USOO7699767B2
`
`(12) United States Patent
`(10) Patent No.:
`US 7,699,767 B2
`
`Mueth et al.
`(45) Date of Patent:
`Apr. 20, 2010
`
`(54) MULTIPLE LAMINAR FLOW-BASED
`PARTICLE AND CELLULAR SEPARATION
`WITH LASER STEERING
`
`(75)
`
`Inventors: Daniel Mueth, Chigago, IL (US);
`Joseph Plewa, Park Ridge, IL (US);
`Jessica Shireman, Kansas City, MO
`(US); Amy Anderson, Prospect Heights,
`IL (US); Lewis Gruber, Chicago, IL
`(US); Neil Harris Rosenbaum, Chicago,
`IL (US)
`
`(73) Assignee: Arryx, Inc., Chicago, IL (US)
`
`(58) Field of Classification Search ..................... 494/3,
`494/5, 36, 37, 38, 45; 250/251; 210/782,
`210/786, 787, 252, 259, 360.1; 435/2, 7,
`435/173.1, 372, 372.3; 422/72, 82.05, 82.06,
`
`(56)
`
`422/8211, 101, 104; 436/177
`See application file for complete search history.
`References Cited
`U.S. PATENT DOCUMENTS
`
`3549329 A
`
`3/1972 RandOIPh
`(Continued)
`
`EP
`
`FOREIGN PATENT DOCUMENTS
`0679325
`11/1995
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U~S-C~ 1540’) by 0 days
`
`.
`(Continued)
`OTHER PUBLICATIONS
`
`(21) Appl. No.: 12/213,109
`
`(22) Filed.
`
`Jun. 13, 2008
`
`(65)
`
`Prior Publication Data
`US 2009/0032449 A1
`F b 5 2009
`e ‘
`’
`
`Related US Application Data
`(60) Division of application No. 11/543 773 filed on Oct.
`6 2006
`P t N 7 402 131
`If h',
`d'
`.
`.
`f
`s
`.
`,.now a '
`0'
`s
`5
`SW 10
`15 a “71510110
`application N0~ 10/934,597, filed 011 Sep. 3, 2004, HOW
`Pat. No. 7,1 18,676, which is a continuation-in—part of
`application No. 10/867,328, filed on Jun. 13, 20043
`now Pat. No. 7 150 834 which is a continuation-in-
`part of application 1110 510/630 904 filed on Jul 31
`2003
`P t N 7 2'41 988 ’
`’
`'
`’ now a ‘
`0’
`’
`’
`‘
`(60) Provisional application No. 60/399,386, filed on Jul.
`31, 2002, provisional application No. 60/435,541,
`filed on Dec 20 2002
`.
`’
`
`’
`
`.
`
`(51)
`
`Int Cl
`(2006 01)
`B0:13 7/08
`(52) US. Cl.
`........................................... 494/36; 494/45
`
`Stephen P. Smith et a1., Inexpensive Optical Tweezers for Under-
`graduate Laboratories, Am. J. Phys. vol. 67, Jan. 1999.
`
`(Continued)
`
`Primary ExamineriKiet T Nguyen
`(74) Attorney, Agent, or Firmilean C. Edwards, Esq.;
`Akerman Senterfitt
`(57)
`
`ABSTRACT
`
`The invention provides a method, apparatus and system for
`separating bIPOd and other types ofcellular components,.and
`can be combined w1th holographic optical trapping manipu-
`lation or other forms of optical tweezmg. One of the exem-
`plary methods includes providing a first flow having a plural-
`ity ofblood components; providing a second flow; contacting
`the first flow with the second flow to provide a first separation
`region; and differentially sedimenting a first blood cellular
`component of the plurality of blood components into the
`second flow while concurrently maintaining a second blood
`cellular component of the plurality of blood components in
`the first fl0W~ The SGPOHd flQW having the first b100d cellular
`component is then differentially removed from the first flow
`having the second blood cellular component. Holographic
`optical traps may also be utilized in conjunction with the
`various flows to move selected components from one flow to
`another, as part of or in addition to a separation stage.
`29 Claims, 22 Drawing Sheets
`
`INPUT
`SOLUTIDN
`
`BUFFER
`SOLUTION
`
`SELECTED
`
`WASTE
`
`$113110"
`SOLUITIDN
`
`BUFFER TESERVDIIT
`SELECTED SDLUTIIN a
` RESERVOIR
`
`
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`
`3.103919"?
`
`155155
`
`CHANEL
`
`I
`
`SELECTION H.041
`DEPTH
`WASTE FLW
`FEGTON DEPTH
`
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`
`now ”gm
`
`,
`
`man
`15mm
`SOHTBT
`
`WASTE
`now
`ntemu
`
`1m
`
`mm; mm
`
`some name
`some srsten
`
`Purim amen
`
`Exhibit No. 1020
`
`PGR of US. Patent 8,933,395
`
`

`

`US 7,699,767 B2
`
`Page 2
`
`US. PATENT DOCUMENTS
`
`3,960,449 A
`4,325,706 A
`
`6/1976 Carleton et a1.
`4/1982 Gershman et a1.
`
`4,409,106 A
`4,424,132 A
`4,660,971 A
`4,667,830 A
`5,007,732 A
`5,100,627 A
`5,180,065 A
`5,194,909 A
`5,229,297 A
`5,483,469 A
`5,620,857 A
`5,674,743 A
`5,752,606 A
`5,800,690 A
`5,837,115 A
`5,849,178 A
`5,879,625 A
`5,966,457 A
`6,053,856 A
`6,071,422 A
`H1960 H
`
`6,368,871 B1
`6,432,630 Bl
`6,451,264 B1
`6,506,609 B1
`6,524,860 B1
`
`10/1983 Furuta et a1.
`1/1984 Iriguchi
`4/1987 Sage et a1.
`5/1987 Nozaki et 31.
`4/1991 Ohki et 31.
`3/1992 Buican et a1.
`1/1993 Touge et 31.
`3/1993 T cko
`7/1993 SZhnipelsky et a1.
`1/1996 Van den Engh et a1.
`4/1997 Weetallet a1.
`10/1997 Ulmer
`5/1998 Wilson etal.
`9/1998 Chow etal~
`11/1998 Austin et a1.
`12/ 1998 Holm et a1.
`3/1999 Roslaniec et a1.
`10/1999 Lemelson
`4/2000 HlaVinka
`6/2000 HlaVinka et a1.
`6/2001 Conrad et a1.
`
`4/2002 Christel et a1.
`8/2002 BlankenStein
`9/2002 Bhullar etal.
`1/2003 Wada et a1.
`2/2003 Seidel et a1.
`
`6,637,463 B1
`6,727,451 B1
`6,815,664 B2
`6,833,542 B2
`6 838 056 B2
`’
`’
`6,944,324 B2
`7,029,430 B2
`7,241,988 B2
`2002/0058332 A1
`2002/0176069 A1
`2003/0032204 A1
`2003/0047676 A1
`2003/0186426 A1
`2005/0061962 A1
`2005/0121604 A1
`2006/0058167 A1*
`2006/0152707 A1
`
`10/2003 Lei et a1.
`4/2004 Fuhr et a1.
`“/2004 Wang et a1.
`12/2004 Wang et a1.
`“2005 Foster
`
`9/2005 Tran-et a1.
`4/2006 H1aV1nka et 31.
`7/2007 Gruber et a1.
`5/2002 Quake et a1.
`11/2002 Hansen et a1.
`2/2003 Walt et a1.
`3/2003 Grier et a1.
`10/2003 Brewer et a1.
`3/2005 Mueth et a1.
`6/2005 Mueth et a1.
`3/2006 Ragusa etal.
`7/2006 Kanda
`
`.................. 494/5
`
`FOREIGN PATENT DOCUMENTS
`
`JP
`JP
`W0
`
`06327494
`2002453260
`WO 01/18400
`
`11/ 1994
`590”
`”001
`
`PaulO.P.Ts’0,“BasicPrinciplesinNucleicAcidChemistry”,
`National Library of Medicine 1974, pp. 311-387, Academic
`press Inc., NewYork, NY.
`
`* cited by examiner
`
`

`

`U.S. Patent
`
`Apr. 20, 2010
`
`Sheet 1 of 22
`
`US 7,699,767 B2
`
`

`

`U.S. Patent
`
`Apr. 20, 2010
`
`Sheet 2 of 22
`
`US 7,699,767 B2
`
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`
`
`
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`
`

`

`U.S. Patent
`
`Apr. 20, 2010
`
`Sheet 3 of 22
`
`US 7,699,767 B2
`
`<22:
`
`.mmdzz m.
`
`.mHm
`
`
`

`

`U.S. Patent
`
`Apr. 20, 2010
`
`Sheet 4 of 22
`
`US 7,699,767 B2
`
`FIG.
`
`4
`
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`

`

`U.S. Patent
`
`Apr. 20, 2010
`
`Sheet 5 of 22
`
`US 7,699,767 B2
`
`MUMHmwmoz
`
`mth<hom
`
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`

`US. Patent
`
`Apr. 20, 2010
`
`Sheet 6 of 22
`
`US 7,699,767 B2
`
`500
`
`FIG . BA
`
`PROVIDE A FIRST FLOV HAVING A PLURALITY
`OF COMPONENTS
`
`PROVIDE A SECOND FLOR
`
`CONTACT THE FIRST FLOV HITH THE SECOND FLOV
`To PROVIDE A FIRST SEPARATION REGION
`
`OIFFERENTIALLY SEDIMENT A FIRST COMPONENT
`OF A PLURALITY OF COMPONENTS FROM THE FIRST
`FLOW INTO THE SECOND FLOW
`
`CONCURRENTLV MAINTAIN A SECOND COMPONENT OF THE
`PLURALITV OF COMPONENTS IN THE FIRST FLOR
`
`DIFFERENTIALLV REMOVE THE SECOND FLOR HAVING
`THE FIRST COMPONENT FROM THE FIRST FLOV HAVING
`THE SECOND COMPONENT
`
`835
`
`505
`
`81°
`
`515
`
`520
`
`525
`
`530
`
`SENAIRIATNSN
`
`YES
`
`PROVIDE A THIRD FLOR
`
`"0
`0
`
`CONTACT THE FIRST FLOV HITH THE THIRD FLOR
`TO PROVIDE A SECOND SEPARATION REGION
`
`“0
`
`545
`
`SEPARATION?
`
`YES
`
`‘
`
`550
`
`HOLOBRAPHIC
`MANIPULATION 0H
`
`
`
`

`

`U.S. Patent
`
`Apr. 20, 2010
`
`Sheet 7 of 22
`
`US 7,699,767 B2
`
`FIG. BB
`
`GENERATE A PLUHALITY
`OF HOLOGRAPHIC TRAPS
`
`USING THE HOLOGHAPHIC
`TRAPS. DIFFERENTIALLY MOVING
`THE SECOND COMPONENT FROM
`THE FIRST FLOW INTO THE
`
`THIRD FLOW
`
`555
`
`580
`
`885
`
`DIFFERENTIALLY
`SEDIMENTINB THE
`SECOND COMPONENT
`FROM THE FIRST
`FLOW INTO THE
`THIRD FLOW
`
`CONCURRENTLY MAINTAINING A THIRD COMPONENT
`OF THE PLURALITY 0F COMPONENTS IN THE
`FIRST FLOW
`
`570
`
`DIFFERENTIALLY REMOVING THE THIRD FLOW
`HAVING THE SECOND COMPONENT FROM THE
`FIRST FLOW HAVING THE THIRD COMPONENT
`
`575
`
`RETURN
`
`EBO
`
`

`

`US. Patent
`
`Apr. 20, 2010
`
`Sheet 8 of 22
`
`US 7,699,767 B2
`
`FIG.
`
`7A
`
`704
`
`703
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`
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`
`700
`
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`705
`
`701
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`
`701
`
`
`
`

`

`US. Patent
`
`Apr. 20, 2010
`
`Sheet 9 of 22
`
`US 7,699,767 B2
`
`FIG. 8
`
`PHO T0 IMAGE
`
`FIG. 9
`
`PHO T0 IMAGE
`
`FIG. 10
`
`PHOTO IMAGE
`
`

`

`U.S. Patent
`
`Apr. 20, 2010
`
`Sheet 10 0f 22
`
`US 7,699,767 B2
`
`3:
`
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`US. Patent
`
`Apr. 20, 2010
`
`Sheet 11 of 22
`
`US 7,699,767 B2
`
`FIG. 12
`
`1203
`
`
`
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`
`IMAGING AND TRAPPING SYSTEM
`
`1204
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`
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`
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`
`
`
`

`

`U.S. Patent
`
`%
`
`US 7,699,767 B2
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`US. Patent
`
`Apr. 20, 2010
`
`Sheet 13 of 22
`
`US 7,699,767 B2
`
`FIG. 15
`
`gmSOLUTION
`2
`
`gm
`
`SOLUTION
`
`INPUT
`
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`
`SOLUTION
`
`

`

`US. Patent
`
`Apr. 20, 2010
`
`Sheet 14 of 22
`
`US 7,699,767 B2
`
`FIG. 16
`
`DIVIDERS
`[2.9. COVER GLASS)
`
`SYRINBE
`NEEDLES
`OF! TUBING
`
`1500
`
`HALL
`MATERIALS
`
`CHANNEL-DEFINING
`MATERIAL
`
`
`
`DEPTH
`
`FIG. 17
`
`DIVIDERS
`
`
`
`I
`
`SOHTINB
`REGION
`WIDTH
`
`2 TUBES OH
`NEEDLES
`(ABOVE EACH
`OTHER)
`
`
`
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`15°"
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`some
`‘
`
`LENGTH
`
`

`

`US. Patent
`
`Apr. 20, 2010
`
`Sheet 15 of 22
`
`US 7,699,767 B2
`
`FIG. 18
`
`BUFFER
`
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`
`
`
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`
`
`SOLUTION
`FLAT SOHTERS
`
`IN DEVICE
`
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`
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`
`
`
`
`
`

`

`US. Patent
`
`Apr. 20, 2010
`
`Sheet 16 of 22
`
`US 7,699,767 B2
`
`FIG. 20
`
` SELECTION
`
`FLOW
`
`WASTE FLOW
`REGION DEPTH
`
`
`
`

`

`US. Patent
`
`Apr. 20, 2010
`
`Sheet 17 of 22
`
`US 7,699,767 B2
`
`FIG. 22
`
`
`
`FIG. 23
`
`
`
`

`

`US. Patent
`
`Apr. 20, 2010
`
`Sheet 18 of 22
`
`US 7,699,767 B2
`
`FIG. 24A
`
`INPUTS
`
`
`
`
`
`
`
`
`
`
`
`
`OUTPUTS
`
`

`

`U.S. Patent
`
`Apr. 20, 2010
`
`Sheet 19 0f 22
`
`US 7,699,767 B2
`
`mvm.me
`
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`
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`

`

`U.S. Patent
`
`Apr. 20, 2010
`
`Sheet 20 0f 22
`
`US 7,699,767 B2
`
`25%Bsudmififiz:
`
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`US. Patent
`
`Apr. 20, 2010
`
`Sheet 21 of 22
`
`US 7,699,767 B2
`
`FIG. 26
`
`2830
`
`2535 OUTPUT
`
`HESEHVOIR(S)
`
`

`

`U.S. Patent
`
`Apr. 20, 2010
`
`Sheet 22 0f 22
`
`US 7,699,767 B2
`
`azouuw
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`
`
`
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`
`

`

`US 7,699,767 B2
`
`1
`MULTIPLE LAMINAR FLOW-BASED
`PARTICLE AND CELLULAR SEPARATION
`WITH LASER STEERING
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS AND PRIORITY CLAIMS
`
`The present application is a divisional application ofparent
`US. patent application Ser. No. 11/543,773, filed Oct. 6,
`2006, which is a divisional application of US. patent appli-
`cation Ser. No. 10/934,597, filed Sep. 3, 2004, now US. Pat.
`No. 7,118,696, which is a continuation-in-part ofU.S. patent
`application Ser. No. 10/867,328, filed Jun. 13, 2004, now US.
`Pat. No. 7,150,834, which is a continuation-in-part of US.
`patent application Ser. No. 10/630,904, filed Jul. 31, 2003,
`now US. Pat. No. 7,241,988, and claims priority from US.
`Provisional Patent Application No. 60/399,386, filed Jul. 31,
`2002, and 60/435,541,
`filed Dec. 20, 2002, commonly
`assigned herewith, the contents of all of which are incorpo-
`rated by reference herein, with priority claimed for all com-
`monly disclosed subject matter (the “first related applica-
`tions”).
`The present invention is related to Jessica Shireman et al.,
`US. Provisional Patent Application Ser. No. 60/571,141,
`filed May 14, 2004, entitled “System and Method of Sorting
`Blood Cells Using Holographic Laser Steering”, commonly
`assigned herewith, the contents of which are incorporated by
`reference herein, with priority claimed for all commonly dis-
`closed subject matter (the “second related application”).
`The present invention is related to and a conversion to a full
`utility application of Daniel M. Mueth, US. Patent Applica-
`tion Ser. No. 60/499,957, filed Sep. 4,2003, entitled “Passive
`Fluidic Sorter”, commonly assigned herewith, the contents of
`which are incorporated by reference herein, with priority
`claimed for all commonly disclosed subject matter (the “third
`related application”).
`The present invention is related to and a conversion to a full
`utility application of Daniel M. Mueth, US. Patent Applica-
`tion Ser. No. 60/511,458, filed Oct. 15, 2003, entitled “Pas-
`sive Fluidic Sorter”, commonly assigned herewith, the con-
`tents of which are incorporated by reference herein, with
`priority claimed for all commonly disclosed subject matter
`(the “fourth related application”).
`The present invention is related to Lewis Gruber et al., US.
`patent application Ser. No. 10/630,904, filed Jul. 31, 2003,
`entitled “System and Method of Sorting Materials Using
`Holographic Laser Steering”, commonly assigned herewith,
`the contents of which are incorporated by reference herein,
`with priority claimed for all commonly disclosed subject
`matter (the “fifth related application”).
`
`FIELD OF THE INVENTION
`
`The present invention relates generally to techniques and
`systems for separation of particulate or cellular materials
`such as blood, semen and other particles or cells into their
`various components and fractions, using multiple laminar
`flows which further may be coupled with laser steering such
`as holographic optical trapping and manipulation.
`
`BACKGROUND OF THE INVENTION
`
`There are several categories of blood cells. Erythrocyte or
`red blood cell (RBC) counts are for women 4.8 million cells/
`ul and men 5.4 million cells/ul. RBCs make up 93% of the
`solid element in blood and about 42% of blood volume.
`
`Platelets are 2 um-3 pm in size. They represent 7% ofthe solid
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`elements in blood and about 3% of the blood volume, corre-
`sponding to about 1.5 to 4><10ll cells per liter. There are 5
`general types of white blood cells (WBCs) or leukocytes
`accounting for about 1.5 to 4><109 cells per liter. The WBCs
`comprise: 50-70% Neutrophils (12-15 pm in size); 2-4%
`Eosinophils (12-15 pm in size); 0.5-1% Basophils (9-10 pm
`in size); 20-40% Lymphocytes (25% B-cells and 75% T—cells)
`(8-10 pm in size); and 3-8% Monocytes (16-20 pm in size).
`They comprise 0.16% of the solid elements in the blood, and
`approximately 0.1% of the blood volume corresponding to
`around 4 to 12><109 per liter. A subject with an infection might
`have a WBC count as high as 25><109 per liter.
`Platelets are the smallest cells in the blood and are impor-
`tant for releasing proteins into the blood that are involved in
`clotting. Patients with immune diseases that cause lower
`counts (such as cancer, leukemia and other chemotherapy
`patients) sometimes need platelet transfusions to prevent their
`counts from becoming too low. The platelet count in adults is
`normally between 140,000-440,000 cells/pl, and this number
`should not fall below 50,000 cells/ML because platelets play
`an integral role in blood clotting.
`Blood separation techniques have traditionally employed
`discrete centrifugation processes. More particularly, a certain
`volume ofblood is removed from a donor at a particular time.
`That volume of blood is then subjected to different levels of
`centrifugation to provide corresponding blood fractions for
`blood components such as plasma, platelets, red blood cells,
`and white blood cells. This process is discrete, rather than
`continuous, such that if more blood from the donor is to be
`processed, another volume is removed from the donor, and
`the process is repeated.
`The steps in platelet collection are: collection of blood
`from donor: addition of anticoagulant; separation via cen-
`trifugation; return of red cells, leukocytes and plasma to the
`donor. A collection normally contains about 200-400 ml of
`plasma, which is reduced to avoid incompatibility. This col-
`lection normally contains about 8 to 8.5><1010 platelets. A
`donor normally gives approximately 10% of his/her platelets
`with no loss in clotting ability, although a larger number of
`platelets could be separated from the blood. These platelets
`must be used within five days of collection.
`Plateletpheresis, called apheresis, is a state of the art pro-
`cess by which platelets are separated [Haemonetics Compo-
`nent Collection System (CCS) and Multi Component System
`(Multi)(Haemonetics, Braintree, Mass.)]. This automated
`machine separates platelets from blood over a period of 1.5 to
`2 hours (assuming 10% donation). This process is faster than
`traditional approaches and is completely automated and can
`be used for single or double platelet doses. Nevertheless, the
`process is slow relative to the patience of donors and is
`capable of improvement for the purity of the separated plate-
`let fraction.
`
`Other procedures are also time consuming, often taking
`several hours, particularly when unused blood fractions are to
`be returned to the donor. For example, platelet donation make
`take several hours, as whole blood is removed from the donor,
`fractionated through centrifugation to obtain the platelets,
`and the remaining blood components are then injected back
`into the donor. This centrifugation process is also compara-
`tively harsh, also can result in damage to a proportion of the
`harvested cells, effectively reducing the usable yield of the
`blood fractions.
`
`Other types of separations are also either time consuming
`or cannot process large-volumes of material in a timely fash-
`ion. For example, sperm sorting, in which viable and motile
`sperm are isolated from non-viable or non-motile sperm, is
`often a time-consuming task, with severe volume restrictions.
`
`

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`US 7,699,767 B2
`
`3
`As discussed below in greater detail in describing the
`present invention, manipulations of particles, such as that
`described in the second and fifth related applications, may
`also be part ofa novel separation technique. One conventional
`technique in manipulating microscopic objects is optical trap-
`ping. An accepted description of the effect of optical trapping
`is that tightly focused light, such as light focused by a high
`numerical aperture microscope lens, has a steep intensity
`gradient. Optical traps use the gradient forces of a beam of
`light to trap a particle based on its dielectric constant.
`To minimize its energy, a particle having a dielectric con-
`stant higher than the surrounding medium will move to the
`region of an optical trap where the electric field is the highest.
`Particles with at least a slight dielectric constant differential
`with their surroundings are sensitive to this gradient and are
`either attracted to or repelled from the point of highest light
`intensity, that is, to or from the light beam’s focal point. In
`constructing an optical trap, optical gradient forces from a
`single beam of light are employed to manipulate the position
`of a dielectric particle immersed in a fluid medium with a
`refractive index smaller than that of the particle, but reflect-
`ing, absorbing and low dielectric constant particles may also
`be manipulated.
`The optical gradient force in an optical trap competes with
`radiation pressure which tends to displace the trapped particle
`along the beam axis. An optical trap may be placed anywhere
`within the focal volume of an objective lens by appropriately
`selecting the input beam’s propagation direction and degree
`of collimation. A collimated beam entering the back aperture
`of an objective lens comes to a focus in the center of the lens’
`focal plane while another beam entering at an angle comes to
`a focus off-center. A slightly diverging beam focuses down-
`stream of the focal plane while a converging beam focuses
`upstream. Multiple beams entering the input pupil of the lens
`simultaneously each form an optical trap in the focal volume
`at a location determined by its angle of incidence. The holo-
`graphic optical trapping technique uses a phase modifying
`diffractive optical element to impose the phase pattern for
`multiple beams onto the wavefront of a single input beam,
`thereby transforming the single beam into multiple traps.
`Phase modulation ofan input beam is preferred for creating
`optical traps because trapping relies on the intensities of
`beams and not on their relative phases. Amplitude modula-
`tions may divert light away from traps and diminish their
`effectiveness.
`
`When a particle is optically trapped, optical gradient forces
`exerted by the trap exceed other radiation pressures arising
`from scattering and absorption. For a Gaussian TEMOO input
`laser beam, this generally means that the beam diameter
`should substantially coincide with the diameter of the
`entrance pupil. A preferred minimum numerical aperture to
`form a trap is about 0.9 to about 1.0.
`One difliculty in implementing optical trapping technol-
`ogy is that each trap to be generated generally requires its own
`focused beam of light. Many systems of interest require mul-
`tiple optical traps, and several methods have been developed
`to achieve multiple trap configurations. One existing method
`uses a single light beam that is redirected between multiple
`trap locations to “time-share” the beam between various
`traps. However, as the number oftraps increases, the intervals
`during which each trap is in its “off” state may become long
`for particles to diffuse away from the trap location before the
`trap is re-energized. All these concerns have limited imple-
`mentations of this method to less than about 10 traps per
`system.
`Another traditional method of creating multi-trap systems
`relies on simultaneously passing multiple beams of light
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`through a single high numerical aperture lens. This is done by
`either using multiple lasers or by using one or more beam
`splitters in the beam of a single laser. One problem with this
`technique is that, as the number oftraps increases, the optical
`system becomes progressively more and more complex.
`Because of these problems, the known implementations of
`this method are limited to less than about 5 traps per system.
`
`In a third approach for achieving a multi-trap system, a dif-
`fractive optical element (DOE) (e.g., a phase shifting holo-
`gram utilizing either a transmission or a reflection geometry)
`is used to alter a single laser beam’s wavefront. This invention
`is disclosed in US. Pat. No. 6,055,106 to Grier et al. The
`wavefront is altered so that the downstream laser beam essen-
`
`tially becomes a large number of individual laser beams with
`relative positions and directions of travel fixed by the exact
`nature of the diffractive optical element. In effect, the Fourier
`transform of the DOE produces a set of intensity peaks each
`of which act as an individual trap or “tweezer.”
`Some implementations of the third approach have used a
`fixed transmission hologram to create between 16 and 400
`individual trapping centers.
`A fixed hologram has been used to demonstrate the prin-
`ciple ofholographic optical trapping but using a liquid crystal
`grating as the hologram permitted ‘manufacture’ of a separate
`hologram for each new distribution of traps. The spatially
`varying phase modulation imposed on the trapping laser by
`the liquid crystal grating may be easily controlled in real time
`by a computer, thus permitting a variety of dynamic manipu-
`lations.
`
`Other types of traps that may be used to optically trap
`particles include, but are not limited to, optical vortices, opti-
`cal bottles, optical rotators and light cages. An optical vortex
`produces a gradient surrounding an area of zero electric field
`which is useful to manipulate particles with dielectric con-
`stants lower than the surrounding medium or which are
`reflective, or other types of particles which are repelled by an
`optical trap. To minimize its energy, such a particle will move
`to the region where the electric field is the lowest, namely the
`zero electric field area at the focal point of an appropriately
`shaped laserbeam. The optical vortex provides an area of zero
`electric field much like the hole in a doughnut (toroid). The
`optical gradient is radial with the highest electric field at the
`circumference of the doughnut. The optical vortex detains a
`small particle within the hole of the doughnut. The detention
`is accomplished by slipping the vortex over the small particle
`along the line of zero electric field.
`The optical bottle differs from an optical vortex in that it
`has a zero electric field only at the focus and a non-zero
`electric field in all other directions surrounding the focus, at
`an end of the vortex. An optical bottle may be useful in
`trapping atoms and nanoclusters which may be too small or
`too absorptive to trap with an optical vortex or optical twee-
`zers. (See J. Arlt and M. J. Padgett. “Generation of a beam
`with a dark focus surrounded by regions of higher intensity:
`The optical bottle beam,” Opt. Lett. 25, 191 -193, 2000.)
`The light cage (US. Pat. No. 5,939,716) is loosely, a mac-
`roscopic cousin of the optical vortex. A light cage forms a
`time-averaged ring of optical traps to surround a particle too
`large or reflective to be trapped with dielectric constants
`lower than the surrounding medium.
`
`When the laser beam is directed through or reflected from the
`phase patterning optical element, the phase patterning optical
`element produces a plurality of beamlets having an altered
`phase profile. Depending on the number and type of optical
`traps desired, the alteration may include diffraction, wave-
`front shaping, phase shifting, steering, diverging and con-
`
`

`

`US 7,699,767 B2
`
`5
`verging. Based upon the phase profile chosen, the phase pat-
`terning optical element may be used to generate optical traps
`in the form of optical traps, optical vortices, optical bottles,
`optical rotators, light cages, and combinations oftwo or more
`of these forms.
`
`Researchers have sought indirect methods for manipulat-
`ing cells, such as tagging the cells with diamond micro-
`particles and then tweezing the diamond particles. Cell
`manipulations have included cell orientation for microscopic
`analysis as well as stretching cells. Tissue cells have also been
`arranged with tweezers in vitro in the same spatial distribu-
`tion as in vivo.
`
`In addition to the cells themselves, optical tweezers have
`been used to manipulate cellular organelles, such as vesicles
`transported along microtubules, chromosomes, or globular
`DNA. Objects have also been inserted into cells using optical
`tweezers.
`
`Accordingly, as an example of new types of sorting using
`laser steered optical traps, a method of cell sorting using a
`technique which isolates valuable cells from other cells, tis-
`sues, and contaminants is needed. Further, a way of achieving
`a unique contribution of optical trapping to the major indus-
`trial needs of blood cell sorting and purification is required.
`Still further, there is a need to separate sperm cells in the
`animal husbandry market.
`As a consequence, a need remains for a separation tech-
`nique and apparatus which is continuous, has high through-
`put, provides time saving, and which causes negligible or
`minimal damage to the various components for separation. In
`addition, such techniques should have further applicability to
`biological or medical areas, such as for separations of blood,
`sperm, other cellular materials, as well as viral, cell organelle,
`globular structures, colloidal suspensions, and other biologi-
`cal materials.
`
`SUMMARY OF THE INVENTION
`
`The exemplary embodiments of the present invention pro-
`vide for separating components in a mixture, such as separat-
`ing the various blood components of whole blood into corre-
`sponding fractions, such as a platelet fraction, a red blood cell
`fraction, a white blood cell fraction, and a plasma fraction.
`The various embodiments of the present invention provide
`separation of components on a continuous basis, such as
`within a continuous, closed system, without the potential
`damage and contamination of prior art methods, particularly
`for fractionation of blood components. The continuous pro-
`cess of the present invention also provides significant time
`savings and higher throughput for blood fractionation. In
`addition, the various embodiments may also include addi-
`tional means for separating and manipulating the compo-
`nents, particularly holographic optical manipulation and
`separation. The various embodiments may also be applied to
`separations of other types ofcellular and biological materials,
`such as sperm, viruses, bacteria, cell debris, cell organelles,
`globular structures, colloidal suspensions, cellular debris,
`and other biological materials.
`As used herein, “Particle” refers to a biological or other
`chemical material including, but not limited to, oligonucle-
`otides, polynucleotides, chemical compounds, proteins, lip-
`ids, polysaccharides, ligands, cells, antibodies, antigens, cel-
`lular organelles, lipids, blastomeres, aggregations of cells,
`microorganisms, peptides, cDNA, RNA and the like.
`An exemplary method of separating blood into compo-
`nents includes providing a first flow having a plurality of
`blood components; providing a second flow; contacting the
`first flow with the second flow to provide a first separation
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`region; and differentially sedimenting a first blood cellular
`component of the plurality of blood components into the
`second flow while concurrently maintaining a second blood
`cellular component of the plurality of blood components in
`the first flow. The second flow having the first blood cellular
`component is then differentially removed from the first flow
`having the second blood cellular component.
`The various sedimentation steps of the present invention
`may be rate zonal or isopycnic. In addition, the first flow and
`the second flow are substantially non-turbulent, and may also
`be substantially laminar.
`In a selected embodiment, the first blood cellular compo-
`nent is a plurality of red blood cells and a plurality of white
`blood cells, and the second blood cellular component is a
`plurality of platelets. For the first blood cellular component,
`the plurality of white blood cells may be holographically
`separated (through laser steering) from the plurality of red
`blood cells. Other holographic manipulations of the present
`invention include holographically removing a plurality of
`contaminants from the first flow, holographically separating
`biological debris from the first flow, and holographically
`separating a plurality of second blood cellular components
`from the first flow.
`
`Additional separation stages may also be included, with the
`exemplary method providing a third flow; contacting the first
`flow with the third flow to provide a second separation region;
`and differentially sedimenting the second blood cellular com-
`ponent of the plurality of blood components to sediment into
`the third flow while concurrently maintaining a third blood
`component of the plurality of blood components in the first
`flow. In selected embodiments, the second blood cellular
`component is a plurality of platelets and wherein the third
`blood component is plasma.
`A plurality of separation stages may also be combined to
`form more complicated structures having multiple separation
`stages, connected in series, connected in parallel, or in com-
`binations of both.
`
`A second exemplary method of separating a fluid mixture
`into constituent, non-motile components, in accordance with
`the present invention,
`includes: providing a substantially
`laminar first flow having the fluid mixture, the fluid mixture
`having a plurality of components, the plurality ofcomponents
`having a corresponding plurality of sedimentation rates; pro-
`viding a substantially laminar second flow; contacting the
`first flow with the second flow to provide a first separation
`region, the first flow and the second flow having a substan-
`tially non-turbulent interface within the separation region;
`differentially sedimenting from the first flow a first compo-
`nent of the plurality of components into the second flow to
`form an enriched second flow and a depleted first flow, while
`concurrently maintaining a second component ofthe plurality
`of components in the first flow, the first component having a
`first sedimentation rate of the plurality of sedimentation rates
`and the second component having a second sedimentation
`rate of the plurality of sedimentation rates, wherein the first
`sedimentation rate is comparatively greater than the second
`sedimentation rate; differentially removing the enriched sec-
`ond flow from the depleted first flow; and holographically
`manipulating the second component in the depleted first flow.
`The second exemplary method may also include additional
`separation stages, such as a holographic separation, includ-
`ing: providing a third flow; contacting the depleted first flow
`with the third flow to provide a second separation region; and
`holographically t