WO1992008120A1 - Pulsed laser flow cytometry - Google Patents

Pulsed laser flow cytometry Download PDF

Info

Publication number
WO1992008120A1
WO1992008120A1 PCT/AU1991/000498 AU9100498W WO9208120A1 WO 1992008120 A1 WO1992008120 A1 WO 1992008120A1 AU 9100498 W AU9100498 W AU 9100498W WO 9208120 A1 WO9208120 A1 WO 9208120A1
Authority
WO
WIPO (PCT)
Prior art keywords
interaction region
beam
flow
cell
light
Prior art date
Application number
PCT/AU1991/000498
Other languages
French (fr)
Inventor
James Austin Piper
Joseph Alan Narai
Donald James Ramsay
Original Assignee
Macquarie University
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 to AUPK3093 priority Critical
Priority to AUPK309390 priority
Application filed by Macquarie University filed Critical Macquarie University
Publication of WO1992008120A1 publication Critical patent/WO1992008120A1/en

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Electro-optical investigation, e.g. flow cytometers
    • G01N15/1429Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its signal processing
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Electro-optical investigation, e.g. flow cytometers
    • G01N15/1425Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its control arrangement
    • G01N15/1427Electro-optical investigation, e.g. flow cytometers 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Electro-optical investigation, e.g. flow cytometers
    • G01N15/1456Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459Electro-optical investigation, e.g. flow cytometers 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N2021/1789Time resolved
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00Features of devices classified in G01N21/00
    • G01N2201/06Illumination; Optics
    • G01N2201/069Supply of sources
    • G01N2201/0696Pulsed
    • G01N2201/0698Using reference pulsed source
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00Features of devices classified in G01N21/00
    • G01N2201/08Optical fibres; light guides
    • G01N2201/0893Using fibres for resolution in time

Abstract

Flow cytometry comprising the steps of: producing a flow of cells of velocity (v), in single file, through an interaction region (12) of length (d), where (d) is defined by the height of a pulsed laser source (11) of height (d), which beam shines onto the flow of cells which beam defines the interaction region; pulsing the laser source at a repetition rate of (f) onto the flow of cells where the time between pulses (l/f) is approximately equal to the transit time (t) of a cell in the interaction region; collecting (15) and detecting (16) the light scattered by or fluoresced from a cell in the interaction region; analysing (17) the detected light to determine the presence or other characteristics of said cell.

Description

PULSED LASER FLOH CYTOMETRY TECHNICAL FIELD

The invention pertains to flow cytometry and more particularly to a pulsed laser light source in a flow cytometer. BACKGROUND ART

Flow cytometry is a technique for rapid measurement of biological and physical properties of cells and particles. It involves analysis of directly scattered or Stokes-shifted light (fluorescence) from cells in a fast-flowing fluid stream, illuminated by a strong light source (usually a laser). Information concerning physical properties of the cells including, shape and size may be derived from the directly scattered light. Cells may be labelled with fluorescent probes to determine biological properties such as, DNA, RNA and protein content. A variety of properties may be studied simultaneously using multiple wavelength excitation. The technology associated with flow cytometry has arisen over the last twenty years and recently many new applications have evolved including AIDS, Hepatitis B and Cancer detection. A flow cytometer is now a standard feature of many hospitals, research and clinical laboratories. A flow cytometer consists of several components, each of which is described below. 1. A flow system which causes particles 1n a fluid to be hydrodynamically focused and transported single file through an analysis region, where they are irradiated with an intense light beam. The resulting scattered and/or fluorescent light gives details about particle characteristics. 2. A light source and focusing system providing an intense light beam (usually a laser) focused to the analysis region within the fluid stream, so that each particle is irradiated as it passes through the beam.

3. A detection system to capture, the light either scattered or fluoresced from each particle, then to generate a corresponding electrical signal.

4. An analysis system to process the electrical signals received and determine the desired information about the particles characteristics.

All current laser flow cytometers, including systems available commercially from a number of international suppliers, employ continuous-wave lasers as the excitation source. Most commonly these are continuous wave argon or krypton 1on lasers operating in the green, red or sometimes ultraviolet, or low power helium-cadmium lasers operating in the violet or ultraviolet. The main problems associated with these laser sources are the high initial purchase and installation costs and high operating costs arising from the large power and cooling water services required. Another problem is the relatively short laser tube lifetime especially for ultraviolet operation of the argon and krypton ion lasers. Even small scale air-cooled argon ion lasers (used in small bench-top flow cytometers) are expensive and have large power supply requirements and a short operating life. Multiple wavelength excitation requires two or more ion lasers and is extremely problematic in terms of system reliability, cost and alignment and there 1s little practical prospect of extending the wavelength capabilities of these devices.

A second disadvantage of present technology is the difficulty in alignment. The laser tube must be aligned with the focusing optics and an slight movement in any of the components can cause large variations in the data obtained. For multiple lasers, this difficulty in alignment is magnified by the number of lasers used. For information regarding cytometry, the reader 1s referred to Practical Flow Cytometry by H.M. Shapiro (1985), Flow Cvtometrv: Instrumentation and Data Analysis by Marvin A. Van Dllla ^t.sl. (eds.) (1985), and Cvtometrv The Journal of the International Society for Analytical Cytology, which works are incorporate by reference herein.

DISCLOSURE OF THE INVENTION

It is an object of the invention to substantially ameliorate some of the disadvantages of the prior art.

Accordingly, a flow cytometer having a flow system is provided characterized by a pulsed laser light source, beam processing optics, collection optics and a detection device, the beam processing optics adapted to deliver a beam whose size and profile are tailored to a transit time of a cell sample in the flow system and the pulse rate of the laser. In addition a method of cytometry is disclosed, comprising the steps of producing a flow of cells, In single file, of velocity (v) through a region of length (d); shining a pulsed laser beam of pulse repetition frequency (f) and diameter (d) onto the region; collecting the scattered or fluoresced light emitted by the cells in the region; and analysing the collected light with a pulse height analyser. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of pulsed laser flow cytometry according to the present invention. Figure 2 is a schematic diagram illustrating one embodiment of the present invention including fibre optic delivery of the laser beam.

Figure 3 is a schematic diagram Illustrating lens beam processing according to the present Invention. Figure 4 illustrates prism/diode array detection which is utiUzable with the teachings of the present invention.

Figure 5 is a schematic diagram Illustrating grating/diode array detection.

Figure 6 is a schematic diagram illustrating temporally multiplexed laser flow cytometry according to the present invention.

Figure 7 is a schematic diagram illustrating another embodiment of temporally multiplexed laser flow cytometry according to the present invention.

BEST MODE AND OTHER EMBODIMENTS OF THE INVENTION Progress in the development of practical high repetition rate pulsed lasers have allowed the inventors to consider the applications of these sources to laser flow cytometry. Immediately available are high pulse rate (5-20kHz) copper vapour lasers generating in the green, yellow and, by various wavelength conversion techniques, the ultraviolet and red. Even more importantly, all-solid-state lasers using semiconductor diode lasers to pump crystalline solid state (eg neodymlum: YAG) lasers whose output may be frequency-doubled to generate high repetition rate pulsed output in the visible are now becoming available 1n small Integrated packages. These latter devices are expected to Increasingly dominate in laser applications requiring long-term reliability 1n the low-power regime; their compact size and projected low cost are especially appropriate to applications requiring multiple sources as in flow cytometry. On a more fundamental level, high repetition rate pulsed lasers have particular advantages in application as flow cytometry sources including high instantaneous Illumination intensities (giving high signal levels), relaxed beam focussing requirements, good beam uniformity and especially wavelength versatility which can be effectively utilised by temporal multiplexing techniques.

Application of pulsed lasers to flow cytometry requires a significant change in the approach to the basic physical problem, however. Since the optical pulse duration of appropriate pulsed lasers (eg. copper laser or diode-pumped solId-state lasers) is typically only a few tens of nanoseconds and the duty cycle even for high pulse rate lasers is only 1 in

4

10 , a major difficulty apparently arises in ensuring each cell is illuminated as 1t passes through the interaction region. This difficulty has previously been taken to rule out the use of pulsed lasers for flow cytometry since sophisticated cell position-detecting electronics appeared to be necessary. However, the inventors have demonstrated that pulsed lasers operating at high pulse rates (~ 10 kHz) can be quite simply

4 matched to the droplet flow rates (also ~ 10 per second) usually employed, given appropriate adjustments in focal spot size of the illuminating beam at the interaction region. In essence, only approximate synchronisation of the laser pulse rate and droplet flow rate is necessary if an extended focal spot size is used so that the transit-time across the interaction region 1s of the order of the interpulse period. The uncertainty in the position of the cell when illuminated does not lead to significant problems with scattered light detection. In general for a cel or particle of velocity v moving through an interaction region of length d (equal to the beam diameter, also d) the transit time t equals d/v. The time between pulses 1/f (where f 1s the laser repetition rate) is preferably equal to the transit time t. Therefore the beam diameter d is preferably equal to vt, the cell velocity v multiplied by the time between pulses. As shown 1n Figure 1, a pulsed laser flow cytometer 10 utilises a pulsed laser source 11 to illuminate cells or particles in an interaction region 12. The cells or particles are delivered to the Interaction region 12 by a flow nozzle 13. Any conventional flow nozzle may be utilized, for example a standard flow nozzle in which the sample stream 1s surrounded by a buffer sheath before ejection from the nozzle, which flow nozzle utilise a simple hypodermic pump system. Beam processing optics 14 are interposed between the pulsed laser source 11 and the interaction region 12. The bea processing optics may be based on lenses but is preferably a fibre optic system. Both lens based and fibre optic beam processing optics will be explained in further detail. The light emerging from the interaction region 12 is collected by collection optics 15. The collection optics may be in the form of a 50 mm diameter, 50 mm focal length lens with a horizontal beam stop 5 mm high, across the collection lens to stop the incident beam from being collected. The light collected by this lens is therefore generated by the particles or cells flowing in the stream carrie by the flow nozzle 13. The collection optics 15 images the collected ligh onto a detection device 16 such as a photomultiplier tube where an electrical signal is generated for each incident pulse. The electrical signals generated by the detection device 16 are processed to generate useful information, such as a standard pulse height analyser package in an IBM™ compatible computer 17. It will be understood that the combination of linear array detectors and digital electronics can be used to provide data from which various characteristics of the cell flow can be ascertained. Cell presence, cell position, cell size, cell shape and nuclear volume for example, can all be ascertained from the scattered or fluoresced light. Information regarding the application of array detectors is available from manufacturers such as Spirlcon, B1g Sky and Exltech. The results obtained may be plotted on a graph showing the pulse height (or intensity of the light pulse) along the horizontal axis and the number of counts (or number or pulses at the corresponding intensity). The system depicted in Figure 1 may also be utilized to collect light at angles other than directly on axis with the incident beam. Typically, a standard flow cytometer will have collection at 0 and 90° and multiple fluorescence collection at 90°. Other collection angles may be used along or in combination.

Figure 2 depicts a pulsed laser flow cytometry system in more detail. In this instance, a copper vapour laser 20 generates 30 nanosecond pulses at a repetition rate of 10kHz. A fibre coupler 21 1s used to couple the beam into a 100 micrometer optical fibre 22. The light emerging from the fibre 22 1s focused onto a 1 mm plnhole 23 using a graded Index lens 24. The plnhole removes stray or unwanted light emerging from the fibre. The light emerging from the plnhole 23 is collected by a 25 mm diameter, 60 mm focal length achromatic lens 25 and imaged onto the flow stream 26 in the Interaction region 27. As previously mentioned, the light emerging from the interaction region 27 is collected via a 50 mm diameter, 50 mm focal length lens 28 with a horizontal beam stop across the collection lens to stop the Incident beam from being collected. The collection lens 28 images the light onto a photomultipHer 29 where an electrical signal is generated for each Incident pulse, which signals are processed by a standard pulse height analyser package in a computer 30. It should be understood that diode pumped lasers such as a NdYAG laser would be considered a suitable alternative to the copper vapour laser 20. It is also worth noting that the photo ultiplier tube 29 may be replaced with photo diodes or linear photodlode arrays, which alternatives will be further described. The optical fibre 22 disclosed with reference to Figure 2, transfers the light from the laser to the interaction region 27 and gives a uniform beam profile. For multimode fibre, the emerging profile is "top hat" and is ideal for pulsed and continuous wave flow cytometry. For single mode fibre, the emerging profile is gaussian which is suitable for continuous wave cytometry only. In the alternative to the fibre optic processing means 22 disclosed in Figure 2, a lens beam system is also a viable alternative. Such a system is disclosed in Figure 3. In a lens beam system, a condensing lens 31 focuses the light from the laser source 20 onto a mask or pinhole 32 of a suitable diameter. The mask prevents unwanted or stray light from reaching the interaction region and gives a uniform "top hat" profile. An imaging lens 33 focuses the emerging light onto the sheath in the interaction region 27.

At least two distinct detection systems may be used to gain spectral information from pulsed laser flow cytometry. A prism/diode array detection system is depicted in Figure 4. A grating/diode array detection system is depicted in Figure 5. With reference to the prism/diode array detection system depicted in Figure 4, light from the interaction region is focused by a collection lens 40 onto a linear array detector 41 via a prism 42. The prism 42 will disperse the light. This means that different wavelengths will be focused onto different elements of the linear array 41. For each laser pulse, the array will be read out yielding information regarding the spectral (wavelength) content of the collected light. With reference to the grating/diode array detection system depicted in Figure 5, light from the Interaction region 27 1s focused by the collection lens 50 onto one or more array detectors 51 by a diffraction grating 52. The defraction grating 52 will reflect light in different directions depending upon the wavelength and the Incident angle. The light reflected from the zeroth order (the central) reflection will be directed to the same position independent of the wavelength and a single element detector can be used to collect the light. The light reflected from the first order (first reflection either side of the center) reflection will be directed to differing positions depending on the wavelength. Thus, linear arrays can be positioned here and operated as for the prism example. The adaptation of pulsed lasers and fibre optic light delivery to flow cytometry also makes possible temporal multiplexing as schematically illustrated In Figures 6 and 7. As shown in Figure 6, a pulsed laser flow cytometer may incorporate separate pulsed lasers 60, one for each wave length desired. They are synchronised by an external trigger 61, delaying each source by an appropriate time so that each wavelength will arrive at the interaction region 27 sequentially. Separate fibres 62 for each pulsed laser source 6 take each lasers output and couple the various outputs Into a single optical delivery system 63. The delivery optics 68 take the emerging ligh and focus it into the interaction region 27. The collection optics 64 deliver the emitted light to a gated detection device 65. The gated detection device 65 also receives a signal from the trigger box 61, which synchronisation signal 66 is used to Initialise the gated detection device and syncronise its readout.

As shown in Figure 7, a single pulsed multi wavelength laser source 70 such as a copper vapour laser 1s utilised. The copper vapour laser can emit at 511 and 578 nm. The emitted light passes through two or more dichroic beams splitters 71 each of which reflect only a particular wavelength. The light is sent through couplers 72 Into separate optical fibre delay lines 73. Each of the optical fibre delay lines is preferably of a different length so that the optical signal reaches the interaction region 27 at different times. The differing length delays each of the pulses by 3.3 nanoseconds from every metre of fibre length. The collection optics 64 and gated detection device arrangement 65 are substantially similar to the arrangement disclosed with reference to Figure 6.

The advantage of temporally multiplexing the lasers rather than physically separating them is that only a single detection and Illumination system is required. With existing multiple wavelength systems, each of the laser sources, focusing optics and detection optics must be aligned individually. This can take several hours even for an experienced operator. This individual alignment is not required in a temporally multiplexed system. Only one optical system is required for illumination and one for detection.

As a further aspect of the invention, there may be optionally provided, an array detector to detect beam intensity variations (temporal and spacial) and an array detector to detect light emerging from the analysis region or interaction region 27. The signal from the array detector compensates the beam profile detector signal, producing a signal independent of the illuminating beam profile. In this way, beam intensity variations, both spatially (across the analysis region) and temporally (jitter from the laser light source), can be simply compensated for in real time. This reduces the need for exact alignment and high quality beam processing optics. For each of the abovementioned systems, the preferred source beam is of low divergence and "top hat" profile. Both of these factors contribute to the resolution of the system. Linear array detectors can be used to correct for irregularities 1n the beam profile. It will be understood tha an ideal "top hat" profile is never exactly achieved. The actual beam profile however can be measured using the linear array detector. This measurement can be compared with, subtracted from or otherwise used to offset the scattered or fluoresced readings so as to yield a compensated o corrected measurement. The linear array can also be used to yield a one dimensional image of the cell as It passes through the Interaction region or, to give an indication of cell position for cell sorting later in the cell flow stream. A two dimensional linear array can be used to yield a two dimensional image of the cell.

While the Invention has been described with reference to particular components, instruments and details of construction these should be understood as having been provided by way of example and not as limitation to the scope or spirit of the Invention. INDUSTRIAL APPLICABILITY

The device of the present invention 1s Ideally suited to cell counting, cell sorting and cell identification 1n hospitals, research facilities and clinical laboratories.

Claims

CLAIMS :
1. A method of flow cytometry comprising the steps of: producing a flow of cells of velocity (v), in single file, through an interaction region of length (d), where (d) 1s defined by the height of a pulsed laser source of height (d), which beam shines onto the flow of cells and which beam defines the interaction region; pulsing the laser source at a repetition rate of (f) onto the flow of cells where the time between pulses (1/f) is approxiamtely equal to the transit time (t) of a cell in the interaction region; collecting and detecting the light scattered by or fluoresced from a cell in the interaction region; and analysing the detected light to determine the presence or other characteristics of said cell.
2. The method of claim 1, further comprising the step of: adjusting the source height (d) to be approximately equal to the cell velocity (v) multiplied by the time between pulses (1/f).
3. The method of either of claims 1 or 2 wherein: the source has a top hat profile.
4. The method of any of claims 1 to 3 further comprising the step of: compensating for a source profile by using one or more array detectors which detect the unscattered beam profile and scattered or fluoresced light signal after which a detection signal may be corrected according to the beam profile.
5. The method of claim 4, further comprising the step of: using one or more linear array detectors to generate a signal indicative of cell position in the interaction region.
6. The method of claim 4, further comprising the step of: using the one or more array detectors are used to provide Information as to the cross-sectional intensity of a cell in the interaction region.
7. The method of any of claims 1 to 6 wherein prior to entering the interaction region, the laser source comprises two or more combined, pulsed laser beams synchronised to pass through the region at different times; and further comprising the steps of: using a gated detection device to detect the scattered or fluoresced light; and synchronising the two or more pulsed beams, using a trigger with the gated detection device.
8. The method of claim 7, wherein: the two or more laser beams are of different wavelength.
9. The method of either of claims 7 or 8, wherein: the two or more laser beams are triggered simultaneously and fed into separate fibre optic delay and delivery optics.
10. The method of any one of claims 1 to 6 wherein: the laser sourc comprises a multi-wavelength laser whose beam is split using a beam splitter into separate beams of individual wavelengths, then introduced into fibres or delay lines, then recombined before shining onto the interaction region; whereby the scattered or fluoresced light is detected with a one or more gated devices which are synchronised with the multi wavelength lasers.
11. The method of any of claims 1 to 10 wherein: collecting is accomplished by collecting optics which further comprise a diffraction grating or prism to separate collected wavelengths and dispense same onto a one or more gated array detectors.
12. An apparatus for flow cytometry comprising: one or more pulsed laser light sources, the beam or combined beams of which are fed through beam processing optics, which optics lead the pulses into a cell flow system comprising an interaction region; collection optics for gathering scattered or fluoresced light; one or more detection devices; and a signal processor for analysing the signals produced by the detection device or devices.
13. The device of claim 12, where: the length of the Interaction region is approximately equal to the velocity of cells in the flow system times the time between pulses and where the interaction region is defined by the height of the laser pulses which impinge on the cell flow.
14. The apparatus of either of claims 12 or 13 wherein: a single multi-wavelength laser source is used and further comprising one or more beam splitters and an equal number of coupled fibre optic delay lines which are recombined prior to delivery into the interaction region; whereby two or more detection devices are gated to be synchronised with the delayed beams emerging from the delay lines.
15. The apparatus of claim 12, wherein: two or more pulsed laser sources, each with its own fibre delay line are recombined prior to reaching the interaction region; and - li ¬ the detection devices are gated and synchronised by a trigger device which also operates the two or more pulsed laser sources.
16. The apparatus of claim 15, wherein: the two or more sources are of different wavelength.
17. The apparatus of any one of claims 12 to 16 where the collection optics further comprises a prism or diffraction grating for frequency separating the gathered light and dispensing same to two or more gated linear arrays.
18. A flow cytometer substantially as hereinbefore described with reference to the drawing figures.
PCT/AU1991/000498 1990-10-29 1991-10-29 Pulsed laser flow cytometry WO1992008120A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AUPK3093 1990-10-29
AUPK309390 1990-10-29

Publications (1)

Publication Number Publication Date
WO1992008120A1 true WO1992008120A1 (en) 1992-05-14

Family

ID=3775041

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1991/000498 WO1992008120A1 (en) 1990-10-29 1991-10-29 Pulsed laser flow cytometry

Country Status (1)

Country Link
WO (1) WO1992008120A1 (en)

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997030338A1 (en) * 1996-02-16 1997-08-21 Inphocyte, Inc. System and method for rapid analysis of cells using spectral cytometry
WO2006111641A2 (en) * 2005-04-21 2006-10-26 Horiba Abx Sas Device and method for multiparametric analysis of microscopic elements
WO2006131181A3 (en) * 2005-05-06 2007-03-29 Lt Res Gmbh Adaptive signal interpretation for fbrm measurement apparatus
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
US7723116B2 (en) 2003-05-15 2010-05-25 Xy, Inc. Apparatus, methods and processes for sorting particles and for providing sex-sorted animal sperm
US7758811B2 (en) * 2003-03-28 2010-07-20 Inguran, Llc System for analyzing particles using multiple flow cytometry units
US7820425B2 (en) 1999-11-24 2010-10-26 Xy, Llc Method of cryopreserving selected sperm cells
US7833147B2 (en) 2004-07-22 2010-11-16 Inguran, LLC. Process for enriching a population of sperm cells
US7838210B2 (en) 2004-03-29 2010-11-23 Inguran, LLC. Sperm suspensions for sorting into X or Y chromosome-bearing enriched populations
US7855078B2 (en) 2002-08-15 2010-12-21 Xy, Llc High resolution flow cytometer
US7929137B2 (en) 1997-01-31 2011-04-19 Xy, Llc Optical apparatus
US8137967B2 (en) 2000-11-29 2012-03-20 Xy, Llc In-vitro fertilization systems with spermatozoa separated into X-chromosome and Y-chromosome bearing populations
AU2012200706B2 (en) * 2003-03-28 2012-09-20 Inguran, Llc "Digital sampling apparatus and methods for sorting particles"
CN102735656A (en) * 2011-03-31 2012-10-17 索尼公司 Fine particle analyzing apparatus and fine particle analyzing method
US8486618B2 (en) 2002-08-01 2013-07-16 Xy, Llc Heterogeneous inseminate system
US8497063B2 (en) 2002-08-01 2013-07-30 Xy, Llc Sex selected equine embryo production system
EP2053381A3 (en) * 2007-10-26 2014-10-22 Sony Corporation Optical detection method and optical detection apparatus for a fine particle
US9145590B2 (en) 2000-05-09 2015-09-29 Xy, Llc Methods and apparatus for high purity X-chromosome bearing and Y-chromosome bearing populations of spermatozoa
US9365822B2 (en) 1997-12-31 2016-06-14 Xy, Llc System and method for sorting cells

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1182342A1 (en) * 1980-01-29 1985-09-30 Предприятие П/Я А-7629 Method of determination in dispersion media
US4786165A (en) * 1986-07-10 1988-11-22 Toa Medical Electronics Co., Ltd. Flow cytometry and apparatus therefor
US4900933A (en) * 1986-09-08 1990-02-13 C. R. Bard, Inc. Excitation and detection apparatus for remote sensor connected by optical fiber
EP0369654A1 (en) * 1988-11-16 1990-05-23 Kowa Company Ltd. Particle measurement apparatus
JPH03150445A (en) * 1989-11-07 1991-06-26 Canon Inc Particle analyzing device

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1182342A1 (en) * 1980-01-29 1985-09-30 Предприятие П/Я А-7629 Method of determination in dispersion media
US4786165A (en) * 1986-07-10 1988-11-22 Toa Medical Electronics Co., Ltd. Flow cytometry and apparatus therefor
US4900933A (en) * 1986-09-08 1990-02-13 C. R. Bard, Inc. Excitation and detection apparatus for remote sensor connected by optical fiber
EP0369654A1 (en) * 1988-11-16 1990-05-23 Kowa Company Ltd. Particle measurement apparatus
JPH03150445A (en) * 1989-11-07 1991-06-26 Canon Inc Particle analyzing device

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DERWENT ABSTRACT, Accession No. 86-111827/17, Class S03; & SU,A,1 182 342 (ZHULANOV YUV), 30 September 1985 (30.09.85). *
DERWENT ENGLISH LANGUAGE ABSTRACT S03, 91-233388/32; & JP,A,03 150 445 (CANON K.K.), 26 June 1991 (26.06.91). *

Cited By (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997030338A1 (en) * 1996-02-16 1997-08-21 Inphocyte, Inc. System and method for rapid analysis of cells using spectral cytometry
US8553226B2 (en) 1997-01-31 2013-10-08 Xy, Llc Optical apparatus
US7929137B2 (en) 1997-01-31 2011-04-19 Xy, Llc Optical apparatus
US8975035B2 (en) 1997-01-31 2015-03-10 Xy, Llc Method of analyzing cells
US9365822B2 (en) 1997-12-31 2016-06-14 Xy, Llc System and method for sorting cells
US9422523B2 (en) 1997-12-31 2016-08-23 Xy, Llc System and method for sorting cells
US7820425B2 (en) 1999-11-24 2010-10-26 Xy, Llc Method of cryopreserving selected sperm cells
US9145590B2 (en) 2000-05-09 2015-09-29 Xy, Llc Methods and apparatus for high purity X-chromosome bearing and Y-chromosome bearing populations of spermatozoa
US10208345B2 (en) 2000-05-09 2019-02-19 Xy, Llc Method for producing high purity X-chromosome bearing and Y-chromosome bearing populations of spermatozoa
US7771921B2 (en) 2000-11-29 2010-08-10 Xy, Llc Separation systems of frozen-thawed spermatozoa into X-chromosome bearing and Y-chromosome bearing populations
US8652769B2 (en) 2000-11-29 2014-02-18 Xy, Llc Methods for separating frozen-thawed spermatozoa into X-chromosome bearing and Y-chromosome bearing populations
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
US9879221B2 (en) 2000-11-29 2018-01-30 Xy, Llc Method of in-vitro fertilization with spermatozoa separated into X-chromosome and Y-chromosome bearing populations
US8137967B2 (en) 2000-11-29 2012-03-20 Xy, Llc In-vitro fertilization systems with spermatozoa separated into X-chromosome and Y-chromosome bearing populations
US8486618B2 (en) 2002-08-01 2013-07-16 Xy, Llc Heterogeneous inseminate system
US8497063B2 (en) 2002-08-01 2013-07-30 Xy, Llc Sex selected equine embryo production system
US7855078B2 (en) 2002-08-15 2010-12-21 Xy, Llc High resolution flow cytometer
US8691584B2 (en) 2003-03-28 2014-04-08 Inguran, Llc Sperm processing methods
US7943384B2 (en) 2003-03-28 2011-05-17 Inguran Llc Apparatus and methods for sorting particles
EP2306173A3 (en) * 2003-03-28 2011-08-03 Inguran, LLC Apparatus and methods for providing sex-sorted animal sperm
US10100278B2 (en) 2003-03-28 2018-10-16 Inguran, Llc Multi-channel system and methods for sorting particles
US8198092B2 (en) * 2003-03-28 2012-06-12 Inguran, Llc Digital sampling apparatus and methods for sorting particles
US8198093B2 (en) 2003-03-28 2012-06-12 Inguran Llc Methods for sorting particles
US8206988B2 (en) 2003-03-28 2012-06-26 Inguran Llc Method and apparatus for orienting sperm in a fluid stream
US8206987B2 (en) 2003-03-28 2012-06-26 Inguran Llc Photo-damage method for sorting particles
US8241914B2 (en) 2003-03-28 2012-08-14 Inguran Llc EPI-damage apparatus and methods for sorting particles
AU2012200706B2 (en) * 2003-03-28 2012-09-20 Inguran, Llc "Digital sampling apparatus and methods for sorting particles"
US20120244610A1 (en) * 2003-03-28 2012-09-27 Inguran, Llc Photo-damage apparatus for sorting particles
US7758811B2 (en) * 2003-03-28 2010-07-20 Inguran, Llc System for analyzing particles using multiple flow cytometry units
EP2306173B1 (en) 2003-03-28 2017-08-16 Inguran, LLC Apparatus and methods for providing sex-sorted animal sperm
US9377390B2 (en) 2003-03-28 2016-06-28 Inguran, Llc Apparatus, methods and processes for sorting particles and for providing sex-sorted animal sperm
US9040304B2 (en) 2003-03-28 2015-05-26 Inguran, Llc Multi-channel system and methods for sorting particles
US8748183B2 (en) 2003-03-28 2014-06-10 Inguran, Llc Method and apparatus for calibrating a flow cytometer
US8535938B2 (en) * 2003-03-28 2013-09-17 Inguran, Llc Photo-damage apparatus for sorting particles
US7799569B2 (en) 2003-03-28 2010-09-21 Inguran, Llc Process for evaluating staining conditions of cells for sorting
US8709817B2 (en) 2003-03-28 2014-04-29 Inguran, Llc Systems and methods for sorting particles
US8617904B2 (en) 2003-03-28 2013-12-31 Inguran, Llc Sperm cell processing methods
US8623658B2 (en) 2003-03-28 2014-01-07 Inguran, Llc Methods for processing sperm cells
US8623657B2 (en) 2003-03-28 2014-01-07 Inguran, Llc Flow cytometer apparatus and method
US8637318B2 (en) 2003-03-28 2014-01-28 Inguran, Llc Methods for sorting particles
US8709825B2 (en) 2003-03-28 2014-04-29 Inguran, Llc Flow cytometer method and apparatus
US8664006B2 (en) 2003-03-28 2014-03-04 Inguran, Llc Flow cytometer apparatus and method
EP2306173A2 (en) * 2003-03-28 2011-04-06 Inguran, LLC Apparatus and methods for providing sex-sorted animal sperm
US8609422B2 (en) 2003-03-28 2013-12-17 Inguran, Llc Method and apparatus for sorting particles
US20130007903A1 (en) * 2003-05-15 2013-01-03 Xy, Llc Efficient haploid cell sorting flow cytometer systems
US7723116B2 (en) 2003-05-15 2010-05-25 Xy, Inc. Apparatus, methods and processes for sorting particles and for providing sex-sorted animal sperm
US7838210B2 (en) 2004-03-29 2010-11-23 Inguran, LLC. Sperm suspensions for sorting into X or Y chromosome-bearing enriched populations
US7892725B2 (en) 2004-03-29 2011-02-22 Inguran, Llc Process for storing a sperm dispersion
US7833147B2 (en) 2004-07-22 2010-11-16 Inguran, LLC. Process for enriching a population of sperm cells
WO2006111641A2 (en) * 2005-04-21 2006-10-26 Horiba Abx Sas Device and method for multiparametric analysis of microscopic elements
FR2884920A1 (en) * 2005-04-21 2006-10-27 Horiba Abx Sa Sa multiparametric device and method for analyzing microscopic elements
WO2006111641A3 (en) * 2005-04-21 2007-01-04 Horiba Abx Sas Device and method for multiparametric analysis of microscopic elements
US7777869B2 (en) 2005-04-21 2010-08-17 Horiba Abx Sas Device and method for multiparametric analysis of microscopic elements
WO2006131181A3 (en) * 2005-05-06 2007-03-29 Lt Res Gmbh Adaptive signal interpretation for fbrm measurement apparatus
EP2053381A3 (en) * 2007-10-26 2014-10-22 Sony Corporation Optical detection method and optical detection apparatus for a fine particle
JP2012215458A (en) * 2011-03-31 2012-11-08 Sony Corp Fine particle analyzer and fine particle analysis method
CN102735656A (en) * 2011-03-31 2012-10-17 索尼公司 Fine particle analyzing apparatus and fine particle analyzing method

Similar Documents

Publication Publication Date Title
US6256096B1 (en) Flow cytometry apparatus and method
Hernandez et al. Laser-induced fluorescence and fluorescence microscopy for capillary electrophoresis zone detection
US6838680B2 (en) Multiplexed fluorescent detection in microfluidic devices
EP0501005B1 (en) Flow imaging cytometer
US5548395A (en) Particle analyzer
US4728190A (en) Device and method for optically detecting particles in a fluid
US5159412A (en) Optical measurement device with enhanced sensitivity
US6309886B1 (en) High throughput analysis of samples in flowing liquid
US6563583B2 (en) Multipass cavity for illumination and excitation of moving objects
US6654119B1 (en) Scanning spectrophotometer for high throughput fluroescence detection
JP3830167B2 (en) Method and apparatus for determining a given characteristic of target particles in the sample medium
Kamentsky et al. Microscope‐based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data
EP1347285B1 (en) Method and apparatus for measuring fluorescence luminance
US5594544A (en) Flow type particle image analyzing method and apparatus
JP3375203B2 (en) Cell analyzer
US4989977A (en) Flow cytometry apparatus with improved light beam adjustment
EP1942333B1 (en) Optical autofocus for use with microtiter plates
US6800860B2 (en) Optical architectures for microvolume laser-scanning cytometers
EP0421736B1 (en) Method and apparatus for measuring optical properties of biological specimens
US5815262A (en) Apparatus for parallelized two-photon fluorescence correlation spectroscopy (TPA-FCS), and the use thereof for screening active compounds
JP4286447B2 (en) Digital imaging system for assays in well plates, gels and blots
US20090122311A1 (en) Flow Cytometer and Flow Cytometry
CN1846126B (en) Light emitting diode based measurement systems
US20010006416A1 (en) Ribbon flow cytometry apparatus and methods
US6974938B1 (en) Microscope having a stable autofocusing apparatus

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR CA CH DE DK ES FI GB HU JP KP KR LK LU MC MG MN MW NL NO PL RO SD SE SU US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BF BJ CF CG CH CI CM DE DK ES FR GA GB GN GR IT LU ML MR NL SE SN TD TG

NENP Non-entry into the national phase in:

Ref country code: CA

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642