WO2007119263A2 - Système d'acquisition d'images en deux dimensions et de reconstruction tomographique en trois dimensions de cellules/particules uniques dans un support de flux (laminaire ou non) - Google Patents

Système d'acquisition d'images en deux dimensions et de reconstruction tomographique en trois dimensions de cellules/particules uniques dans un support de flux (laminaire ou non) Download PDF

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Publication number
WO2007119263A2
WO2007119263A2 PCT/IT2007/000299 IT2007000299W WO2007119263A2 WO 2007119263 A2 WO2007119263 A2 WO 2007119263A2 IT 2007000299 W IT2007000299 W IT 2007000299W WO 2007119263 A2 WO2007119263 A2 WO 2007119263A2
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WO
WIPO (PCT)
Prior art keywords
cell
particles
flow
images
laminar
Prior art date
Application number
PCT/IT2007/000299
Other languages
English (en)
Other versions
WO2007119263A3 (fr
Inventor
Umberto Martinelli
Giancarlo Fausto Abate
Giuseppe Castello
Pasquale Daponte
Original Assignee
Società Tecnologie Biomediche S.R.L.
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
Priority claimed from ITCE20060008 external-priority patent/ITCE20060008A1/it
Priority claimed from ITCE20060009 external-priority patent/ITCE20060009A1/it
Priority claimed from ITCE20060011 external-priority patent/ITCE20060011A1/it
Priority claimed from ITCE20060010 external-priority patent/ITCE20060010A1/it
Application filed by Società Tecnologie Biomediche S.R.L. filed Critical Società Tecnologie Biomediche S.R.L.
Priority to EP07736803A priority Critical patent/EP2111539A2/fr
Publication of WO2007119263A2 publication Critical patent/WO2007119263A2/fr
Publication of WO2007119263A3 publication Critical patent/WO2007119263A3/fr

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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
    • G01N15/1425—Optical investigation techniques, e.g. flow cytometry using an analyser being characterised by its control arrangement
    • G01N15/1427—Optical investigation techniques, e.g. flow cytometry using an analyser being characterised by its control arrangement with the synchronisation of components, a time gate for operation of components, or suppression of particle coincidences
    • 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
    • G01N15/1429—Signal processing
    • 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
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • 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
    • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
    • 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
    • G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
    • G01N15/147—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle 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
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/016—White blood cells
    • 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
    • G01N2015/1027—Determining speed or velocity of a particle
    • 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
    • G01N2015/144—Imaging characterised by its optical setup
    • G01N2015/1445—Three-dimensional imaging, imaging in different image planes, e.g. under different angles or at different depths, e.g. by a relative motion of sample and detector, for instance by tomography

Definitions

  • FC Flow-Cytometry
  • the signals detected by these instruments are classified as light pulses which ones were simultaneously originated by single cells carried by a laminar sheath fluid flow so to flow though a measurement micro-chamber, where the cells are hit by a laser beam (excitation source) which, in turn, is focused at the centre of the cylindrical fluid envelope (stream core)
  • each single flowing cell becomes the primary light pulses emission source and the pulse amplitudes are proportional, respectively, to the cell dimension (0° scatter), to the cell granularity (90°. scatter) and to the Fluorescence/es emitted by fluorochrome molecules (PE,
  • the flow-cytometer is able to detect and measure the dimension and granularity of each flowing cell and more to typize and classify each cell as to belong, with certainty (95%), to a functional classified cellular sub-population (cellular typization); for instance, the cytometer detects and identifies a flowing particle and classifies it as a Lymphocyte basing on dimension and granularity measured for this particle and, more, to classify it as belonging to the "T-Lymphocytes" sub-population and, again, to classify this cell as a "T-Helper Lymphocyte” and so on.
  • the cellular or tissue sampling suspension is injected by a very simple pneumo-fluidic device, in a conical shaped chamber and here the suspended particles are enveloped and carried out by a laminar fluid sheath flow trough the square or rectangular section micro- channel (flow channel) and the four optically treated walls (optical flat surfaces) of a quartz measurement chamber.
  • optical lens assemblies used in conventional flow-cytometers are structured with plano-concave lenses to get the excitation laser spot magnification (2X-3X) and, then, to collimate it onto plano-convex lenses (PCX), aspherical or cylindrical, to focus the spot (circular or elliptical spot) at the centre of the fluid envelope (stream core) which carries the sampling cells as a "mono-distributed" sequence running at almost constant speed (laminar sheath flow).
  • This laser focusing method to hit flowing particles is known as “hydrodynamic focusing” by producing a light “spot” 25 to 50 micron in diameter at the "intercept point".
  • the laser beam normally, is stopped by a thin black bar so to avoid the stray light hits the photo-transducers when there are no particles at the intercept point and, hence, there are no scatter and fluorescence pulses (background).
  • E PNEUMO-FLUIDIC SUB-SYSTEM (Air pump, Air Filter, Tanks, Regulators)
  • F FLOW- CELL SUB-SYSTEM (Cell, Holder and Ports, Obscuration Bar)
  • G LASER SOURCE SUB-SYSTEM (Laser Head, Collimation/focusing optics)
  • H OPTICS SUB-SYSTEM, PIN DIODE, I/V CONVERTER (Collection Angle: 0°)
  • A OPTICS SUB-SYSTEM, PIN DIODE, I/V CONVERTERS (Collection Angle: 90°)
  • B PULSE PROCESSING, PEAK/HOLD, MUX INPUTS
  • BITS SUB-SYSTEM D DEDICATED WORKSTATION SUB-SYSTEM
  • the first application and industrial utilization, for which it is claim international patent, is the new OPTO-ELECTRONIC system (as better described later on). It was realized (Hw/Sw) an automatic Cyto-Tomograph unit, structured to integrate, in a single module, the functionality of an advanced (front-end) flow-cytometer and the acquisition system (image capturer) at Hi-sensitivity, Hi-speed and Hi-Resolution of 2D cell , sections and of 3D cyto-tomography re-constructed images from flowing cells carried by a laminar fluid flow (true innovation). Therefore, it was designed and realized all the main sub-systems: mechanical, pneumatic, fluidic, optical and electronics.
  • CBMN Test was performed using the realized Cyto-Tomograph System, as shown in Fig 5/5, and experimentation was carried out to identify cell's Micronuclei, generated during the mitosis phases.
  • BFC Specifically designed to get cells image measurement chamber with micro-channel
  • TP Opt-Electronic Trigger Generator by Paw feedback and time controller
  • EP Hi-Resolution Tomographic System to acquire 2D and 3D still images of flowing cells
  • MC Experimental multichannel based detector with integrated optical de-mixing components
  • the sub-systems A, B, C, D are similar to those of conventional cytometer, but they were designed to enhance the performances as required today:
  • [A] Optical bench equipped with integrated stepper motor (X,Y,Z) driven micro adjustment stages.
  • [B+C+D] Parallel, Hi-speed Hi-Resolution multi-channel acquisition and multiple A/D converters integrated software controlled, continuous sampling electronics, as described hereafter. To get excellent results as pulse shape fidelity with respect to the original analog pulses, PCI board performing 8 simultaneous channels signal acquisition and multiple A/D conversion at 3 .Msmple/Sec/channel in continuous sampling (NO Peak and Hold) were used.
  • the OPTO-ELECTRONIC digital system In order to acquire high-resolution images of flowing cells and/or particles Hi-Resolution and Hi-Speed device fully controlled in timing and operational sequences by the optical trigger generator (TP in Legend), the OPTO-ELECTRONIC digital system was developed and realized. This OPTO-ELECTRONIC system make possible to perform the functional control of the Cyto-Tomograph and the acquisition of all cytometric signals (0°, 90° scatter and the emitted fluorescence intensity), together with the simultaneous capture of Tomographic images for each single cell flowing through the special measurement micro- chamber (BFC in Legenda)
  • the OPTO-ELECTRONIC system able to acquire standing still high resolution 2D and 3D Tomographic images of flowing cells/particles carried by a laminar/un-laminar flow, for which one we claim a patent, is composed by 3 components, already described in Fig.5/5 Legend and as- per Priority Claims stated in the previous 08 patents applications for industrial inventions and utility models n°.
  • Component n 1: [BFC] Measurement chamber with micro-channel specifically designed for flowing ''cell/particles imaging" (CE2006A000009 IT e CE2006U000004 IT):
  • This component- has been designed as an unique integrated system made of micro-cell Fig.1/5 of synthetic quartz (as claimed in the reported patent application n.CE2006A000009 IT e CE2006U000004 IT) and of the PMMA laminar flow generator to exactly allocate the micro-cell (up holder), the champion injection needle (down holder) as well as two inlet fluidic ports (side sheath flow ports).
  • the improvement of the quartz micro-chamber comprises a proper shape, which allows to a common (4OX, lOOX) DIN microscope objectives to get in contact with the micro-cell and, more exclusively, comprises a proper thickness of the micro-cell wall in front of the microscope objective to focus in the middle of the flow stream, i.e.170 micrometer, exactly like a slide microscope cover glass.
  • the proposed solution clears up the "Working Distance" WD problem for such objective of large magnification M and hi-numerical aperture NA, which normally is less then 400micrometer.
  • the thickness of the walls in conventional flow-chambers is about 2000 micron, which forbidden the use of large magnification microscope objectives.
  • n 2 [TP] optoelectronic Trigger generator based on event classification and delay feedback control (CE2006A000011 IT and CE2006U000006 IT):
  • the device based on a RISC microprocessor, generates a TTL synchronization pulse of the I-CCD trigger input.
  • the improvement comprises that the trigger input it is released only after the RT calculation is made by the processor of the speed of a target flowing cells, which were classified as "event of interest" at the intercept point of the main cytometric detection area.
  • the system performs the simultaneous measurements of Pulse Area and Pulse Width for the fluorescence emitted by stained nuclei and compares these parameters with respect to those ones measured on a mono-nucleated (normal) lymphocyte (window comparators).
  • the system classifies only BNC as event of interest and the processor performs the RT calculation of the speed of that BNC by taking memory of its Pulse Width.
  • the reference parameter used for the system can generate the TTL trigger pulse for I- CCD, is the distance in between the cytometric intercept point and that image capture point where it is focused a structured sheet or lamina of light (UV, VIS, NIR), and this distance was experimentally optimised to be about 1000 micrometer.
  • the device might be made SW or HW, but in the best example was designed and realized as hardware circuit to avoid the indeterministic timing control, typically of the operating system platforms not reliable when managing time intervals less then 250 mSec.
  • the improvement comprises that the moving cells at the "intercept point" will pass through the lamina of light building 2D section images of the cell/particles of the same thickness as the lamina. In other words, for a given cell it will be reconstructed tomographically the 3D images of the analysed cell and this was never done before.

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  • 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)
  • Engineering & Computer Science (AREA)
  • Signal Processing (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

La présente invention concerne la conception, le développement et la réalisation (matériel et logiciel) d'un système optoélectronique innovant, qui incorpore un module unique, conjuguant simultanément la fonctionnalité de capture d'images en deux/trois dimensions de cellules/particules transportées dans un flux laminaire ou non, conjointement avec sensibilité, résolution et acquisition à grande vitesse. La fonctionnalité de capture d'images en deux/trois dimensions de cellules/particules uniques transportées dans un flux laminaire ou non a été démontrée et appliquée à l'échelle industrielle, dans un équipement cyto-tomographique. Le logiciel de commande, d'acquisition et d'analyse de signal et d'images dérivées d'une cellule unique dans un flux laminaire a été développé avec un degré élevé de simplicité de fonctionnement et de fiabilité dans l'analyse automatique de données, facilitant l'approche interprétative des résultats.
PCT/IT2007/000299 2006-04-19 2007-04-19 Système d'acquisition d'images en deux dimensions et de reconstruction tomographique en trois dimensions de cellules/particules uniques dans un support de flux (laminaire ou non) WO2007119263A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP07736803A EP2111539A2 (fr) 2006-04-19 2007-04-19 Système d'acquisition d'images en deux dimensions et de reconstruction tomographique en trois dimensions de cellules/particules uniques dans un support de flux (laminaire ou non)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
ITCE2006A000008 2006-04-19
ITCE20060008 ITCE20060008A1 (it) 2006-04-19 2006-04-19 Sistema tomografico con rete neurale per l'acquisizione di immagine 3d di singole cellule e/o particolati in movimento tramite trasporto in flusso laminare
ITCE20060009 ITCE20060009A1 (it) 2006-04-20 2006-04-20 Camera di misura con microcelletta specificatamente disegnata per immagini di cellule e/o particolati in flusso laminare.
ITCE20060011 ITCE20060011A1 (it) 2006-04-20 2006-04-20 Generatore di segnali di avvio optoelettronico (trigger) tramite classificazione dell'evento e controllo del ritardo.
ITCE2006A000010 2006-04-20
ITCE2006A000009 2006-04-20
ITCE2006A000011 2006-04-20
ITCE20060010 ITCE20060010A1 (it) 2006-04-20 2006-04-20 Sistema di illuminazione per la generazione e l'acquisizione di immagini nascenti su ed attraverso lamina di luce per tomografi di cellule e/o particolato in flusso laminare.

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WO2007119263A2 true WO2007119263A2 (fr) 2007-10-25
WO2007119263A3 WO2007119263A3 (fr) 2007-11-29

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014085360A1 (fr) * 2012-11-29 2014-06-05 Fluid Imaging Technologies, Inc. Système et procédé pour dilution d'échantillon et imagerie de particules
EP3144662A1 (fr) * 2015-09-18 2017-03-22 Sysmex Corporation Dispositif d'imagerie de particules et procédé d'imagerie de particules
US9983115B2 (en) 2015-09-21 2018-05-29 Fluid Imaging Technologies, Inc. System and method for monitoring particles in a fluid using ratiometric cytometry

Citations (4)

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Publication number Priority date Publication date Assignee Title
EP1070952A2 (fr) * 1999-07-23 2001-01-24 Eauxsys (UK) Limited Procédé et moyens pour la mesure des particules
US20020141625A1 (en) * 2001-03-28 2002-10-03 Nelson Alan C. Apparatus and method for imaging small objects in a flow stream using optical tomography
US20040260157A1 (en) * 2003-06-20 2004-12-23 Montes Miguel A. Method for automated screening of cervical/endocervical malignant and premalignant epithelial lesions using flow cytometry with HPV DNA fluorescent in-situ hybridization ( FISH) technology
WO2005050558A2 (fr) * 2003-11-18 2005-06-02 University Of Washington Procede et appareil de radiographie destines a des applications de tomographie optique

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1070952A2 (fr) * 1999-07-23 2001-01-24 Eauxsys (UK) Limited Procédé et moyens pour la mesure des particules
US20020141625A1 (en) * 2001-03-28 2002-10-03 Nelson Alan C. Apparatus and method for imaging small objects in a flow stream using optical tomography
US20040260157A1 (en) * 2003-06-20 2004-12-23 Montes Miguel A. Method for automated screening of cervical/endocervical malignant and premalignant epithelial lesions using flow cytometry with HPV DNA fluorescent in-situ hybridization ( FISH) technology
WO2005050558A2 (fr) * 2003-11-18 2005-06-02 University Of Washington Procede et appareil de radiographie destines a des applications de tomographie optique

Non-Patent Citations (2)

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Title
ALTENDORF E ET AL: "Differential blood cell counts obtained using a microchannel based flow cytometer" 1997 INTERNATIONAL CONFERENCE ON SOLID-STATE SENSORS AND ACTUATORS. DIGEST OF TECHNICAL PAPERS. TRANSDUCERS 97. CHICAGO, IL, JUNE 16 - 19, 1997. SESSIONS 3A1 - 4D3. PAPERS NO. 3A1.01 - 4D3.14P, INTERNATIONAL CONFERENCE ON SOLID-STATE SENSORS AND ACTU, vol. VOL. 2, 16 June 1997 (1997-06-16), pages 531-534, XP010240530 ISBN: 0-7803-3829-4 *
GRIMALDI D ET AL: "Hardware and software improvements in flow-cytometry measurements" MEASUREMENT, INSTITUTE OF MEASUREMENT AND CONTROL. LONDON, GB, vol. 36, no. 2, September 2004 (2004-09), pages 111-119, XP004576357 ISSN: 0263-2241 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014085360A1 (fr) * 2012-11-29 2014-06-05 Fluid Imaging Technologies, Inc. Système et procédé pour dilution d'échantillon et imagerie de particules
EP3144662A1 (fr) * 2015-09-18 2017-03-22 Sysmex Corporation Dispositif d'imagerie de particules et procédé d'imagerie de particules
JP2017058352A (ja) * 2015-09-18 2017-03-23 シスメックス株式会社 粒子撮像装置および粒子撮像方法
US20170082531A1 (en) * 2015-09-18 2017-03-23 Sysmex Corporation Particle imaging device and particle imaging method
CN106546528A (zh) * 2015-09-18 2017-03-29 希森美康株式会社 粒子拍摄装置和粒子拍摄方法
AU2016225885B2 (en) * 2015-09-18 2018-01-18 Sysmex Corporation Particle imaging device and particle imaging method
US10732095B2 (en) 2015-09-18 2020-08-04 Sysmex Corporation Particle imaging device and particle imaging method
CN106546528B (zh) * 2015-09-18 2020-08-25 希森美康株式会社 粒子拍摄装置和粒子拍摄方法
US9983115B2 (en) 2015-09-21 2018-05-29 Fluid Imaging Technologies, Inc. System and method for monitoring particles in a fluid using ratiometric cytometry

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Publication number Publication date
WO2007119263A3 (fr) 2007-11-29
EP2111539A2 (fr) 2009-10-28

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