MOTIONUS 20170234874A1 LAWNMN TANTUM IN ( 19) United States (12 ) Patent Application Publication (10 ) Pub. No. : US 2017/ 0234874 A1 Adams et al. (43 ) Pub. Date : Aug . 17 , 2017 ( 54 ) INTEGRATED VISUAL MORPHOLOGY AND Related U . S . Application Data CELL PROTEIN EXPRESSION USING (60 ) Provisional application No . 62 / 238 ,605 , filed on Oct . RESONANCE -LIGHT SCATTERING 7 , 2015 . (71 ) Applicant: Clearbridge BioPhotonics Pte Ltd ., Singapore (SG ) Publication Classification (51 ) Int . CI. (72 ) Inventors : Thomas H . Adams, Rancho Santa Fe, GOIN 33 / 569 ( 2006 .01 ) CA (US ) ; Stephen Roman Fait , GOIN 15 / 14 ( 2006 .01 ) Carlsbad , CA (US ) ; Eric Scott A61B 5 /00 ( 2006 .01 ) McCampbell , Carlsbad , CA (US ) ; GOIN 21 /552 ( 2006 .01 ) Michelle Brooke McCampbell , Rancho (52 ) U . S . Cl. Santa Fe, CA (US ); Edward Jablonski, CPC . GOIN 33 /56966 (2013 .01 ) ; GOIN 21/ 553 Escondido , CA (US ) ; Robert Earl ( 2013 .01 ) ; GOIN 15 / 1456 ( 2013 .01 ) ; A61B Klem , Rancho Santa Fe, CA (US ) 5 /0082 ( 2013 .01 ); G06T 7 / 0012 ( 2013 .01 ) ( 57 ) ABSTRACT (21 ) Appl . No. : 15 / 286 , 340 The invention relates to detecting cell biomarker signatures and integrated cell biomarker- morphological profiles by detecting resonance - light scattering of functionalized nano (22 ) Filed : Oct. 5 , 2016 particles. General Protocol for successive displacements of labelled nanoparticles ( free ) + 1st oligo Rev . compl. ds - hybridization relatively lower Tm ds- hybridization to 1st oligo relatively higher Tm next Ab -np Rev. compl. Add 3rd oligo to 3rd oligo .. Add 1st Ab - no to displace to cell Scan / image np -oligo 1st Ab -op cell cell cell First plurality of functionalized nanoparticle species comprises 1st Ab -np Then , add next Ab -np to interrogate next antigen (serially or in parallel) , repeat process n times . Patent Application Publication Aug. 17 , 2017 Sheet 1 of 25 US 2017 / 0234874 A1 edi **** *** * cell *** * ets * nextAb-np * 1stoligo itti (free) .. .. cell nextantigen(seriallyorinparallel), relativelyhigherTm Then,addnextAb-nptointerrogate Add3rdoligo times.processnrepeat ds-hybridization todisplace no-oligo Scan/image cell relativelylowerTm Add1stAb-np Fig.1-GeneralProtocolforsuccessivedisplacementsoflabellednanoparticles ds-hybridization tocell cell comprises1stAb-np 1stAb-np Firstpluralityoffunctionalized nanoparticlespecies Rev.compl Ab Rev.compl to1stoligo to3rdoligo Patent Application Publication Aug. 17 , 2017 Sheet 2 of 25 US 2017 / 0234874 A1 (free) *** * *. it relativelyhigherTm ds-hybridization, 919+* ware (free) inute "M * * * ** **** * * * relativelyhigherTm * oligo-B ds-hybridization, * *** ** oligo-A Fig.2-Alternativedisplacementembodiments OR relativelylowerTm ds-hybridization, Patent Application Publication Aug. 17 , 2017 Sheet 3 of 25 US 2017 /0234874 A1 T! (free) relativelyhigherTm ds-hybridization, *** * * ** *" Fig.3-Alternativedisplacementembodiments:displacingbridgingoligo Addoligo-D relativelylowerTm ds-hybridization, Patent Application Publication Aug. 17 , 2017 Sheet 4 of 25 US 2017 / 0234874 A1 **** * * * * * * * * * * * * * * SAV=streptavidin b=biotin SAV +SAV 1111111111111111111*1* 2)Wash(PBS COOH1)EDC/NHS Fig.4-Nanoparticlefunctionalizationdirectlylinked Biotin-NHS OR Maleimide-BiotinBiotin-Maleimide du S NH SH Hindistinctio I**!! !! ! ! !! ! ! Patent Application Publication Aug. 17 , 2017 Sheet 5 of 25 US 2017 /0234874 A1 +q Capunreacted SAVwithfreebiotin +biotin-oligo SAVou"SOMEDCNHS +amine-oligo +SAV Fig.5a-Nanoparticlefunctionalizationoligonucleotideoverlap OR 2)Wash(PBS) COOH1)EDC/NHSNHL 2)Wash(PBS COOH1)EDC/NHS du tot Patent Application Publication Aug . 17 , 2017 Sheet 6 of 25 US 2017 / 0234874 A1 W 5°)':3 53' 1111111111iiiiii*** +)du Fig.5b-Nanoparticlefunctionalizationoligonucleotideoverlap +Maleimideoligo Mar SH Patent Application Publication Aug. 17 , 2017 Sheet 7 of 25 US 2017 / 0234874 A1 OR . incinin w \5' w 3' (bridgingoligo) +AV 111* !!! ! ! 3:115 :11 :31 :1111 :111 ) Fig.6-Nanoparticlefunctionalizationoligonucleotidebridge +Maleimideoligo ???? SH Patent Application Publication Aug . 17 , 2017 Sheet 8 of 25 US 2017 /0234874 A1 wnios FIG.7 Patent Application Publication Aug . 17 , 2017 Sheet 9 of 25 US 2017 / 0234874 A1 . ' . ' . ' . ' . ' . ! ! . 50um FIG.8A Patent Application Publication Aug . 17, 2017 Sheet 10 of 25 US 2017 /0234874 A1 . : : 8 .', . FIG.8B Patent Application Publication Aug . 17, 2017 Sheet 11 of 25 US 2017 /0234874 A1 FIG.9 Patent Application Publication Aug . 17, 2017 Sheet 12 of 25 US 2017 /0234874 A1 FIG.10 Patent Application Publication Aug . 17, 2017 Sheet 13 of 25 US 2017 /0234874 A1 50um FIG.11 :: :: :: : :: : : : : : : : : : : : : : : : : : : :: : : : : : :: : : : : : : : : : : : : :: Patent Application Publication Aug . 17, 2017 Sheet 14 of 25 US 2017 /0234874 A1 Green Blue Yellow : 5um FIG.12 Patent Application Publication Aug . 17 , 2017 Sheet 15 of 25 US 2017 /0234874 A1 FIG.13 Patent Application Publication Aug . 17, 2017 Sheet 16 of 25 US 2017 /0234874 A1 . !', . ! . .! . ,'. .' . .' . 2 . .. .' ,' . ..'" Hrrrrrrrrr.: . .' .'" . .' . .',. .' . ..',. '. Di . ... !. :. .: . .'" . .1777 .. I . .' . : I . .' . 7 . 2017. .'. .: . ! . ! . ' :5 .' . ', . Green : .' ', . : . mintain . ', . ', . : . .' :. :: : "'. : . .: . : . 7 . 199 . :. 11:. : . Blue Yellow . ': . '. ' . ' . :::: : . ' . ' . ' . ' . .. : . : . : . ! ' . LLLLLLLLLLLLLLLL.!:' . .I.',. .. .! . : . 13: . : . .'" . : .' . .' . .' . .' : . : .' . .' . : . IIIIIIIIIIIIIIIIIIIIII.'IIIIIIIIIIIIIIIIII.", . : . ... : . : . "'. : . Orange . .' . ! '.,', . .' . ,'. .' . .: .' . : : . IIIIIIIIIII.:' . - . 5um . ',iiii. * . ..,' ' .!' . ., . ' . .: FIG.14 . :' : . i.iii : . 1:32 . .. - : ' . ' ' ' ' ' ' ' ' ' ' ' : : : . Patent Application Publication Aug . 17, 2017 Sheet 17 of 25 US 2017 /0234874 A1 FIG.15 Patent Application Publication Aug . 17, 2017 Sheet 18 of 25 US 2017 /0234874 A1 FIG.16 Patent Application Publication Aug . 17, 2017 Sheet 19 of 25 US 2017 /0234874 A1 FIG.17 Patent Application Publication Aug . 17, 2017 Sheet 20 of 25 US 2017 /0234874 A1 FIG.18 Patent Application Publication Aug . 17, 2017 Sheet 21 of 25 US 2017 /0234874 A1 FIG.19B Auanti-CD3(yellow)andAgCD4 particlesfieldnothe-lymphocytesin (blue)particlesbindtothefour bindtoneutrophils . : : : : : : : : . : . .. .. : .- :',1 .. 7 . .! . .IIIIII', .'. : : : YYYYYYYYYYYYY . : . .. W . 1 . : FIG.190 FIG.19A . .: . .': .. : .. ." . .... .: . u . .. .. IIIIIII : . 77777777777777777777777777777 . : 1111111111 : : : : : : : : . : : . 1 . 1 . 1 . 1 !. 1 . 23: . : . 23:31 .. .! : . - . iiiiiiiiiiiM . : 7 .. 2 . : < . : . : - . FIG.19D .'viri. !'. ' . .' . .. : . '. ) . .' . .. I V iscsiiiiiiiiiiiiiiiiiiiiiiii . Patent Application Publication Aug . 17, 2017 Sheet 22 of 25 US 2017 /0234874 A1 CD4+,CD3 CellType t CD3-,CD4 Blue/darker THelperCell Yellow/lighter CD3,CD4 Neutrophil particles particles TCell : : :: : : . .' : : : : .. '. : . :-: . : : : : :: . : . :: :: . .' : :: : :: : : : :: :: : :: : :: : . .' : : : : : : : . .' : : : : :: ',. .' : : :: . .' : : : . .' . .' : . .' . * .' . * . 1 . Darkfield .' .' 13 : : : : . : .' : : :: FIG.197 : : : : : : : : : : : :: : . : . : . : . : . : . : . : . : FIG.19H FIG.191 . Brightfield FIG.191 FIG.19EFIG.19E FIG.19G Patent Application Publication Aug . 17 , 2017 Sheet 23 of 25 US 2017 /0234874 A1 to13outof14anti-CD3(yellow)bindAu noparticleslymphocytesinthefield- neutrophilstobind FIG.20B . LLLLLLLLLLLLL 3 . : 1 . IIII , , . 15 : FIG,200 : . : 1 9.. FIG.20A 20DFIG. E Patent Application Publication Aug . 17, 2017 Sheet 24 of 25 US 2017 /0234874 A1 FIG.21B : FIG.21AFIG.21A . ( . Patent Application Publication Aug . 17, 2017 Sheet 25 of 25 US 2017 /0234874 A1 . .: . : Green/darker . : . Yellow/lighter FIG.22 US 2017 /0234874 A1 Aug . 17 , 2017 INTEGRATED VISUAL MORPHOLOGY AND using the resonant light scattering properties of the particles CELL PROTEIN EXPRESSION USING are : ( a ) the nanoparticles can be detected and imaged at RESONANCE -LIGHT SCATTERING magnifications as low as 10x using a simple illuminator, such as a white light illuminator , with dark field illumina RELATED APPLICATIONS tion , ( b ) the nanoparticles provide a non -bleaching signal, ( c ) the color of scattered light can be changed by changing [0001 ] This application claims the benefit of U . S . Provi nanoparticle size and / or composition for multicolor multi sional Application Ser . No . 62 /238 ,605 , filed on Oct . 7 , plexing , ( d ) the nanoparticles can be conjugated with bio 2015 , the entire contents of which is hereby incorporated be marker - binding moieties for specific analyte detection to reference in its entirety . create functionalized nanoparticles, ( e ) biological samples contacted with the functionalized nanoparticles are archiv FIELD able , and ( f ) the functionalized nanoparticles exhibit a [0002 ] The presently disclosed subject matter relates to greater range of linearity of detection when present on a cell compositions and methods for integrated visualmorphology because the particles do not self- quench . In some embodi and cell protein expression analysis . ments , the methods of this invention are useful in obtaining images of cell - functionalized nanoparticle complexes under INTRODUCTION AND SUMMARY OF THE ambient conditions which do not require use of a darkroom , INVENTION in contrast to fluorescent labeling systems. In some embodi [0003 ] Cellular analysis is an important tool in histopa ments , the samples may be viewed on a microscope in a thology to aid in diagnosing