US 8,691,164 B2

Page 2
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* cited by examiner

US. Patent

Apr. 8,2014

Sheet 1 0151

FIG. 1

US 8,691,164 B2

US. Patent

Apr. 8,2014

Sheet 2 0151

US 8,691,164 B2

Sheath ?ow: 50/50 splitting of cells

Buffer >

<- Buffer

FIG. 2

US. Patent

Apr. 8, 2014

Sheet 3 0151

US 8,691,164 B2

Skewed particle flow via differential sheath ?ow rate

Fluorescence-Negative/Optical Switch not triggered

37

(10

32

,JL
0

Buffer ~>

<'Buffer

(lower ?ow rate)

:Hli

(higher ?ow rate)

I i

a1

I
i

\54

'1 E
fE

55

0 l
, l

.A
35
Fluorescence Negative

37
Fluorescence Positve

Non-target Cells

Target Cells

FIG. 3a

US. Patent

Apr. 8,2014

Sheet 4 0151

US 8,691,164 B2

Skewed particle ?ow via differential sheath ?ow rate

Fluorescence-Positive/Optical Switch triggered

Buffer 0i

<~ Buffer

(higher ?ow rate)

(lower ?ow rate)

Fluorescence Negative

Fluorescence Positve

Non-target Cells

Target Cells

FIG. 3b

US. Patent

Apr. 8,2014

Sheet 5 0151

US 8,691,164 B2

Skewed particle flow via differential channel width

Fluorescence-Negative/Optical Switch not triggered

40
47

\k rj/
l e{

Buffer >

<-Buffer

'1 i
I I
\

E \44

Lew-45
E
l

'
.

i
l
I

, /\
45

47

Higher ?ow rate

Lower ?ow rate

Fluorescence-Negative
Non-target Cells

Fluorescence-Positve
Target Cells

FIG. 4a

US. Patent

Apr. 8, 2014

Sheet 6 0f 51

US 8,691,164 B2

Skewed particle ?ow via differential channel width

Fluorescenee-Positive/Optical Switch triggered

Buffer ~>

< Buffer

Higher ?ow rate

Lower ?ow rate

Fluorescence-Negative
Non-target Cells

Fluorescence-Positve
Target Cells

FIG. 4b

US. Patent

Apr. 8, 2014

8305621I.'Y|l%5
i.52:s5?

@2300

a
0
0
a

US 8,691,164 B2

Sheet 7 0f 51

s
Q

nH?3uoz\xi2mrgY/!5e0
6
0

u>QsonmEigw?zlcZ20

e
o

aHuSE?shmlarv1!.m

2$6EBwH3muh0D?kOobZmazh

m65m

US. Patent

Apr. 8, 2014

US 8,691,164 B2

Sheet 8 0f 51

hMQOGHUlkI"am!DBiF-UT
5li:ant5i:.

2B1E5D

llmuJ
omw
3@8 56?$0z2:I.'/3w0
6
Q

huS?Estimz[la1Av!.m

O
G

0
o

gsDw>EmoZc320i
gumZcho?s
gEmobuaqi

EZBaEwOy-cPo

m65%

2836.052
EwbO?w

US. Patent

Apr. 8, 2014

Sheet 9 0151

US 8,691,164 B2

~Particles enter on "In" side

-Laser pattern is turned on at appropriate

locatien for appropriate amount of time
depending on which channel particle is to be sent
to on out side

'Coneept can be extended to MxN switching

instead of 1xN
> Flow Streams

In

/ Optical Switch

@2322 ill!
/l\

Out

FIG. 7a

US. Patent

Apr. 8, 2014

Sheet 10 0151

US 8,691,164 B2

-Input of arbitrarin positioned particles and

multiple arbitrarily drawn laser beams to sort
them
In

Q?
o

<3

</

WW

\> AnalyS1s
/

Region

---> Flow Streams

/ Optical Switch
0 o 0

Out

FIG. 7b

US. Patent

Apr. 8,2014

Sheet 11 0151

Cells

US 8,691,164 B2

Cells

Fluorescence detector > -e-1'i

l

Optical Switch

i-
<Fluorescence detector
l
l

.1 6/ Optical Switch
l

Fluidic split plane

Higher flow rate

Fluorescence-Negative

Higher ?ow rate

Fluorescence-Negative
Lower ?ow rate

Fluorescence-Positive

FIG. 70

US. Patent

Apr. 8, 2014

US 8,691,164 B2

Sheet 12 0f 51

Cylindrical lens

A.)

LCM

PBS

PBS

B.)

AOM

Cylindrical lens

c.)

AOM

[1:]

LCM

PBS

FIG. 8

PBS

US. Patent

Apr. 8, 2014

Sheet 13 of 51

US 8,691,164 B2

Fluorescence Positive/Optical Switch triggered

Cel ls
o

Buffer w>

<Buffer

/Fluorescence detector
'

rOptical Switch

/.
Fluorescence Negative
Non-target Cells

G
Fluorescence Positve
Target Cells

FIG. .93

US. Patent

Apr. 8,2014

Sheet 14 0151

US 8,691,164 B2

Fluorescence Positive/Optical Switch triggered

Cells

l
Buffer >

<-~Buffer

A/Fluorescence detector
Q)

<-- Optical Switch

Q)

o /\ e
Fluorescence-Negative

Fluorescence-Positve

Non-target Cells

Target Cells

FIG. 9b

US. Patent

Apr. 8, 2014

Sheet 15 0151

US 8,691,164 B2

Method for timing and triggering optical switch laser

-Single laser and separate detectors used to detect particle and trigger switch laser

Detector Arrangement:
Sort parameter negative/ Optical Switch not triggered
Cells

let

Buffer >

I
______

1'

<-Butfer

__..______._

Detectors:
Fluorescence and Forward scatter w"
measured from single laser source

t1

. -=

t
1

i
0

i /

Negative: No sort

FIG. 10a

Error check detector

Positve

US. Patent

Apr. 8, 2014

Sheet 16 of 51

US 8,691,164 B2

Method for timing and triggering optical switch laser

- Single laser and separate detectors used to detect particle and trigger switch laser

Timing/trigger diagram:
Sort parameter negative/Optical Switch not triggered
,

Forward scatter

/////

///,/

er atelinit

ppg

signal

. v

/
Fluorescence

..

Lower gate limt

,// / /, /

/,

Upper gate llnit

Lower gate hnit

/\

signal

'

Optical Switch
Error check

detector signal
t1

t1

FIG. 10b

time 7

US. Patent

Apr. 8, 2014

Sheet 17 0151

US 8,691,164 B2

Method for timing and triggering optical switch laser

- Single laser and separate detectors used to detect particle and trigger switch laser

DetectOr Arrangement:
Sort parameter Positive/Optical Switch triggered
Cells

Buffer h+

kBUfer

Particle detectors:

Fluorescence and Forward scatter I ah-"r"?

_

'

I |

measured trom Single laser source

Opncal SWHCh lasgr set

'

H,

dlsmnce X: from _ 1

{2

Fluorescence/particle detector

ll

Optical Switch

l
l3

/0

Negative

Error check detector

Positve: Sort

FIG. 1 00

US. Patent

Apr. 8, 2014

Sheet 18 of 51

US 8,691,164 B2

Method for timing and triggering optical switch laser

- Single laser and separate detectors used to detect particle and trigger switch laser

Timing/trigger diagram:
Sort parameter positive/Optical Switch triggered (t 2 is a pre-set delay)

Forward scatter
detector srgnal
Fluorescence

Upper gate 1th

l

i/

J l\
jf/

/ /

Upper gate unit

Lower gate linit

detector signal

Optical Switcn mi
Trigger

Error check

it

detector signal
t1< At > t2

FIG. 10d

113

time

US. Patent

Apr. 8, 2014

Sheet 19 0151

US 8,691,164 B2

Method for timing and triggering optical switch laser

- Separate lasers and detectors used to detect particle, determine particle ?ow rate and trigger switch laser

Detector Arrangement:
Sort parameter negative/Optical Switch not triggered
Cells
.

My

Buffer --
.

l{

Forward scatter #1 measured

...

from a laser source

Buffer
---------~

m,

H
a.
H 1

by distance d

t
t3

it i

Particle detectors:

51

Fluorescence and Forward scatter #2

'

measured from other laser source

E
'
O

t4

/\/

Negative: N0 sort

Error check detector

Positvc

FIG. Ila