111111
`
`1111111111111111111111111111111111111111111111111111111111111
`US008653442B2
`
`c12) United States Patent
`Mueth et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 8,653,442 B2
`Feb.18,2014
`
`(54) MULTIPLE LAMINAR FLOW-BASED
`PARTICLE AND CELLULAR SEPARATION
`WITH LASER STEERING
`
`(75)
`
`Inventors: Daniel Mueth, Chicago, IL (US);
`Joseph Plewa, Park Ridge, IL (US);
`Jessica Shireman, Kansas City, MO
`(US); Amy Anderson, Palatine, IL (US);
`Lewis Gruber, Chicago, IL (US); Neil
`Harris Rosenbaum, Chicago, IL (US)
`
`(73) Assignee: Premium Genetics (UK) Limited,
`Cheshire (GB)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 46 days.
`
`(21) Appl. No.: 13/412,969
`
`(22) Filed:
`
`Mar. 6, 2012
`
`(65)
`
`Prior Publication Data
`
`US 2012/0183947 Al
`
`Jul. 19, 2012
`
`Related U.S. Application Data
`
`(60)
`
`(60)
`
`Continuation of application No. 12/659,277, filed on
`Mar. 2, 2010, now Pat. No. 8,158,927, which is a
`division of application No. 12/213,109, filed on Jun.
`13, 2008, now Pat. No. 7,699,767, which is a division
`of application No. 11/543,773, filed on Oct. 6, 2006,
`now Pat. No. 7,402,131, which is a division of
`application No. 10/934,597, filed on Sep. 3, 2004, now
`Pat. No. 7,118,676, which is a continuation-in-part of
`application No. 10/867,328, filed on Jun. 13, 2004,
`now
`Pat. No.
`7,150,834, which
`is
`a
`continuation-in-part of application No. 10/630,904,
`filed on Jul. 31, 2003, now Pat. No. 7,241,988.
`
`Provisional application No. 60/399,386, filed on Jul.
`31, 2002, provisional application No. 60/435,541,
`filed on Dec. 20, 2002, provisional application No.
`60/571,141, filed on May 14, 2004, provisional
`application No. 60/499,957, filed on Sep. 4, 2003,
`provisional application No. 60/511,458, filed on Oct.
`15, 2003.
`
`(51)
`
`Int. Cl.
`BOJD21101
`(52) U.S. Cl.
`CPC ...................................... BOlD 21101 (2013.01)
`
`(2006.01)
`
`USPC ............ 250/251; 494/36; 494/45; 435/173.1;
`210/732; 210/800; 210/802
`(58) Field of Classification Search
`USPC .................... 250/251; 494/36, 45; 435/173.1;
`210/732, 800, 802
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`3,390,449 A
`3,649,829 A
`
`7/1968 Fox
`3/1972 Randolph
`(Continued)
`
`FOREIGN PATENT DOCUMENTS
`
`DE
`EP
`
`5/2001
`19952322 A1
`8/1982
`0057907 A1
`(Continued)
`
`OTHER PUBLICATIONS
`
`Hori M. eta!., "Cell fusion by optical trapping with laser-involves
`contacting different cells .
`. ", WPI/Thomson, Dec. 27, 1991,
`Abstract.
`
`(Continued)
`
`Primary Examiner- Kiet T Nguyen
`(74) Attorney, Agent, or Firm- Jean C. Edwards; Edwards
`Neils PLLC
`
`ABSTRACT
`(57)
`The invention provides a method, apparatus and system for
`separating cellular components, and can be combined with
`holographic optical trapping manipulation or other forms of
`optical tweezing. One exemplary method includes providing
`a first flow having a plurality of components; providing a
`second flow; contacting the first flow with the second flow to
`provide a first separation region; and differentially sediment(cid:173)
`ing a first cellular component of the plurality of components
`into the second flow while concurrently maintaining a second
`cellular component of the plurality of components in the first
`flow. The second flow having the first cellular component is
`then differentially removed from the first flow having the
`second 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.
`
`64 Claims, 22 Drawing Sheets
`
`

`

`US 8,653,442 B2
`Page 2
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,325,706 A
`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
`5,800,690 A
`5,837,115 A
`5,849,178 A
`5,879,625 A
`5,966,457 A
`6,053,856 A
`6,071,442 A
`6,185,664 B1
`H1960 H
`6,368,871 B1
`6,416,190 B1
`6,432,630 B1
`6,451,264 B1
`6,506,609 B1
`6,524,860 B1
`6,637,463 B1
`6,727,451 B1
`6,833,542 B2
`6,838,056 B2
`6,944,324 B2
`7,029,430 B2
`7,241,988 B2
`
`4/1982 Gershman et a!.
`10/1983 Furuta et al.
`111984 Iriguchi
`4/1987 Sage eta!.
`5/1987 Nozaki, Jr. eta!.
`4/1991 Ohki eta!.
`3/1992 Buican eta!.
`111993 Touge eta!.
`3/1993 Tycko
`7/1993 Schnipelsky et a!.
`111996 Van den Engh et a!.
`4/1997 Weetall et al.
`10/1997 Ulmer
`9/1998 Chow eta!.
`1111998 Austin eta!.
`12/1998 Holmet al.
`3/1999 Roslaniec eta!.
`10/1999 Lemelson
`4/2000 Hlavinka
`6/2000 Dean eta!.
`2/2001 Jeddeloh
`6/2001 Conrad et al.
`4/2002 Christel et a!.
`7/2002 Grier eta!.
`8/2002 Blankenstein
`9/2002 Bhullar et al.
`112003 Wada et al.
`2/2003 Seidel eta!.
`10/2003 Lei eta!.
`4/2004 Fuhr et al.
`12/2004 Wang et al.
`112005 Foster
`9/2005 Tran eta!.
`4/2006 Hlavinka et a!.
`7/2007 Gruber eta!.
`
`7,472,794 B2 *
`7,482,577 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
`
`112009 Oakey et al ................... 210/420
`112009 Gruber et a!.
`5/2002 Quake eta!.
`1112002 Hansen eta!.
`2/2003 Walt et al.
`3/2003 Grier et a!.
`10/2003 Brewer et al.
`3/2005 Mueth eta!.
`6/2005 Mueth eta!.
`3/2006 Regusa et a!.
`7/2006 Kanda
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`FR
`JP
`JP
`JP
`JP
`JP
`JP
`wo
`wo
`wo
`
`0679325 A1
`2798557 A1
`57-131451 A
`58-090513 A
`06-327494
`07-024309
`2002-153260
`2005-502482 A
`99/39223 A1
`01118400 A1
`2004/012133 A2
`
`1111995
`3/2001
`8/1982
`5/1983
`1111994
`111995
`5/2002
`1/2005
`8/1999
`3/2001
`2/2004
`
`OTHER PUBLICATIONS
`
`S. Takayama et a!., "Patterning cells and their environments using
`multiple laminar fluid flows ... ", Proc. Nat!. Acad. Sci. USA, May
`1999, pp. 5545-5548, vol. 96.
`Paul O.P. Ts'O, "Basic Principles in Nucleic Acid Chemistry",
`National Library of Medicine, 1974, pp. 311-387, Academic Press
`Inc., New York, NY.
`Stephen P. Smith et a!., Inexpensive Optical Tweezers for Under(cid:173)
`graduate Laboratories, Am. J. Phys., Jan. 1999, vol. 67.
`* cited by examiner
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 1 of22
`
`US 8,653,442 B2
`
`

`

`U.S. Patent
`
`Feb. 18,2014
`
`Sheet 2 of22
`
`US 8,653,442 B2
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 3 of22
`
`US 8,653,442 B2
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 4 of22
`
`US 8,653,442 B2
`
`.FIG. 4
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 5 of22
`
`US 8,653,442 B2
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 6 of22
`
`US 8,653,442 B2
`
`FIG. SA
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 7 of22
`
`US 8,653,442 B2
`
`FIG. SB
`
`( ...... · ....-sa~
`"· Hfrtm.M
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 8 of22
`
`US 8,653,442 B2
`
`~Ml.._ :
`.
`l~ ~~ ..
`
`FIG. lA
`
`FIG. 78
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 9 of22
`
`US 8,653,442 B2
`
`FIG.
`
`tJ
`
`F!G. 9
`
`FIG.
`
`iO
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 10 of 22
`
`US 8,653,442 B2
`
`· - ->,. .. ~
`_......,;jj...,..,.~eeeeee*~:?,HH} ~~'** ~~
`~ •··········
`~\
`...
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 11 of 22
`
`US 8,653,442 B2
`
`FIB. 12
`
`~ .. · . · ·
`
`~~ OlSl!D.friON Ulst
`
`·············
`
`..
`
`FIG. 13
`
`I
`
`/ 0
`
`

`

`U.S. Patent
`U.S. Patent
`
`Feb.18,2014
`Feb. 18, 2014
`
`Sheet 12 of 22
`Sheet 12 of 22
`
`US 8,653,442 B2
`US 8,653,442 B2
`
`MMMMMMMMMMMMMM
`
`WMMMMMMMMMMM...MMMMM
`
`MMMMMMMMMMMMWM
`
`
`
`
`Mwwwmwmm”$memeMMMMMMMMMM.Mwwwwmm
`
`
`
`MMMMMWMMM.HWWMWMWMMMMMMMMMMMMMMMMMMMMMMMMMMMif”.M.
`
`
`
`
`
`
`
`MMMMMMMMMMMM
`
`
`
`
`
`MMMMMMMMMMM..MMMM.MMMMM
`
`.
`
`!I
`
`MwMMMMMMM
`MMme3.5
`
`
`
`
`
`FxMMMMMMMMMMMMMMMMMM_M...MMMMMMMMMMM
`
`
`MMMMMMM,WMMMMMMMMMMMMMMMMMMWMMMMMW
`
`
`
`MWMMMWMWWWM,MMMMMMMMMMMMMMMM
`
`MMMMMMM
`
`MMMWMMMMMMmMMM
`
`!
`
`
`
`
`
`MMHMMMMMMMWMMMMMMMMWMMMMMMMwfifiwwwwwwwMMMMMMMMMMM
`....5555255,}35.55::it???
`
`
`
`
`
`
`
`
`
`mewwwMmMMMMMme
`
`'•! l
`ffi -· t.~
`' t:; I ~ :,...t I!
`
`
`
`
`
`
`
`533333333....iiiiiiiiia.M...VVVVVV»
`
`.........
`
`
`
`
`

`

`U.S. Patent
`U.S. Patent
`
`Feb.18,2014
`Feb. 18, 2014
`
`Sheet 13 of 22
`Sheet 13 of 22
`
`US 8,653,442 B2
`US 8,653,442 B2
`
`~· ............ :49"
`
`~: :
`
`r;
`1
`
`fifiifiéfi
`
`
`mg" "
`* "
`3
`
`
`'
`
`.. /
`
`%$?a¥£%$§
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 14 of 22
`
`US 8,653,442 B2
`
`FIG. il
`OlVt!aS
`~\..
`
`.:.·.·
`
`-
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 15 of 22
`
`US 8,653,442 B2
`
`FIG. 18
`
`vnwur
`t SOLVWJM
`
`M Of tWff
`H.IOO--~tT .. :ftAU!J
`Fb\T SOOT'EHS
`1~ ut¥ICE
`
`· YMTE
`..,. ..
`............
`I S!ltff-t~
`
`saftTI~
`~----~~----------~~--WW4~
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 16 of 22
`
`US 8,653,442 B2
`
`FIG. 20
`
`i i
`SELEttlOO
`,~~·fUM
`!}fY"lli
`·>•>•> •' •>·~~y.y NNW·~~ . . . . . - · ~~~~«~_1 • ~.Jt.,L
`·~m·»»>y f~.-~t~l w· ~~~
`
`~=*·~~~ y,, .. , .. ,
`
`FIG. 21
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 17 of 22
`
`US 8,653,442 B2
`
`FIG. 22
`
`FIG, 23
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 18 of 22
`
`US 8,653,442 B2
`
`FIG. 24A
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 19 of 22
`
`US 8,653,442 B2
`
`.... ~
`
`-.r-
`
`·~ ~
`
`~r"
`
`f.
`~ ...
`
`.·~
`~
`~ .
`j
`!
`l
`
`:
`·"'!-"
`
`t""
`
`r
`
`"' ~
`
`·-'·r"
`
`!---
`
`+·-
`
`M>-
`
`,.....
`
`.,,....
`
`~;,...
`
`!-'
`
`r-
`
`,,......
`
`!
`
`,....
`
`f.-
`
`r- l
`! .1
`"'"' I
`·~-- 1
`
`(~~----~------~
`11:~~ t~. ~:
`~i I
`I~ T t
`l ~l 1 ~
`II! T t
`I f ~
`
`·~
`
`~=
`
`~
`
`1 !
`
`tQ
`~
`
`t'\J .
`~
`N u..,
`
`Y Y X l. Y ~( l·- l
`), A ~ 1\. A , _ ·-- 1\
`XXXXXXXXi
`YI\VJYYYY
`A .J ,AJ.AA
`)
`\
`................. ~\l
`
`

`

`U.S. Patent
`U.S. Patent
`
`Feb.18,2014
`Feb.18,2014
`
`Sheet 20 of 22
`Sheet200f22
`
`US 8,653,442 B2
`US 8,653,442 B2
`
`1· .. . .
`
`
`
`

`

`U.S. Patent
`
`Feb.18,2014
`
`Sheet 21 of 22
`
`US 8,653,442 B2
`
`FIG. 26
`
`}2$~
`?.~,j···· ~!s:~· !t::., =~·~~R-.OW~R.-A.....,IE1=,. ·=· =¢4t
`!
`,
`~
`
`!
`
`[
`
`......
`
`. . I
`
`!'UWfSl
`' .
`'
`
`'l
`
`OOIM
`ff:OiO!lHS!
`
`/"~$-4!* '
`
`

`

`U.S. Patent
`US. Patent
`
`Feb. 18, 2014
`Feb.18,2014
`
`Sheet 22 of 22
`Sheet 22 of 22
`
`US 8,653,442 B2
`US 8,653,442 B2
`
`Fig?2??"
`
`

`

`US 8,653,442 B2
`
`1
`MULTIPLE LAMINAR FLOW-BASED
`PARTICLE AND CELLULAR SEPARATION
`WITH LASER STEERING
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS AND PRIORITY CLAIMS
`
`2
`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/
`fll and men 5.4 million cells/fll. RBCs make up 93% of the
`solid element in blood and about 42% of blood volume.
`Platelets are 2 f.tm-3 f.tm in size. They represent 7% of the solid
`elements in blood and about 3% of the blood volume, corre(cid:173)
`sponding to about 1.5 to 4x10 11 cells per liter. There are 5
`general types of white blood cells (WBCs) or leukocytes
`accounting for about 1.5 to 4x109 cells per liter. The WBCs
`comprise: 50-70% Neutrophils (12-15 f.tm in size); 2-4%
`Eosinophils (12-15 f.tm in size); 0.5-1% Basophils (9-10 fll in
`size); 20-40% Lymphocytes (25% B-cells and 75% T-cells)
`(8-10 f.tm in size); and 3-8% Monocytes (16-20 f.tm 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 12x 1 09 per liter. A subject with an infection might
`have a WBC count as high as 25x109 per liter.
`Platelets are the smallest cells in the blood and are impor(cid:173)
`tant for releasing proteins into the blood that are involved in
`25 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/fll, and this number
`30 should not fall below 50,000 cells/f.LL 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.
`35 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
`40 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(cid:173)
`trifugation; return of red cells, leukocytes and plasma to the
`45 donor. A collection normally contains about 200-400 ml of
`plasma, which is reduced to avoid incompatibility. This col(cid:173)
`lection normally contains about 8 to 8.5x10 10 platelets. A
`donor normally gives approximately 10% of his/her platelets
`with no loss in clotting ability, although a larger number of
`50 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(cid:173)
`cess by which platelets are separated [Haemonetics Compo(cid:173)
`nent Collection System (CCS) and Multi Component System
`55 (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
`60 process is slow relative to the patience of donors and is
`capable of improvement for the purity of the separated plate(cid:173)
`let fraction.
`Other procedures are also time consuming, often taking
`several hours, particularly when unused blood fractions are to
`65 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,
`
`The present application is a continuation application of
`parent U.S. patent application Ser. No. 12/659,277, filed Mar.
`2, 2010, which is a divisional application of U.S. patent 10
`applicationSer. No. 12/213,109, filed Jun. 13,2008, now U.S.
`Pat. No. 7,699,767, which is a divisional application of U.S.
`patent application Ser. No. 11/543,773, filed Oct. 6, 2006,
`now U.S. Pat. No. 7,402,131, whichisadivisionalapplication 15
`ofU.S. patent application Ser. No. 10/934,597, filed Sep. 3,
`2004, now U.S. Patent No. 7,118,676, which is a continua(cid:173)
`tion-in-part of U.S. patent application patent application Ser.
`No. 10/867,328, filed Jun. 13, 2004, now U.S. Pat. No. 7,150,
`834, which is a continuation-in-part ofU.S. patent application 20
`Ser. No. 10/630,904, filed Jul. 31, 2003, now U.S. Pat. No.
`7,241,988, and claims priority via U.S. patent application No.
`10/630,904, to U.S. Provisional Patent Application No.
`60/399,386, filed Jul. 31,2002, and 60/435,541, filed Dec. 20,
`2002, and claims priority via U.S. patent application Ser. No.
`10/934,597, to U.S. Provisional Patent Application No.
`60/571,141, filed May 14, 2004, U.S. Provisional Patent
`Application No. 60/499,957, filed Sep. 4, 2003, and U.S.
`Provisional Patent Application No. 60/511,458, filed Oct. 15,
`2003, commonly assigned herewith, the contents of all of
`which are incorporated by reference herein.
`The present invention is related to Jessica Shireman eta!.
`U.S. 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(cid:173)
`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, U.S. Patent Applica(cid:173)
`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, U.S. Patent Applica(cid:173)
`tion Ser. No. 60/511,458, filed Oct. 15, 2003, entitled "Pas(cid:173)
`sive Fluidic Sorter", commonly assigned herewith, the con(cid:173)
`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 Gruberet a!., U.S.
`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
`
`

`

`US 8,653,442 B2
`
`3
`and the remaining blood components are then injected back
`into the donor. This centrifugation process is also compara(cid:173)
`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(cid:173)
`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.
`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 of a novel separation technique. One conventional
`technique in manipulating microscopic objects is optical trap(cid:173)
`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(cid:173)
`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 25
`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 oflight are employed to manipulate the position
`of a dielectric particle immersed in a fluid medium with a 30
`refractive index smaller than that of the particle, but reflect(cid:173)
`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 35
`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' 40
`focal plane while another beam entering at an angle comes to
`a focus off-center. A slightly diverging beam focuses down(cid:173)
`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 45
`at a location determined by its angle of incidence. The holo(cid:173)
`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 of an input beam is preferred for creating
`optical traps because trapping relies on the intensities of
`beams and not on their relative phases. Amplitude modula(cid:173)
`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 TEM00 input
`laser beam, this generally means that the beam diameter
`should substantially coincide with the diameter of the 60
`entrance pupil. A preferred minimum numerical aperture to
`form a trap is about 0.9 to about 1.0.
`One difficulty in implementing optical trapping technol(cid:173)
`ogy is that each trap to be generated generally requires its own
`focused beam of light. Many systems of interest require mul- 65
`tiple optical traps, and several methods have been developed
`to achieve multiple trap configurations. One existing method
`
`4
`uses a single light beam that is redirected between multiple
`trap locations to "time-share" the beam between various
`traps. However, as the number of traps 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(cid:173)
`mentations of this method to less than about 10 traps per
`system.
`Another traditional method of creating multi-trap systems
`10 relies on simultaneously passing multiple beams of light
`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 of traps increases, the optical
`15 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
`diffractive optical element (DOE) (e.g., a phase shifting holo-
`20 gram utilizing either a transmission or a reflection geometry)
`is used to alter a single laser beam's wavefront. This invention
`is disclosed, in U.S. Pat. No. 6,055,106 to Grier eta!. 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(cid:173)
`lations.
`Other types of traps that may be used to optically trap
`particles include, but are not limited to, optical vortices, opti(cid:173)
`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(cid:173)
`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
`50 shaped laser beam. 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
`55 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(cid:173)
`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 (U.S. Pat. No. 5,939, 716) is loosely, a mac(cid:173)
`roscopic cousin of the optical vortex. A light cage forms a
`
`

`

`US 8,653,442 B2
`
`5
`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,
`wavefront shaping, phase shifting, steering, diverging and
`converging. Based upon the phase profile chosen, the phase
`patterning 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 of two
`or more of these forms.
`Researchers have sought indirect methods for manipulat(cid:173)
`ing cells, such as tagging the cells with diamond micro(cid:173)
`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(cid:173)
`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(cid:173)
`sues, and contaminants is needed. Further, a way of achieving
`a unique contribution of optical trapping to the major indus(cid:173)
`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(cid:173)
`nique and apparatus which is continuous, has high through(cid:173)
`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 40
`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(cid:173)
`vide for separating components in a mixture, such as separat(cid:173)
`ing the various blood components of whole blood into corre(cid:173)
`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(cid:173)
`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(cid:173)
`tional means for separating and manipulating the compo(cid:173)
`nents, particularly holographic optical manipulation and
`separation. The various embodiments may also be applied to
`separations of other types of cellular and biological materials,
`such as sperm, viruses, bacteria, cell debris, cell organdies,
`globular structures, colloidal suspensions, cellular debris,
`and other biological materials.
`
`6
`As used herein, "Particle" refers to a biological or other
`chemical material including, but not limited to, oligonucle(cid:173)
`otides, polynucleotides, chemical compounds, proteins, lip(cid:173)
`ids, polysaccharides, ligands, cells, antibodies, antigens, cel(cid:173)
`lular organelles, lipids, blastomeres, aggregations of cells,
`microorganisms, peptides, eDNA, RNA and the like.
`An exemplary method of separating blood into compo(cid:173)
`nents includes providing a first flow having a plurality of
`blood components; providing a second flow; contacting the
`10 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
`15 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
`20 the second flow are substantially non-turbulent, and may also
`be substantially laminar.
`In a selected embodiment, the first blood cellular compo(cid:173)
`nent is a plurality of red blood cells and a plurality of white
`blood cells, and the second blood cellular component is a
`25 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
`30 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
`35 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(cid:173)
`ponent of the plurality ofblood 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
`45 form more complicated structures having multiple separation
`stages, connected in series, connected in parallel, or in com(cid:173)
`binations of both.
`A second exemplary method of separating a fluid mixture
`into constituent, non-motile components, in accordance with
`50 the present invention, includes: providing a substantially
`laminar first flow having the fluid mixture, the fluid mixture
`having a plurality of components, the plurality of components
`having a corresponding plurality of sedimentation rates; pro(cid:173)
`viding a substantially laminar second flow; contacting the
`55 first flow with the second flow to provide a first separation
`region, the first flow and the second flow having a substan(cid:173)
`tially non-turbulent interface within the separation region;
`differentially sedimenting from the first flow a first compo(cid:173)
`nent of the plurality of components into the second flow to
`60 form an enriched second flow and a depleted first