CA1326556C - Single molecule tracking - Google Patents

Single molecule tracking

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Publication number
CA1326556C
CA1326556C CA000578710A CA578710A CA1326556C CA 1326556 C CA1326556 C CA 1326556C CA 000578710 A CA000578710 A CA 000578710A CA 578710 A CA578710 A CA 578710A CA 1326556 C CA1326556 C CA 1326556C
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Prior art keywords
spatial
detected
coordinates
emissions
train
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CA000578710A
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French (fr)
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Edgar Brooks Shera
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University of California
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University of California
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1456—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Electro-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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N2015/0038—Investigating nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0205—Investigating particle size or size distribution by optical means, e.g. by light scattering, diffraction, holography or imaging
    • G01N15/0211—Investigating a scatter or diffraction pattern
    • G01N2015/0222—Investigating a scatter or diffraction pattern from dynamic light scattering, e.g. photon correlation spectroscopy
    • G01N2015/1027—

Abstract

SINGLE MOLECULE TRACKING

ABSTRACT OF THE DISCLOSURE
A detection system is provided for identifying individual particles or molecules having characteristic emission in a flow train of the particles in a flow cell.
A position sensitive sensor is located adjacent the flow cell in a position effective to detect the emissions from the particles within the flow cell and to assign spatial and temporal coordinates for the detected emissions. A
computer is then enabled to predict spatial and temporal coordinates for the particle in the flow train as a function of a first detected emission. Comparison hardware or software then compares subsequent detected spatial and temporal coordinates with the predicted spatial and temporal coordinates to determine whether subsequently detected emissions originate from a particle in the train of particles. In one embodiment, the particles include fluorescent dyes which are excited to fluoresce a spectrum characteristic of the particular particle. Photons are emitted adjacent at least one michrochannel plate sensor to enable spatial and temporal coordinates to be assigned. The effect of comparing detected coordinates with predicted coordinates is to define a moving sample volume which effectively precludes the effects of background emissions.

Description

SINGLE MOLECULE TRACKING

BACI~G~O~ O~ ~ INv~rIoN
~hi~ i~Y~ntio~ ~ g~n~ally r~lat~ to the dst~ctio~
0:~ alliC~O~GOp~ art~clo8 andO llOC~ pa~rtic~ rly, lto ehc ~tlo~tio~ an~ ti~ica~io~ o ~i~gl~ ~o~cul~.
T~o capability ~or ~ot0cti~g 1aiero~copi~ part~
pEocæ~di~g to~r~ ~aal~er p~ ttclo~. ~or ~y a~ppl1ci~t~o~l~ it ~ 2~ntial1 tllat ai cEof~eopic par~iele~ ~
~t~t~d isl 8 liqui~-~h2~ e~triro~t. Ex~ei~g to~hs~igu~, u~ in ~ lig~ pha~ ro~n , ~r~
10 b~e~ o~l o~ti~al ~ppi~g ~lld oll ~lo~ ~parae~o~ u~ing hy~rody~ cally Po~u~ 10~8 . Eloleeul~lr i~aelf ication ~y la~er-in~u~od f luor~c~ has ~ u~d ~d~t~ :
hyaeoay~ cally ~o~ d ~lo~ to p~r~lt th~ d~t~ctio~ of largs ~ hi~y t1UOE~ t laol~ule~ u~i~g co3~rent~0nal 15 photo1al2le~pli~r t~b~ eo d~e~t th~ ~01e~:U1g~ ~luore~co~ce~
O~tical ~rapp~g a~d a~an~ulatlo~ ç~ ru~fls a~a ~act~ri~ a~e t~ught i~ A. A~hkl~ et allo ~ ~opeis~ Tr~ppi~g t an~ ~Sa~p~latio~ o~ Vin~Og~e~ a~ ~acteria, " S~ ce 23~, 1517 11987). ~yl~îg~l~ a~ lzo~ par~icl~
par~ iz~ ra~g~ fro~ about 10 ~ do~ to a~stro~, haY~ bee~ trapp~ usi~st opt~ca1 o~c~ ~o ~O~ Q~ ~ho pareicl~s . $ho o~ly 3~ethoa of ideneif i~alt~ o~

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taught by Ashkin e~ al. appear6 to be a ~ize de~er~i~ation from a ~cattering compari60n with a ~E~here of known size.
Purther, a larqe number of particle6 arle trapped.
A hydrodyna~ically focu~ed flow ~y6tem i~ taught by D. C. Nguyen et al., ~Ultra6en6itive La~er-I~duced PluoreRcence De~ection in Hydrodynamically Pocu~ed Plo~,~
J. Op~0 Soc. Am. B4, 138 (1987), and D. C. Nguyen et al., "Detection of Single ~olecule6 of Phycoerythrin i~
Hydrodynamically Focu~ed Flows by Lasee Induced Fluoreficence~ n Anal. Chem. 59, 2158 l1987). As taught therein, improvements in the optics and reductions in the size of the pxobe volume provide a ~en~itivity effective to detect a ~ingle specie6 containing the fluore~cence equivalent of eight rhodamine-6G chromophores. The detection of 6ingle ~olecules of the hig~ly f luore~cent ~pecies phycoeryt~rin i6 reported.
A variety of modiication~ are reported to enhance the detection ~en~itivity of the device, with the impcovement~
being related to conv*ntional opti~6 and flow dynamic~, and with a sample volu~e reduction from 11 pL ~o 0.6 pL
produci~g a concomitant reduction in de~ected background radiation. The reported sensitivi~ies do not, however.
enable the devi~e to detect individual ~olecules that ~i~ht typically be of intere~t, ~uch afi fluorophore-tagged ver~ions o the ba~e oolecule6 that ~ake up the DNA
polymer.
Thus, available method~ and apparatu6 for detecti~g particle~ i~ a flow 6tream do not provide the gen~itivity for detec~ing individual ~olecules that migh~ typi~ally be encoun~ered in i~munofluore6cence as6ay, flo~ cytometrY.
liquid chromatogEaphy, and ~imllar application6. An ,~, . .
.;
.:: . . , -.~ .
: ,. .~.... .; ., . . .

132~

agglo~ tio~ of ~lecll~e~ ~igh~ be d~t~te~. but ~ole~ule~ ~ould ~ot tl~en ~e identlf ~d. Th~s la~k of c~ablliti~ the are i~ overcome by t~e p~ nt lnve~ltio~ a~d ilsprov~d ~tbod ~d appalra~u~ l~r~ proYided 5 for dete~ti~g a ~i~gl~ ~ode~tly ~luo~Q~eo~t Z~lolecule.
A~cordingly, ~t 1~ an ob~ect of the pre6 E!lDlt i~ tio~
to ~eliably detect a sin~le fluore~een~ Elole~ule.
A~oth~r obje~t i8 to reliably tlet~ct 3i~gle f luores~e~t ~olecule~ b~ith ~ f luore~cenoe ~qu~ralen~ to 10 luore~cently-lab~lea versio~s of the b~6es orlZairlg the ~N~ poly~er.
Y~t another ob~ec~ i~ to provide a~ increa~ed capability of re~ec~in~ b3ckgEou~d radiatio~.
On~ othe~ ob~e~t i~ to ~ini~ize ~he re~olutio~
15 11mitation~ lnher0nt i~ co~l~ec~cio3al opeic~ while ~ai~t2i~ing a lar~e ~ield of v1e~.
~U~Y OF T~ INV~NTION
To achieve the f oregoi~7 ~d ot~er o~ ject~, a~d in a~cordance vi~h th~ purpo~e~ of the pre~ellt i~ve~on. a~
20 e~bodied ~nd broaaly de~eibed herei~, the apparatus of this invention snay coD~prise a alolecu1e detection sy~t~
~or id~Qtifying ~ndi~idua1 1ao1ecu1ar cha~acteristic emi~1O~s in a train of ~olecule~ a f1OV ee11.
po~ition s~n~itive sen~or ~ea~ located ef~ect~re to 25 de~ct e~is~ion~ fro~ uolec~le~ ~ithin the ~lov cell alld to a~1gn ~pa~i~1 a~d te~poral coordi~ates îor the detec~ed e~i~sion~. A co~put~r predicts ~;pat~a~ and tempora1 coordinate~ o~ a Ro1~cu1e ~n l:he 1a~ ar ~1O~? as a fu~ctio~ o~ the d~cected ~oordi~a~e~ of a ir~t deteet~d 30 ~olisYio~. Co~pacl~o~ eanG the~ co~pares det~Gted ~patial aad te~poral coordi~a~e~ ~n~h tlle predicted spatia1 al~d ~e~po~al coocdi~atQ~ to a~ter~i~e ~rhe~her a detect~

.

. ~2~5~J~

em~ion oliçlnated fro~ a~ ~cited ~olecule i~ t~e tr~i~
of l~oleculeE~ Thufl~ ~oleGular e~ lo~ .ca~ be di~ti~qui~h@d fro~ back~round ~ Dn~ and i~e~t~fied wieh a particular ~ol~eul~ i~ the ~equence.
In a~o~her characterizat~on of the present ihven~io~, a detect~on ~ethoæ i~ pro~ided for identi~yi~g i~dividual ~olecules ~ithi~ a ~lo~ cell ro~ characterist~ ~olecula~
emi~sio~s. ~ole~ular e~ isn~ from ~ e ~lov eell are d~tected vie~ a po~it~o~ ~en~i~ive ~en~og. Spatial and temporal coordinate~ are the~ as3igned to the detect~d emissio~. Ba~ed on k~o~ 10~ charac~eri~tic~ i~ the ~lov cell, ~p2tial and t~poral coordinate~ ~re pr~dic.ted for a ~olecule in the flow as a functio~ of a ~ir~t detected e~i~3~0n vithi~ the flow cell. Ttle dete~d ~atial a~d teDIporal coo~ a~QE of ~ub~egue~t e~ ions are ~o~pa~ed vith p~edicted ~pati~l a~d ee~poral coordi~ates to d~ter~ine ~hethar a dete~ted ~ lo~
o ~gi~te~ fro~ a ~olecule in the l;rai~ of D~olecule~.
Th~ olec~l~r e~is~ion~ ar@ distingui~hed fro~
ba~kqround eve~e~ a~d a single aoleeule can be i~e~l~iied duriGg pas~age t~rsugh the f lo~ sell .
BRIE~ DESC~IP~ION OF THE DRAWINGS
The ~ccompa~ying drawi~s, ~hi~h are i~o~porated i~
an~ ~o~ a part of ~he speciicatio~, illu~trate an ~bodi~ent of ~he p~e6e~ ea~ioa and, together ~ith the de6criptio~ 6er~e to ~plai~ the prin~iple~ of ehe i~ve~tio~ the dra~i~gg:
~IGU~E 1 ~s a block diag~a~ ~chematic o the presen~
i~vention.
~IGU~ 2 i~ ~ detail o~ the sy~te~ flo~ cell in pictorial ~or~. .

, ' 13~

~ IaUl~E 3 1~ f lov chart f or d l ~ti~ hir~ d ev~luaei~y individual ~olec~len iQ tbe f lo~ ~
~5~1~ ~ I~TION
l~eferri~ ov eo Figure 1, ~chere i~ æ~o~n in block diagra~ ~c~e~atic ~ora a a~ol~cule deteceion ey~t~
accordi~g to t~e ~re~ent inveneio~. The laser e~c:leatio~
~yste~ iB gene~ally ~ ovn and de~cribed in tll~ 2aguye~
et al . article~, hereinabove ref erenced . La~er 10 i8 sele~ted with a wavele~gth eff0ctive 'co fluore~ce a 10 selected fluorophore for id~neifying the ~lole~ule to be detected. The output fro~ la~er 10 i~ co~reD.tio~ally ~a~sed throu~h half-~,ra~e plate 12 and polarizinq 1p2i8111 14, wher~in the outl?ut power o~ la~er 10 ~a~ be ad ~u~ted by varying the a~gle of pl2lte 1~ ~ith re~p~ct ~o pr~s~
15 The la~er output powes arld the polarlza~cio~ can be aa3u~ed to ~ini-lize background cou~t~3.
~ ir~or 16 direct~ the laser b~am th~ough l~u }~ to foeu~ Yitltlil~ flow cell 22 for activating fluorophore~
attac~ed to ~olecul~ in the ~a~ple ~t~ea~. A~ ~o~
20 Figure 2, ~a~ple ~erea~ g2 is ortho~o~al to focused laser beaa 46. ~a~ple ~trea~ ~2 passe~ withi~ a su~rou~ding hydraulic sheath ~4 to p~ovide ~ydrodynamie focu~ g of the flov Yithin flow cell 22.
Ref~rri~g again ~o Figure 1, the output ~ro~ iElow 25 cell 22 i~ optical ~ig~al 20 Yith iafor~ation on tbe fluore~cinq ~olecule~ hin flow cell 22. Opei~al ~ignal 20 i~ ocus~d by Dllicro~opic ob j~cti~e len~ 24 a~d fil~e-ed by spectEal filter 25 to re~ove ~avelen~th~ vhi~
are not of i~tsrest. The output opocical ~iqnal i~
30 ~o~rided ~o a po~itio~ ensitiY~ se~sDr 26. 1~ o~
eDlbodi~en~c~ positioQ-~e~itive detec~or ~6 i~ fo~med f~o~
a mic~oclla~nel plats po~ieio~-~ensi~ive deeec~cor (~ICP) ~d opera~io~ he~einaf ~er di~cu~sed with eespoct to a ~qCP.

A ~os~eio~-sel~iti~ ~ete~tor or ~CP 26 output~ a ~ig~ hich i~ i~di~ati~ of tbe oocurre~ce o~ a pboto~
ev~nt bri~hin ~lo~r cell 22 a~d al~o lo~tlon d~tal functionally rel~ted to the ~pa~ial coordinate~ of the ~hoto~ ~ven~. Spatial eoord~nates are p~o~ided to d~ gitizer 2~ and co~bined ~ith a te~poral input rom tl~er 32 to provide at least a ehre~-di~en~ional (~c, y, t~
lo~eion for the photo~ 0v~llt. Photon ~vent coo~dinate6 a~e outpu~ fro~ d~gl~izer 2~ 'co ~neoory 34 for ~ub~eque~t proce~8i~g by co~puter sta~cion 36.
Re~erci ng ~ov to po~itio~-sen~itive sen~or 26 . it de~ira~le to ~ave the resolutioll o~ the sy~e~, and related position accusacy, lio~ited ~y the ~y~te~ opti~
rather tha~ a ~CP. CollY2~tio~al ~SCP5 aay ~ave D
po~itional re~olu~io21 o 500-lOOO pi~el~ ~n ~ac~
diDIRn~io~. If t~o p~cels ~o~er ea~h Rayleigh li~ait, resolutio~ ite~ by t~ opti~ a~ld ~ ield of v~ e~ of ~00-200 ~icron~ in ~ ete~ i~ pro~i~ed by ob)ec~i~re le~
2 By vay of e~a~pl~, the ~ayleig~ it at a va~elengeh Of 560 ~ i6 about O . 4 ~icrons . ~hus, a positio~ accura~y of 1 ~i~ron ~equire~ ol~ly a pr~ci~io~ o ~ 2 pi3cel~. A
~uieabl~ ~cP i~ available as model F41q61q rool ITT, El2~tro-Optical Product~ Di~i~io~.
Digit~zer 28 provide~ ~pati~l a~d te~poral coordillate da~a il~ a f oc~at tl~at i~ able f or ~irect ~torag~ in aelRory 3~ ~d caQ opera~ in real time. Collvee~tiol~al a~cP
po~itio~ ~ircuitry digit~ze~ in about 5 lls. 'rhi~
digiti~ing lllter~al ca~l be reduced to abo~t 1 tl~, ~
1es~, vith cu~to~ circuit~y~ i a hig~ data rate ope~aeion i~ d~irable. A 1 ~1~ digitiæing i~ter~ra1 vould eelable a ~a~i~um photoll d~teetioR raee of 1 ~2 srJ e.g.,. 170 ~ho~o~ du~ing the ~aYI~it tilee pr~di~eed by ~quy~ et al.

.

.

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for ~ ~y8tem co~p~rablç eo ~low coll ~2. a~ ~iiscus~ed b~lo~r, the de~ectio~ of only a ~ photoals ca~ p~o~ride ol:
reliable ~loleeule ide~tif ication ~Yell ~ith a relatiYely un~op~ lcated dat~ reduc~lon 819101:ithl~1. The 10 5 velr~city and la~er inten~ity ~a~ readily be ~d~u~ted to provide a data rat~ ~d o~er~ation t~e su~table ~or pa eicul~r applica~io~.
I~ ~ill be ap~reciated tha~ ~he above Ry~tem pro~r~des the po~itio~ accuracy needed to ide~tiîy a photo~ e~ren~
10 ~dithi~ cro~ or leE6. With a ~uitable wiath o~ t2~e la~er bea~ in t~le lo~gitudinal di~ection as, fo~ e~Jcample, by e~ranescent wave illu~i~atio~, this aceuracy ehus produce6 a~ effecti~e ss~ple ~rolum0 o~ 10-l8 1a3, or 10 3 pL, ~ ~ample ~olusle redustio~ by about 500 o~rer the 15 o. 6 pL vaîue di~cu~f~ed in l;l~uyer~ et al . T~e ~fecti~
~a~ple ~olume a110s~s the ~y~te~ to ~ ri~ te agai~t photoa 2ve~t~ ~hich do ~o~ origi~a~ with a 9E1uoreE~ci~lg ~olecul~ ~lnce only a fe~ ~caeto~ g eYeQt~ ill rando~1y occur Wit~ the ~f~ive s~a~ple ~roluD~e. The 1alsinar 20 f10v pro~ le~ own trajQc~orie~ fo~ ~o1ecu1~ haYing a ic~ostn ~elocity. Detected photon eve~t~ cas be co~hpa~ed ~eb predicted molecule coordi~ate~ and photoll e~e~t~
~hicll do not correspond vith pr~d1~ted ~oordiQate~ can be di~regaraed. Thi~ ~apabili~y eec~cive1y prosride~ a 25 ~ovi~g sa~ple voluDle as ~mall al8 10 3 pL ~ithia ~hich th~ p~esewe of a ~olecu1~ can be re11ab1y predic~ced.
The above ~y~te~ ha~ b~ea described a~o~re u~ing a hydrody~la~ic f lo&r regi~e and dl ~E1uorescinq ~ole~ul~ .
E~o~e~r, the fu~c~io~a1 pri~cip1es apply equally to any 30 dy~a~ic sy~teDI ~hic~ caQ ~ai~ta~ a ~redi~cab1e ~lo~ of ~o10cu1e~ or s~a11 I?arti~les in ~eq~ ce throu~h a d~ectol:0 L~ewi~, 1a~er-induced ~E1uo~esce~ce ., :, ' ~32~

co~v~ni~nt t~c~niqu~ to ~ag ~lld ide~tl~y ~olecul~s. ~11 that i~ ~eded~ ~ov~ver~ iB ~1 det~t~ble e~ia~ioa~ fro~ t~e oolecule o~ part~cle. Alternati~rely, o~her slolecular e~i~sion~, such a~ electro~. qamma ray6, and the like.
can be dete~ted by su~able po~it~o~-sencitive dev~
The ~r~ent inven~io~ broadly contemlplat~ hydrody~aaic arld a~rodyna~ic 10~ regi~e~, a~ well a~ olecular emission~ of all ki~d~.
~ aefeeci~g nob~ to ~igure 3, ehere i~ ~ow~ a f lo~r diaqr~lQ for e~emplary ~oftwaYe for dete~ the pre~ence of a ~ole~ule i~ an eSf ective sa~ple Yolu~e . On the o~cucrence of a photon ~vent lla f ro~ itlCP 26 ~Fl~ure 1~, the eYe~t coo~di~ate~ (xe, y~, te) are input 52 and a bic i~ def i~ed 54 vie~ ~oordi~ate~ reD
t~) w~ere t~ nor~alized 53 to the ti&e 2 ~olecule ~a~ng ehe spatial coordi~ teR ~ould ha~ Ql3~s~ed ~
fi~ld o vie~ de~ ed l~y obj~ti~re lel~ 2~ t~ e l~.
The ~e~ def iD~ed bi~ iB co~pared 56 ~ith e~isti~lg ~i~s . Tf ~he ~ev bi~ ~oe~ l~ot e~ , 'ch~ ~e~ bin ~ ~to~ed 58 ~o repre~e~t an initial e~ene a~d tlhe contene6 of Ibia f ile 51 are updated 62.
If ~ ing bin coordinate~ a~commodate the def ined event bi~ coordi~ateg 54, the even~ is as~ignea to tlh~t particul~ bi~ 64. Bi~ ontinue to accu~ulate 64 e~ellts u~til th~ bin te~poral coo~dinate indieate that the bi~
ha~ ~as~ outside elhe ~eld oP ~riev o t~e ~ystela.
Ti~se~ 66 periodically cauee~ the bin~ eo be exalained 8 to dete~ e ~hetl~er a Ibi~ i~ still ~dithi~ t~e field of ~ie~ or whet~er a ~olec~ e ~a~ p~e~ent i~ that birl. B1 that a~cu~ulate a large ~u~ber of e~e~t~ have a higher probability o~ cont~ g a~l a~tual linole~:ula than ~ 18 wi~ll fe~e~ eYe~le~. The ~alall ef~e~ e s~mple volu~e ~L ~ 2 ~

which is prov~ded according to the pre~ent i~ventio~ ca~
produce a cleat ~eparatio~ betveen bi~$ that eontai~
~ol~cule~ and tho~e ~hat corlt~1n onl!;r ra~do~a back~rourld event~O Af ter the bin count i~ proc~e~7 the bin i~
S ~l~aled 74 foe reu~e. I the pr2s~nce of a ~olacul~ 1~
indicated, the fluoresconoe aata can be proee~sed 72 foe the particular deter~i~atio~ bei~g ~ade by the ~yPtem.
~ore co~plex ~ata reduction algo~i~;h~s olight flarther con6ider di~usion ahd otlaer departur~ fro~ lar~inar ~lov 10 that ~an OCCUI in variou6 applicatior~s.
T~e capability to ~raclc and identify an individual slolecule provide~ applieations w~ic~ are not po~3ible u~1 ng con~e~ional pho~o~ul~iplier tubea . In o~e i1aportant application, tlhe system sti~ht be ~dapt~d to 15 deeeet ar~d identi~y inaiYidual base~ for~ g a DNA
seguen~e. A pl~rality of la~eL ~ra~elellgth~, alo~ or eolabi~atio~ vlth separate f i~ter~ 25 an~ ~le~e~to~ Z6 ~rigure 1), coul~ be uged to excit~ di~idual ~aolecule~
a~ they pa~6 through the ~ample f lo~ ll:ell 22 to ~deQt~ir 20 f luore~ce~t ba~e-~pecif ic label~ vhich ar~ a~tached to the molecule~. The t~ack of a ~ole~ule vill alter~ately appear or di6appear to ~nable E~olecule ide~tiicatlo~
during th~ exeitation 6eguence. Se~reral l~olecules ~ay be ~imulta~eou~ly pre6e~t i~ f low cell 22 ~nd be i~dividually 25 traclced for i ae~ificatio~.
~ ahile a ~i~g1e ~5CP 6y~tem ha~ bee~ discu3~ed above, it ~ay be de6irable to p~ov~ide ewo orthogo~ally pla~e~ ~CPe ~o i~lcreaze ~be nu~b~r of photo~ h are collect~d durillg transit of the ~olec~ule through f lov celï ~2 a~d to 30 provide additional ~patial infor~atio~.~ Dee~t~d pho~o~
eve~lt~ would l~e corelated to provide ~o~plete th~ee-di~e~io~al ~patial ~oordi~at~. A d~teeted pho~on ~' ' .

,: , . . . ... ..
3 32~r,~j6 1~

~n eae~ of two o tho~orlally pl~ced detecgor~ w~ n prillciple, enable a tr~ectory to be ~red~ed, suclh tltlat the pre~ence of ~ third photo~ on the co:~puted four-di~en~ional trajectory iB ~vide~e o~E a ~ol~cule 5 pa~s~ge.
lt ~Jill b~ app~e~iated that tlle detection o~ th~e ev pl~oeo~ ~n t~e available ~ran~it ~i~e~; produce~ an inf inite~ilaal probability of l~li8~iD,g a Qolecule entirely.
By ~ay of e~ca~ple. in a 2 ~CP geo~try, and a 170 1~8 10 tran~it ti~e, ~ho~dn by laguyen ~t al ., ~uPra , in qrable 3 , it e~an be e~till2ated that ea~h dete~tor vill accumulate 8 real a~ld 40 b~cl~grou~d photo~ lU8, the 2 ~CPs ~ill detect A ~ea~ ~u~ber of 16 photo~ ~ pro~ridi ~g a p~obability of l~s6 thall 10 ~ of detecti~g few~r tha~
15 the 4 photon~ needed for solecul0 dete~tio~.
T~e so~tw~re discus~ed for Pigure 3 can be pro~ided for eacb ~ICP a~d the bi~ r~ed duri~g t~2 pfoce~ g. A
~erSI~r deter~i~aeio~ eould be ~ade o~ ~he ba~i~ oI the available ~om~on i~forlDation. i.e., tiL~e coordi~a~e6.
20 Bin~ ~ight be e:~a~i~ed f or ~ergi~lg o~ly af ter a ~ uo number of e~r~n'c6 ~re accu~ulated i~ t}~at bin, t~ereby a~suriQg that the ti~ae coordi~ate6 of bo~h bi~6 are æufficie~tly well-dete~ ed to Dlake a ~ralid comparisoQ
fo~ ~Ihe ~erger. ~he bill ~ith t~e large ~um~3~r of 25 a~cumulated e~fe~lts ~ight be ~elected for the ~e;cgea binO
v~er~by all ~ubsequent photo~ ev~nt~ o~ ~che bil~ tra je~cory are a~ig~ed to a Rillgl~ bi~.
Tll~ ~oregoi~g de~criptio~ of the p~eferr~d ~mbodi~e~
o f the i~eD.tio~ ~a~ bee~ ~res~D~ted ~or purpo~e~ 3' 30 illu~tratioa ~nd de~cription. It i~ not i~tended to be ea~hausti~ or to lhait t~e i~e~tio~ ~o th~s pr~ci~ îor~
di~clo~d, and obYiou~ly ~a~y modificatio~l~ al~d ~aria~ion~

....
...

~ 3 ~ g ar~ po~ ble 1~ light of the ~l~o~e geach~g~ Tlle ~mbodiaent bras ehosel~ a~d de~cri~ed ~ order to ~t ~xplain ~he pr~cipl~ o~ the i-~e~tio~ a~d it~ praceical application to thereby ~nable other~ ~killed in t~le ~rt to S befit utilize th~ Ye~ion i~l various ~bodi1~ent~ and with variou~ ~odif icataon~ a~ ~re ~uited to the l?arti~ul~r u e eorltelDplated. lt i8 i~te~ded that the ~cope of ~b.e invention be def ined by the claim6 apperlded hereto.

Claims (12)

1. A molecule detection system, comprising:
a flow cell for passing a train of molecules in laminar flow:
laser means for exciting said molecules to emit photons at a selected wavelength;
position sensitive sensor means effective to detect said emissions within said flow cell and assign spatial and temporal coordinates for said detected photons:
computer means for predicting spatial and temporal coordinates for a molecule in said laminar flow as a function of a first detected photon; and comparison means for comparing said detected spatial and temporal coordinates with said predicted spatial. and temporal coordinates to determine whether a detected photon originated from an excited molecule in said train of molecules.
2. A detection system according to claim 1, wherein said position sensitive sensor means includes at least one microchannel plate sensor for outputting said spatial coordinates.
3. A detection system according to claim 1, wherein said position sensitive sensor means has a position accuracy effective for said computer means to create from said predicted coordinates a moving sample volume effective to functionally eliminate background signals from consideration.
4. A detection system for identifying individual molecules having characteristic emissions in a flow train of molecules in a flow cell, comprising:
position sensitive sensor means effective to detect said emissions from said molecules within said flow cell and assign spatial and temporal coordinates for said detected emissions;
computer means for predicting spatial and temporal coordinates for a molecule in said flow train as a function of a first detected emission; and comparison means for comparing said detected spatial and temporal coordinates with said predicted spatial and temporal coordinates to determine whether subsequently detected emissions originate from an excited molecule in said train of molecules.
5. A detection system according to claim 4, wherein said position sensitive sensor means includes at least one michrochannel plate sensor for outputting said spatial coordinates.
6. A detection system according to claim 4, wherein said position sensitive sensor means has a position accuracy effective for said computer means to create from said predicted coordinates a moving sample volume effective to functionally eliminate background signals from consideration.
7. A detection method for identifying individual molecules having a characteristic emission in a flow train, comprising:
detecting in position sensitive sensor means molecular emissions within said flow train;
assigning spatial and temporal coordinates for said detected emissions;
predicting spatial and temporal coordinates for a molecule as a function of a first detected emission; and comparing said detected spatial and temporal coordinates with said predicted spatial and temporal coordinates to determine whether subsequently detected emissions originate from a molecule in said train of molecules.
8. A method according to Claim 7, wherein detecting emissions on at least one microchannel plate sensor.
9. A method according to Claim 7, wherein said predicting spacial and temporal coordinates further defines a moving sample window for molecule detection effective to discriminate background emission events from molecule emission events.
10. A method according to Claim 7, wherein said emission products are photons.
11. A method according to Claim 10, further including the step of irradiating said molecules with a laser to induce a fluorescence for emitting said photons.
12. A method according to Claim 7, further including the step of hydrodynamically focusing said flow train within a flow cell.
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EP0381694B1 (en) 1994-06-01
US4793705A (en) 1988-12-27
DE3889918D1 (en) 1994-07-07
EP0381694A1 (en) 1990-08-16
WO1989003525A1 (en) 1989-04-20
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EP0381694A4 (en) 1991-08-28

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