EP2864762A1 - Collecte de lumière spatialement corrélée à partir de flux d'échantillons multiples excités à l'aide d'une source lumineuse concentrée sur une ligne - Google Patents

Collecte de lumière spatialement corrélée à partir de flux d'échantillons multiples excités à l'aide d'une source lumineuse concentrée sur une ligne

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Publication number
EP2864762A1
EP2864762A1 EP20130807610 EP13807610A EP2864762A1 EP 2864762 A1 EP2864762 A1 EP 2864762A1 EP 20130807610 EP20130807610 EP 20130807610 EP 13807610 A EP13807610 A EP 13807610A EP 2864762 A1 EP2864762 A1 EP 2864762A1
Authority
EP
European Patent Office
Prior art keywords
sample
streams
particles
flow
light
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.)
Ceased
Application number
EP20130807610
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German (de)
English (en)
Other versions
EP2864762A4 (fr
Inventor
Steven Wayde GRAVES
Pearlson Prashanth AUSTIN SUTHANTHIRARA
Andrew P. Shreve
Gabriel P. Lopez
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UNM Rainforest Innovations
Original Assignee
STC UNM
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 STC UNM filed Critical STC UNM
Publication of EP2864762A1 publication Critical patent/EP2864762A1/fr
Publication of EP2864762A4 publication Critical patent/EP2864762A4/fr
Ceased legal-status Critical Current

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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
    • G01N15/1404—Handling flow, e.g. hydrodynamic focusing
    • 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
    • G01N15/1404—Handling flow, e.g. hydrodynamic focusing
    • G01N2015/142—Acoustic or ultrasonic focussing

Definitions

  • flow cytometry requires significant additional sample preparation steps to be effective in the analysis of very rare cell populations, uses offline particle concentration to analyze particles in large volume samples, and requires special purpose large flow channel cytometers using low linear velocity hydrodynamic focusing in wide channels to analyze particles that are >70 ⁇ in diameter at low analysis rates (200 particle/s).
  • Such limitations severely reduce the effectiveness of flow cytometry in many critical applications including the detection of rare blood cell populations, the detection of pathogens in liquid samples, and large particle high throughput analysis model systems (e.g. multicellular model organisms, cellular spheroids, and one-bead-one-compound chemical libraries).
  • the present disclosure provides an affordable flow cytometry system with a significantly increased analytical rate, volumetric sample delivery and usable particle size compared to previously described flow cytometry systems.
  • the present disclosure provides a system for detecting particles in multiple flow streams.
  • the system may include a light beam that interrogates multiple flow streams and provides excitation across all of the streams, and an optical objective configured to collect light from the sample streams and image the light onto an array detector.
  • the light emitted from each flow stream can be individually identified and distinguished from light emitted from any other flow stream by its location on the array detector.
  • the multiple flow streams are achieved through the use of acoustic saves.
  • FIG. 1 is a schematic illustration of an exemplary system according to an embodiment of the present disclosure.
  • FIG. 2 is a schematic illustration of an exemplary system according to another embodiment of the present disclosure.
  • FIG. 3 is a schematic illustration of an exemplary system according to a further embodiment of the present disclosure.
  • Fig. 4 is a schematic illustration of the use of acoustic focusing to produce multiple sample streams.
  • FIG. 5 is a schematic illustration demonstrating the use of negative acoustic contrast particles.
  • FIG. 6 is a schematic illustration of multiple sample streams according to an embodiment of the present disclosure.
  • Fig. 7 is a schematic illustration of multiple sample streams according to another embodiment of the present disclosure.
  • FIG. 8 is a schematic illustration of multiple sample streams according to a further embodiment of the present disclosure.
  • Fig. 9 is an end-on view of a channel employing either matched acoustic waves or two transducers in order to achieve vertical particle positioning.
  • FIG. 10 is a schematic illustration showing the use of shallow channels as a means of vertical particle position control.
  • Fig. 11 is an illustration of how data might be presented in an exemplary embodiment of the present disclosure.
  • the present disclosure provides an affordable flow cytometry system with a significantly increased analytical rate, volumetric sample delivery and usable particle size compared to previously described flow cytometry systems.
  • the system described herein is able to analyze cells or particles ranging from 1 to 1000 ⁇ in diameter, at flow rates of up to 50 mL/min, and at rates of up to 1 x 10 6 particles/second.
  • the presently described system is able to generate up to 300 focused parallel streams of particles using both acoustically resonant microfabricated channels and multi-node acoustic standing waves. These flow cells focus particles up to 200 ⁇ in diameter at volumetric delivery rates as high as 25 mL/min.
  • light from a laser or other suitable source is shaped, directed, and or focused into a line that extends across an entire array of individually interrogable sample streams so as to provide excitation across the streams.
  • Light emitted from particles excited by the light while traveling in the sample streams is then collected and imaged onto one or more array detectors.
  • the image of the particles being interrogated by the line of light maps onto individual or small numbers of elements in the array detector. This enables elements of the array detector to be correlated with specific sample streams.
  • This further enables the collection of particle by particle optical properties, while recording which sample stream a particle was located in.
  • the continuous collection of signal from elements in the array detector that correlate to spaces between sample streams will offer opportunities to monitor background signal for use in signal processing algorithms.
  • the presently described system can be designed to include sample streams that can easily accommodate larger particles and increased flow rates.
  • the combination of simultaneous interrogation and increased sample stream capacity results in a powerful tool for the field of particle analysis.
  • system 10 includes a laser 12, beam shaper 14, an optical objective 16, a sample stream 18, beamsplitters 20 and one or more array detectors 22.
  • laser 12 is configured to produce a standard circular or elliptically-shaped laser beam.
  • beam shaper 14 may take the form, for example, of a Powell lens (commercially available, for example, from Laser Line Optics Canada, Inc, Ontario, CA), which shapes the light into a line.
  • optical objective 16 which focuses the line of light across the entire width of the sample stream array 18, so as to simultaneously interrogate each sample stream.
  • optical objective 16 also collects light from the sample and images the collected light onto array detector(s) 22. As shown, the collected light may be directed through one or more beamsplitters 20 before reaching the array detector(s).
  • Laser 12 may be a laser of any type. Alternatively, a laser diode bar may be used. Additionally, any intense light source such as an light emitting diode or an arc lamp may also be used.
  • the array detector(s) 22 may be any array of detectors, including multianode PMTs, emCCDs, CMOS, CCD, or other such optical detectors.
  • optical objective 16 may take the form of an infinity corrected lOx objective having an NA 0.45. Objectives fitting these specifications are comparable to that used in commercial cytometers and have been effectively shown to support creation of a 10 ⁇ wide, 1.6 mm long laser line. See e.g., Sinclair et al (2004) Design, construction, characterization, and application of a hyperspectral microarray scanner. Applied Optics 42, 2079-2088. For geometrically longer laser lines and wider fields of view, aspheric lenses such as the AL7560-A from ThorLabs, Inc (Newton, NJ, USA), having 75 mm diameter, 0.62 NA and a focal length of 60 mm might be used.
  • the infinity corrected light path, large diameter, and high NA of this objective make it possible to produce laser lines tens of mm in length when paired with the appropriate Powell lenses. Furthermore, its high NA ensures sensitive optical collection over a wide field of view. Though these lenses give workable examples, any objective may be used here, including single element lenses (such as aspheric lenses), multi element lenses, and objectives with varying specifications.
  • FIG. 2 A second embodiment is shown in Fig. 2.
  • simultaneous interrogation of the streams of sample stream 16 is achieved by the use of a Bessel beam 24.
  • Bessel beams are non- diffracting and self-healing laser beams that are made by focusing light through a conical lens known as an axicon.
  • Bessel beam 24 is positioned so as to interrogate the sample stream orthogonally across the width of the flow channel, such that the self-healing properties of the beam ensure that intervening particles minimally affect the excitation of adjoining streams.
  • FIG. 3 A third embodiment is shown in Fig. 3.
  • telescopes 26 are used to adjust the size of the incoming or outgoing light path.
  • masks 90 may be placed in the image plane or slits 92 made in the focal plane of the array detectors 22 and the configuration/angle of optical objective 16 and sample stream array 18 can be altered according to various design requirements or desires.
  • the system described herein includes a sample stream array.
  • the array is able to separate a sample into a large number, even hundreds, of streams.
  • These streams could be created by numerous approaches including: 1. the use of microfluidic channels that employ hydrodynamic focusing to position particles. 2. Dielectrophoretic focusing that uses dielectrophoresis to position particles. 3. Acoustic focusing that uses standing ultrasonic waves or surface acoustic waves to position particles. 4. Microfluidic channels that use "inertial focusing" or induced flow from microfabricated structures to position particles. Any of these methods could be used to generate a regular array of particle steams flowing past the excitation line.
  • acoustic focusing which uses acoustic standing waves to generate multiple sample streams.
  • the use of acoustic focusing to produce multiple sample streams suitable for use in flow cytometry and other applications is described for example, in co-pending US Patent Application No. 13/103,756 filed July 29, 2011.
  • an acoustic wave generator 30 produces an acoustic wave field 32 across the width of a capillary or flow cell 34.
  • the acoustic wave field may be produced, for example, by a piezoelectric element.
  • a second piezoelectric element 44 may be placed on the opposing side of the first element to pick up feedback signals.
  • an acoustic wave field can be used to produce a large number of streams in a single channel. For example, we have been able to produce up to thirty seven acoustically separate streams in a single channel.
  • a "channel” shall refer to a physical structure that provides division while a "stream” or “flow stream” shall refer to a specific, distinguishable, pathway taken by particles within a moving fluid sample.
  • the system can be multiplexed by producing multiple parallel particle streams via acoustic focusing in multiple channels. Using a combination of channels and acoustically focused streams, it is possible to achieve up to hundreds of streams for interrogation.
  • the number of nodes per channel is defined by equation 1, below, where C m is the speed of sound in the media, N is the number of desired nodes, and W is the width of the channel in the direction that the one-dimensional acoustic wave of frequency v is propagating.
  • C m is the speed of sound in the media
  • N is the number of desired nodes
  • W is the width of the channel in the direction that the one-dimensional acoustic wave of frequency v is propagating.
  • sample streams described herein could be used in conjunction with a high throughput sampling system such as that described in Edwards et al., (2001) HTPS flow-cytometry: a novel platform for automated high throughput drug discovery and characterization. Journal of biomolecular screening 6, 83-90.
  • This system can process a 384- well plate in less than 10 min when connected to a single flow cytometer (See e.g., Edwards et al., (2009) High-content screening: flow cytometry analysis. Methods Mol Biol 486, 151-165 and Edwards et al., Cluster cytometry for high-capacity bioanalysis. Cytometry, Part A : the journal of the international Society for Analytical Cytology 81, 419-429).
  • a 4-probe configuration will enable a 4-fold reduction in single cytometer plate sampling time in combination with an 4-fold increase in sample volume analyzed, which is an important feature for samples in which cells targeted for analysis represent a small fraction of total cells or in which cell concentrations are unavoidably low.
  • acoustic wave field also creates opportunities for the development of assays and experiments that can take advantage of the properties provided by the presence of an acoustic field.
  • positive and/or negative acoustic contrast particles can be used for sorting, isolation, and purification applications.
  • Examples of acoustic contrast particles suitable for use with the systems described herein are disclosed in co-pending U.S. Patent Application No. 13/320,476, which is a U.S. National Stage application of PCT/US 10/34415, filed May 11, 2010.
  • Fig. 5 is a schematic illustration demonstrating the ways in which monodisperse negative acoustic contrast particles may be used as part of a powerful bioanalytical methodology.
  • biospecific negative acoustic contrast particles 120 that have been previously mixed with a biological sample 122 can be separated from other (positive acoustic contrast) particles 124 in the sample by using acoustic pressure radiation fields (e.g. acoustic standing, cylindrical, or spherical wave fields).
  • the acoustic pressure radiation field e.g. acoustic dipole excitation
  • channels can be used to produce the desired number of flow streams, including hydrodynamic focusing channels used for standard flow cytometry.
  • suitable channels include, channels formed from optically thin wall rectangular glass capillaries, channels formed by etching channels in a silicon chip, machined flow cells, and combinations thereof.
  • the system of the present disclosure includes channels formed from thick wall optically clear rectangular capillaries coupled with piezos that are designed to accommodate the specific wall dimensions.
  • the approach has excellent optical accessibility, but in some instances, for example when the channel is used in conjunction with acoustic focusing, thick wall capillaries may not effectively transmit acoustic energy. In this case it may be desirable to use PZT drives with matched impedance to the system.
  • Fig. 6 is a schematic illustration of another specific embodiment in which channels are etched into a glass substrate.
  • the etched glass 50 including multiple channels 52, is covered with a glass top 54.
  • an optional acoustic transducer 56 may be placed below the etched glass.
  • This embodiment is suitable for use in a system such as that shown in Fig. 1, wherein a laser line 58, is directed onto the array.
  • Fig. 7 is a schematic illustration of yet another specific embodiment in which channels 61 are etched all the way through a silicon layer 60.
  • the channels include both a glass top 62 and a glass bottom 64.
  • an optional acoustic transducer 65 may be placed below the etched glass.
  • This embodiment is also suitable for use in a system such as that shown in Fig. 1, wherein a laser line 66, is directed onto the array.
  • Fig. 8 is a schematic illustration of still another specific embodiment.
  • a machined flow cell 70 includes glass sides 72 at the top and bottom which enable epiflourescence excitation and detection suitable for use for the system such as that shown in Fig. 1.
  • machined flow cell 70 may further include a laser window 74, which enables the use of this channel when a Bessel beam 76 (as shown in the embodiment depicted in Fig. 2) is used as the means of particle excitation.
  • the channel sidewalls may further include wide PZT mounting regions that taper to the channel width to focus the acoustic energy into the aqueous media.
  • Fig. 9 is an end-on view of a channel employing either matched acoustic waves or two transducers in order to achieve vertical particle positioning.
  • the flow of the channel is out of the page.
  • matched acoustic waves 80 form a series of vertically constrained nodes.
  • two transducers can be used to create a single vertical node 84. These methods of vertical constraint may be particularly desirable when deep channels are used.
  • shallow channels may be used as a means of vertical position control, as particles are constrained top to bottom by the ceiling and floor of the channel.
  • the inertial lift forces induced by the flow profile in the vertical direction are expected to drive even particle spacing and flow induced particle spacing at high particle concentrations. This spacing increases the maximum analysis rate by eliminating particle coincidences in the analysis area.
  • the system of the present disclosure is able to analyze particles that are less than 70 ⁇ in diameter (the typical maximum diameter for particles analyzed with traditional flow cytometry systems), the system of the present disclosure is also able to easily analyze particles having a diameter of up to 1000 ⁇ , including particles having diameters between 1 and 1000 ⁇ ,
  • the present system can accommodate flow rates of less than 250 ⁇ 7 ⁇ (as are found in traditional flow cytometry systems), the present system can accommodate flow rates of up to 50 mL/min including flow rates of between 0 and 50 mL/min, 250 ⁇ / ⁇ and 25 mL/min, 1 ml and 10 mL/min and any other range in between.
  • the present system can accommodate rates of up to 1 x 10 6 particles/second, including rates between 1 particle/second and 1 x 10 6 particles/s, lxlO 4 and 1 x 10 6 particles/s, lx 10 5 and 1 x 10 6 particles/s and any other range in between.
  • a high throughput flow cell might have 8- etched channels that are 148 ⁇ in width by 40 ⁇ in depth. When driven at 10 MHz each channel will support two acoustically focused flow streams. Using 46 ⁇ walls between the channels will result in 8 channels providing 16 total flow streams over a 1.6 mm total width.
  • the MHz drive frequency will be highly effective for focusing, as the radiation force of the acoustic standing wave is proportional to the drive frequency. Furthermore, >1 MHz acoustic standing waves do not lead to cavitation or cell damage.
  • the 40 ⁇ channel depth will allow for vertical positioning and inertial spacing of cells, to increase analysis precision and rates.
  • Channel number and dimension can vary for applications requiring high volumetric throughput but lower analysis rates or for large particle applications. In these cases, it may be desirable to use multiple large channels. For example, three 500 x 500 ⁇ square channels that are spaced by 50 ⁇ and driven at 1.48 MHz will generate a single node centered both vertically and horizontally in each channel.
  • a flow cell might be designed to include 40 ⁇ deep channels that are 1.6 mm wide and driven at 7.4 MHz, which will result in 16 streams across a 1.6 mm channel. Notably, these channels will be effectively usable at high linear velocities (-10 m/s) as the shallow 40 ⁇ height will limit turbulent flow.
  • a flow cell could be designed to include channels that are 2000 ⁇ wide and 400 ⁇ deep. When driven at 1.48 MHz these channels will generate four flow streams, where the first and fourth stream are 250 ⁇ from their nearest sidewalls and each stream is spaced from each other stream by 500 ⁇ . Such a system could use particles up to roughly 350 ⁇ in diameter and would be effective for large particle and high volume applications.
  • optical collection is performed by means of an epifluorescent collection path.
  • telescopes 26 may be used as beam expanders to enable placement of the optimal image size onto the array detector.
  • masks 90 may be placed in the image plane of the array detectors to limit scatter from the flow cells and cross-talk between adjoining streams.
  • Masks with 20 ⁇ resolution may be constructed, for example, from 500 ⁇ thick silicon wafers using deep reactive ion etching.
  • slits 92 may be made in the focal plane of each detector. Still viewing Fig. 3, it can be seen that the sample stream array and/or optical objective can be angled to accommodate various types of collection paths including epifluorescence, SSC (side scatter), and orthogonal fluorescence.
  • an array detector comprising multiple PMTs may be used.
  • One exemplary detector is the Hamamatsu H7260-20 PMT module (Hamamatsu Phototronics K.K., Hamamatsu City, JP) which has 32 channels, 200 ⁇ spaces between 800 ⁇ wide by 7 mm high sensors, a 200-020 nm spectral range, ns rise times, and a 10 6 fold gain. These properties essentially provide 32 closely spaced PMTs with the performance characteristics that have made the PMT the dominant detector in flow cytometry. The presently described system images the flow stream onto these PMTs such that each flow stream can be correlated to an individual channel of the PMT.
  • the array detector may be or incorporate an emCCD, CCD, or CMOS camera.
  • stream locations may be correlated to specific pixel locations in the cameras.
  • An example emCCD is the Luca S (Andor Technology, PLC Harbor, Northern Ireland), which has a frame rate of 217 frames per second at 256 by 256 resolution.
  • This offers two advantages: first, the higher resolution will enable the measurement of up to 100 flow streams; and second, the camera obviates the need for an acquisition system by directly streaming images that can be processed offline to quantify optical events.
  • Fig. 11 provides a demonstration of the type of data that would be produced by such a system.
  • emCCD, CCD, or CMOS cameras with increased sampling rates (>32,000 frames/second) could be used to increase analytical rates.
  • An exemplary experiment that could be performed using the system described herein is the detection of rare events in blood by simply diluting the blood sample and flowing the diluted sample through the system.
  • the system In order to detect rare events in blood, like, for example, circulating tumor cells, the system should be able to detect 100 target cells per cm 3 of blood that contains 5x109 cells.
  • the gap between the trailing edge of one particle exiting the beam, and the leading edge of a successive particle entering the beam will be -8 ⁇ 8.
  • These models demonstrate that our platform can detect as few as 100 cells/mL of blood using only dilution followed by analysis. This task would take days using a conventional flow cytometer. Instruments with 32 to 100 streams will enable either more forgiving flow rates or even higher analysis rates.

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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)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un système économique de cytométrie en flux caractérisé par une nette augmentation de la cadence d'analyses, de l'amenée d'échantillons volumétriques et de la taille de particule utilisable, et comprenant un faisceau lumineux qui interroge des flux d'écoulement multiples de façon à assurer une excitation sur l'ensemble des flux, ainsi qu'un objectif optique configuré pour recueillir de la lumière provenant des flux d'échantillons et capturer une image de la lumière sur un détecteur en mosaïque.
EP13807610.4A 2012-06-21 2013-03-15 Collecte de lumière spatialement corrélée à partir de flux d'échantillons multiples excités à l'aide d'une source lumineuse concentrée sur une ligne Ceased EP2864762A4 (fr)

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US201261662541P 2012-06-21 2012-06-21
PCT/US2013/032025 WO2013191772A1 (fr) 2012-06-21 2013-03-15 Collecte de lumière spatialement corrélée à partir de flux d'échantillons multiples excités à l'aide d'une source lumineuse concentrée sur une ligne

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EP2864762A1 true EP2864762A1 (fr) 2015-04-29
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JP6110484B2 (ja) 2017-04-05
JP2015524070A (ja) 2015-08-20
WO2013191772A1 (fr) 2013-12-27

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