the desired information about the cell characteristics which can be 0 obtained from the light pulses.
  • a conventional optical detection system is shown schematically i Figure 1, which is a horizontal section through a flow chamber of an FCM.
  • FIG 1 the flow chamber through which the particle stream passes is shown at 2, the section being taken at the point at which the incident light beam intersects the stream.
  • the cell instantaneously exposed to the beam is shown at 4 and the incident light beam is shown at 6.
  • the light pulses which are emitted from the cell 4 are collected perpendicularly to the incident beam 6 within a solid angle ( ) by a lens 8, then passed through a first beam splitter 10a.
  • the light deflected by the first beam splitter 10a is passed through a color filter 12 onto a first photom ltiplier
  • the x light transmitted through the first beam splitter 10a meets a second beam splitter 10b.
  • the light respective ⁇ ly deflected and transmitted by the second beam splitter passes through further color filters 14, 16 to further photomultipliers, PM 2 and PM.,.
  • the light pulses are analyzed in three different parts of the wavelength spectrum.
  • This conventional detection system is disadvan ⁇ tageous in that each part of this system needs to be adjusted for correct location in three dimensions, and even with very experienced operators, initial adjust ⁇ ments and readjustments during measurement may involve several hours work. With systems effecting more than three color analysis, the use of a highly skilled operator is required for operation.
  • suES 5T ⁇ ⁇ SHEET Further, with this conventional system, all analysis is restricted to the two dimensional plane in which the optical system is mounted. An analysis which could be carried on without such restriction would yield more information concerning the light scatter character ⁇ istics of cells, and a higher proportion of the omni ⁇ directional, but normally weak, fluorescent light could be collected.
  • an optical detection system in a flow cytometer comprising an array of optical fibres which are located directly adjacent to the zone where the light from the cell is emitted, whereby the fibres act to collect emitted light.
  • the ends of the fibres will be within a few millimeters from the cell.
  • Figure 1 is a schematic of a conventional prior art optical detection system
  • Figure 2 is a schematic horizontal cross- -section through a flow chamber of a flow cytometer to illustrate the basic principles of the present inven ⁇ tion;
  • Figure 3 is a similar horizontal section of a first practical embodiment of the invention.
  • Figure 4 is a side view of the embodiment of Figure 3;
  • SU25T5TUTS SHEET Figure 5 is a horizontal section of a second practical embodiment of the invention.
  • Figure 6 is a side view of the embodiment of Figure 5.
  • optical fibres can be used to collect directly the fluorescent or scattered light from the cell.
  • a very simple mounting system for the fibres can be used which does not require a high accuracy in setting up.
  • the optical fibres may be held by the hand or fixed with a putty-like substance about 1 mm from the flow chamber and with this form of mounting the readings of scatter and fluorescence signals obtained have been found to have the same order of accuracy as achieved by a conventional optical system when set up in its optimum manner.
  • Figure 2 shows, schematically, a horizontal section through a transparent vertical flow chamber 2 through which the particle stream passes centrally, the section being taken at the point at which the light beam intersects the stream.
  • the excited cell is shown at 4, and the incident light beam is shown at 6.
  • An optical fibre which directly collects the emitted light is shown at 20.
  • the optical fibre 20 collects light emitted from the cell 4 within a solid angle*£ along an axis inclined at an angle ⁇ to the incident beam 6.
  • the measured solid angle£ can be changed; a
  • SUBSTITUTE SHEET similar effect can be obtained by altering the size of the light-acceptance aperture by means of an aperture mask at the end of the fibre.
  • the fibre can also be moved in order to change the angle fi relative to the incident beam 6.
  • a part-spherical shell 22 is mounted around part of the flow chamber 2, the center of the sphere being coincident with the instantaneously excited cell 4 in the chamber 2.
  • the center of the shell 22 is coincident with the point of intersection of the incident light beam 6 with the particle stream.
  • the beam 6 passes through an appropriate opening 23 in the shell 22.
  • Holes 24 are formed through the wall of the shell 22, the axis of each hole 24 lying on a different radial axis of the shell 22 so that each hole 24 faces toward the excited cell 4.
  • a group of optical fibres is provided (not shown) , the fibres leading to one or more photomultipliers.
  • the ends of the fibres can be removably plugged into any one of the holes 24 in the shell 22 to enable readings to be taken at selected points around the cell 4, in other words at different angles of/? with the possible variation of this angle not only being in the plane of Figure 2 but also in planes inclined to that of Figure 2.
  • a compromise has to be made between the desire for high angular resolu ⁇ tion by small solid angles and the need to collect sufficient amounts of light. Therefore, in practice, the solid anglec ⁇ of light collection for each photo-
  • SUBSTITUT ⁇ SHEET multiplier also needs to be variable. Possible methods of varying the solid angle include the following: a. Different sizes of holes 24 for fitting different diameter fibres. This would require a pre ⁇ determination of angles of interest for the scatter light analysis, where the angle of resolution is im ⁇ portant, the remaining angles being free for larger size fibres collecting the omnidirectional fluorescent light. b. Fibre fittings for allowing variation of depth of fibre plugging, thus varying the angle of light
  • the light from different directions may be collected by several fibres and directed into one photomultiplier.
  • the fibres are relatively inexpensive, the fibres may be fixedly mounted in the shell 22. In this
  • each hole 24 is non-removably plugged with a fibre, with the selection of light analysis angles being obtained by plugging the other ends of the relevant fibres into selected photomultipliers. This would facilitate the precision-setting of all fibres on the shell 22 and thus reduce alignment problems.
  • the shell 22 may be supported by a mounting system which allows adjustment of the position of the shell 22 in all directions relative to the flow chamber.
  • the shell 22 may be mounted by a pre ⁇ cision lock in a fixed position relative to the flow chamber, to thereby avoid the necessity of having to align the system subsequent to manufacture.
  • the flow chamber 2 is not of conventional rectangular cross-section, but in the embodiment shown is of circular cross-section, the chamber being of cylindrical form.
  • the chamber may be of spherical form, with the entry and exit areas of the incident light beam being flattened. In this case all non-perpendicular transitions of light through the interface between glass and air would be avoided.
  • the use of a flow chamber is not essential, and the system shown in Figures 3 and 4 can be used in an FCM in which the particle stream moves through open air.
  • the chamber 2 extends through a para ⁇ bolic reflective shell 30 with the instantaneously excited cell 4 being at the focus of the parabola.
  • This parabolic shell 30 is closed by a circular plate 32 the center of which is apertured for passage of the incident light beam 6 onto the cell 4 at the focus of the shell 30.
  • the shell itself is provided with an aperture 33 in alignment with the central aperture in the plate to permit exit of the light beam 6.
  • Holes 34 are formed through the plate 32 in a number of concentric rows. With each hole 34 being directed perpendicularly to the plane of the plate 32, i.e. parallel to the light beam 6.
  • color discrimination filters can be associated with the fibres, the filters preferably being positioned at the point where the fibres enter the housing of the photo ⁇ multipliers.
  • optical fibres to directly collect the emitted light provides enhanced flexibility of measurement in relation to that of a conventional optical system, and permits easier setting up of experi ⁇ ments. More specifically, the main advantages of the described systems are: i. Reduction of optical alignment problems; ii. Reduced need for highly skilled personnel for operating the system; iii. Reduced cost of flow cytometers; iv. Increased versatility for sophisticated non-routine investigations on cell discrimination.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Cytomètre d'écoulement et procédé de détermination des propriétés de cellules isolées ou d'autres particles (40) consistant à faire passer un courant de particles à travers une zone d'analyse où une source lumineuse dirige un faisceau lumineux (6) de sorte qu'il croise perpendiculairement le courant de particules pour n'exposer qu'une seule cellule (4) au faisceau lumineux (6). Un réseau de fibres optiques à proximité de la zone d'analyse collecte la lumière réfractée par les cellules lors du passage de chaque cellule (40) à travers la zone d'analyse. Chaque fibre est reliée à un photomultiplicateur qui convertit la lumière en signaux électriques qui sont analysés par une unité électronique d'analyse pour déterminer les propriétés des particules. L'angle auquel la lumière est collectée par la fibre optique peut être régulé pour permettre de collecter davantage de lumière, afin d'obtenir plus d'informations sur la particule.
EP85901277A 1984-02-29 1985-02-26 Cytometres d'ecoulement Withdrawn EP0172242A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPG384184 1984-02-29
AU3841/84 1984-02-29

Publications (1)

Publication Number Publication Date
EP0172242A1 true EP0172242A1 (fr) 1986-02-26

Family

ID=3770518

Family Applications (1)

Application Number Title Priority Date Filing Date
EP85901277A Withdrawn EP0172242A1 (fr) 1984-02-29 1985-02-26 Cytometres d'ecoulement

Country Status (3)

Country Link
EP (1) EP0172242A1 (fr)
AU (1) AU580032B2 (fr)
WO (1) WO1985004014A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5496767A (en) * 1990-09-20 1996-03-05 Sumitomo Electric Industries, Ltd. Semiconductor laser and manufacturing method of the same
CN114486691A (zh) * 2022-02-16 2022-05-13 上海纬冉科技有限公司 一种便携式能量探测设备及其调试装置

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4500518A (en) * 1984-04-19 1985-02-19 E. R. Squibb & Sons, Inc. Amino thiol dipeptides
JP3375203B2 (ja) * 1994-08-08 2003-02-10 シスメックス株式会社 細胞分析装置
CA2279574C (fr) 1997-01-31 2007-07-24 The Horticulture & Food Research Institute Of New Zealand Ltd. Appareil optique
US6149867A (en) 1997-12-31 2000-11-21 Xy, Inc. Sheath fluids and collection systems for sex-specific cytometer sorting of sperm
US7208265B1 (en) 1999-11-24 2007-04-24 Xy, Inc. Method of cryopreserving selected sperm cells
CA2468772C (fr) 2000-11-29 2013-10-29 George E. Seidel Systeme de dissociation de spermatozoides congeles-decongeles porteurs d'un chromosome x de ceux porteurs d'un chromosome y
US7713687B2 (en) 2000-11-29 2010-05-11 Xy, Inc. System to separate frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations
DE10155142C2 (de) 2001-11-12 2003-09-04 Friz Biochem Gmbh Dunkelfeld-Abbildungsvorrichtung zur ortsaufgelösten Dunkelfeldabbildung einer flächigen Probe
US8486618B2 (en) 2002-08-01 2013-07-16 Xy, Llc Heterogeneous inseminate system
EP1545203B1 (fr) 2002-08-01 2016-10-19 Xy, Llc Systeme de separation de cellules spermatiques basse pression
AU2003265471B2 (en) 2002-08-15 2009-08-06 Xy, Llc. High resolution flow cytometer
US7169548B2 (en) 2002-09-13 2007-01-30 Xy, Inc. Sperm cell processing and preservation systems
DK2305171T3 (da) 2003-03-28 2022-03-21 Inguran Llc Apparat og fremgangsmåder til tilvejebringelse af kønssorteret dyresæd
DK1625203T3 (en) 2003-05-15 2015-07-06 Xy Llc EFFECTIVE SEPARATION OF haploid cells FOR FLOWCYTOMETRISYSTEMER
BRPI0509485A (pt) 2004-03-29 2007-09-11 Monsanto Technology Llc suspensões de esperma para uso em inseminação
MX2007000888A (es) 2004-07-22 2007-04-02 Monsanto Technology Llc Procedimiento para enriquecer una poblacion de celulas de esperma.
WO2022232102A1 (fr) * 2021-04-27 2022-11-03 Life Technologies Corporation Réglage de pas de fibre optique

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3781112A (en) * 1972-12-15 1973-12-25 Technicon Instr Method and apparatus for analysis of leukocytes using light scattered by each leukocyte at absorbing and non-absorbing wavelength
CH592933A5 (fr) * 1976-04-05 1977-11-15 Cerberus Ag
US4101276A (en) * 1976-06-02 1978-07-18 Beckman Instruments, Inc. Method and apparatus for signalling the introduction of chemical reaction components into a chemical analyzing system
GB1602969A (en) * 1977-08-26 1981-11-18 Standard Telephones Cables Ltd Oil-in-water detection system
US4200802A (en) * 1979-03-28 1980-04-29 The United States Of America As Represented By The United States Department Of Energy Parabolic cell analyzer
US4250394A (en) * 1979-07-19 1981-02-10 Akzona Incorporated Apparatus for determining immunochemical substances
US4348107A (en) * 1980-07-18 1982-09-07 Coulter Electronics, Inc. Orifice inside optical element

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8504014A1 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5496767A (en) * 1990-09-20 1996-03-05 Sumitomo Electric Industries, Ltd. Semiconductor laser and manufacturing method of the same
CN114486691A (zh) * 2022-02-16 2022-05-13 上海纬冉科技有限公司 一种便携式能量探测设备及其调试装置

Also Published As

Publication number Publication date
AU580032B2 (en) 1988-12-22
WO1985004014A1 (fr) 1985-09-12
AU3922585A (en) 1986-07-17

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Effective date: 19860130

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Inventor name: BOEHMER, RALPH-MICHAEL