-state image either DrSpl eraged over based on the. 12. rSplit mages nce of ... Black of cells tran. Y but non-tr d with EGF. 0 nM of rapa stribution of was co-expre.
Small near-infrared photochromic protein for photoacoustic multicontrast imaging and detection of protein interactions in vivo Lei Li, Anton A. Shemetov, Mikhail Baloban, Peng Hu, Liren Zhu, Daria M. Shcherbakova, Ruiying Zhang, Junhui Shi, Junjie Yao, Lihong V. Wang and Vladislav V. Verkhusha
Supplementary Materials
1
Supplementary Tables Supplementary Table 1. Spectral and photoacoustic properties of RpBphP1 and DrBphP-PCM in vitro.
PA signalto-noise ratioc
PA switching ratiod
Temporal frequency PA image CNRe
Maximum Maximum Molar Fluoresc PA Photo ON-to-OFF absorption emission extinction ence excitation Protein switching photoswitching wavelength wavelength coefficient quantum wavelengtha state rateb (s-1) 12 –1 –1 (nm) (nm) (M cm ) yield (%) (nm) 0 mm 0 mm 12 mm 0 mm 12 mm mm depth depth depth depth depth depth Pfr (ON)
756
none
78,300
none
Pr (OFF)
678
n.d.
87,500
n.d.
DrBphP- Pfr (ON) PCM Pr (OFF)
750
none
59,200
none
700
720
98,000
2.9%
RpBphP1
780
1.56
501.4 15.2 4.3 ±15.3 ±0.3 ±0.2
2.3 380.3 21.3 ±0.2 ±12.0 ±0.2
780
0.54
636.2 25.4 8.7 ±17.2 ±1.3 ±1.5
4.7 550.4 38.3 ±1.2 ±16.0 ±0.3
The laser fluence was 8 mJ cm–2. The wavelengths were chosen on the basis of the absorption of the proteins and the power spectra of
a
the lasers. bThe ON-to-OFF photoswitching rate is defined as the reciprocal of the time when the PA signal drops to 1/e of its maximum, measured at a a laser fluence of 4 mJ cm–2. cThe protein concentration was 30 µM, and the reduced scattering coefficient of the scattering media was ~10 cm–1. Data are reported as mean ± s.d. dThe switching ratio is the ratio of the PA signal amplitudes acquired in the ON and the OFF states. Data are reported as mean ± s.d. eContrast-to-noise ratio (CNR). The hemoglobin concentration was 2.3 mM. Data are reported as mean ± s.d.
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Supplementary Table 2. Quantified coefficients of the decay functions of the PA signals from cellsa.
High fluencec
Low fluenced
Scattering mediae
Coefficientsb
f g
HEK-293
U87
HEK-293
U87
HEK-293
0.014 0.493 0.488 1.45 1.01 0.005
0 0.968 0.082 1.72 11.8 0.844
0.001 0.515 0.483 2.01 1.07 0.03
0 1.01 0 2.14 100 0.980
0.035 0.500 0.481 0.591 1.04 0.02
HEK-293:U87 U87 mixture with about 1:1 ratio 0 0.266 1.00 0.169 0.0930 0.581 0.596 0.756 10.8 3.44 0.830 0.549
a
The signals from the cells were spatially averaged first, and the calculations were based on their
spatial mean values. bThe decay function can be expressed in the form of ∙
∙
, where
, and the ratio
/
is within the range of 2.2–3.0.
c
The cells were embedded in clear media, and the illumination fluence is 5 mJ/cm2. dThe cells
were embedded in clear media, and the illumination fluence is 0.5 mJ/cm2. eThe cells were embedded in scattering media, and the local fluence is unknown. fAs defined in Online Methods, , ,
, which was used as a criterion to distinguish two types of cells. It can be seen from
Table 2, regardless of local fluence, the
of HEK-293 cells is approximately 1, whereas the
of
U87 cells is much larger, normally greater than 10, to avoid infinity in computation, the upper limit of k was set to 50. gAs defined in Online Methods, we define
to quantify the
concentration of U87 cells if a mixture of the two types of cells was imaged.
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Supplem S entary F Figures Supplem mentary Figu ure 1. Developm ment of a 1.5 5-fold smalleer photochro omic probe ffor differentiial photoacooustic imaginng.
Monomeers (shown) of naturallly dimeric bacterial b phhytochromess (BphPs) sshare a com mmon domain structure. s It is i representeed here by th he photosennsory core m module (PCM M), formed bby the PAS-GA AF-PHY dom main triad, and outputt domains. The biliveerdin (BV) chromophoore is covalentlly bound with w conserrvative cystteine from the PAS domain annd secured to a chromophore-binding g pocket in the GAF domain. The m molecular weight of the BphPs monnomer Da. BphP1 from f Rhodop pseudomona as palustris, called RpBphP1, consists of the reegular is ~80 kD PCM and the outpu ut PAS/PAS S and HOS domains. T The domain organization of BphP from Deinococccus radiodu urans, DrBp phP, is com mprised of thhe PCM andd the outputt histidine kkinase (HisK) domain. d We truncated t fulll-length DrB BphP to its P PCM and called the resuult DrBphP-P PCM, achieving g a 1.5-fold reduction r in the molecullar weight off the PA proobe.
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mentary Figu ure 2. Supplem Quanficaation of the spatial s resolu ution of the RS-SIP-PAC R CT system.
(a) An im mage of threee tungsten wires, w each with w a nominnal diameterr of 50 µm. Scale bar, 2 mm. (b) The PA P amplitud de distributio on along the red line in ((a). (c) The ccontrast-to-nnoise ratio (C CNR) versus th he shift in the sum of thee original lin ne profile shhown in (b) aand the shift fted copy. Thhe inplane resolution, deefined as th he shift corrresponding to 6-dB C CNR, is 1225 µm. (d) The oustic image of a tungsteen wire with h a nominal diameter off 50 µm, proojected on thhe x-z photoaco plane. (e) The line profiles of (d d) at the centter of the rinng (indicatedd in (d) by tthe red solidd line) and at 7.5 5 mm off-ceenter (indicatted in (d) by y the green daashed line).
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mentary Figu ure 3. Supplem Characterizations of DrBphP-PC CM photochrromic properrties.
(a) Molaar extinction n coefficient ratios betw ween the P Pfr and Pr sstates of DrrBphP-PCM M and RpBphP1 1, showing the t superior switching contrast c of D DrBphP-PCM M in the NIR R spectral reegion. (b) Molaar extinction n coefficien nt ratio betw ween the Pfr and Pr sstates of DrrBphP-PCM M and RpBphP1 1 at 780 nm, showing th hat DrBphP--PCM has 2--times betterr switching ccontrast thann that of RpBph hP1 at 780 nm. n (с) Dependence of the t Pr→Pfr photoconveersion on thee 636/20 nm m light intensity. The kinetiic curves caan be fitted with monoeexponential functions (R R2≥0.958 fo for all curves). (d) Dependence of the Pfr→Pr ph hotoconversiion on the 7780/20 nm llight intensity. A i in absorbance a of o the Pr staate was obseerved for alll tested lighht intensities. The gradual increase kinetic curves c can be b fitted wiith monoexp ponential fuunctions (R2 ≥0.958 forr all curves). (e) Dependence of the half-time off light-inducced DrBphP P-PCM Pr→ →Pfr photocoonversion on the intensity of 636/20-n nm light (n = 3; error bars b are s.e.m.). (f) Deppendence off the half-tim me of light-indu uced Pfr→P Pr photoconv version on th he intensity oof 780/20-nm m light (n = 3; error barrs are s.e.m.). 6
mentary Figu ure 4. Supplem Frequenccy lock-in reconstruction n provides beetter contrassts of BphPs..
(a) Norm malized photo oacoustic (P PA) signal off BphPs channges during the illuminaation modulaation. (b) The temporal t freq quency specctrum of the signal in (a)) clearly show ws the moduulation frequuency and its haarmonics. (cc) Frequency y lock-in recconstruction (LIR) proviides ~2–3-foold better CN NR of BphPs th han the diffeerential imag ging method d; error bars are s.e.m. ((n = 40), callculated baseed on the pixel values from m regions of interest. i
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mentary Figu ure. 5. Supplem Comparison of RpBp phP1 and DrrBphP-PCM expression llevels.
(a) Flow cytometry was w used to analyze the fluorescencee intensity ddistribution oof live HeLaa cells pBphP1 or DrBphP-PCM D M with EGF FP downstreaam of the T22A self-cleavvable that co-expressed Rp ( BphP-T2 2A-EGFP co onstructs). The T mean EG GFP fluoresscence intensity is preseented. peptide (in Error barrs, s.e.m. (n = 3; transfecction experim ments). (b) Q Quantificatioon of the data presented in (a). (c) PA im mages of hem moglobin, RpBphP1, R an nd DrBphP-P PCM in the Pfr (ON) sttate (left collumn) and Pr (O OFF) state (middle ( colu umn). LIR im mages of hem moglobin, R RpBphP1, annd DrBphP-PCM (right column). Scalee bar, 2 mm m. (d) Fractio onal changee of PA signnals from boovine blood,, U87 cells expressing eitheer RpBphP1 or DrBphP--PCM duringg 780 nm illuumination.
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mentary Figu ure 6. Supplem Lock-in reconstructio r on of U87 cu ultured cells expressing either DrBphhP-PCM or RpBphP1.
(a) Norm malized PA siignal of U87 7 cells expressing changees in either D DrBphP-PCM M or RpBphhP1 during th he illuminatio on modulatio on. (b) The temporal t freequency specctrum of the signal in (a)) clearly sh hows the mo odulation freequency and its harmoniccs. (c) Frequuency lock-inn reconstrucction (LIR) pro ovides ~2–3-fold better CNR C of U87 7 cells expreessing either DrBphP-PC CM or RpBphP1 than the differential d imaging i metthod; error bars are s.e.m m. (n = 40), ccalculated baased on the ppixel values fro om regions of o interest.
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mentary Figu ure 7. Supplem Separatio on of three types of cells expressin ng different BphPs in sscattering m media at diffferent depths.
(a) Top and a side view ws of the sch hematics of the phantom m containingg the three tyypes of cells (top, U87 cellss expressing g DrBphP-PC CM only, miiddle, HEK--293 cells exxpressing booth DrBphP-PCM and RpBphP1, bottom m, a mixturee of U87 cellls and HEK K-293 cells), which were embedded iin the scattering g media witth different distances to o the phantoom surface. Top illuminnation of 780 nm light wass used. (b) LIR L images of o three typees of cells. (cc) Computedd coefficiennts, b, of the three types of cells. c (d) Co omputed coeffficients, c, of o the three ttypes of cellls. (e) Computed coefficients, k, of thee three types of cells, which w enablles a good sseparation oof each otheer. (f) Compputed coefficien nts, s, of thee three typees of cells, which w illustrrates the conncentrations of the U87 cells inside alll cells well. Scale S bar, 2 mm.
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mentary Figu ure 8. Supplem In vivo PA detection of a brain tu umor expresssing DrBphP P-PCM.
The brain n of a nude mouse m was im maged two weeks w after tthe injectionn of U87 gliooblastoma ceells expressin ng DrBphP-P PCM ~3 mm m underneath h the surfacee. PA image of the mouse brain corteex with DrB BphP-PCM in n its ON statte (a) and OF FF state (b). (c) LIR imaage overlaidd on the brainn cortex vaasculature, sh howing the brain b tumor with high coontrast. Scalee bar, 2 mm.
11
mentary Figu ure 9. Supplem Comparison of photo oswitching properties p of DrSplit andd DrBphP-PC CM in MTLnn3 cells.
(a) Fluorrescence speectra of lysaates of HeL La cells exppressing eithher DrBphP--PCM or DrrSplit (treated with w rapamy ycin). (b) ON N state (left column) andd OFF state (middle collumn) PA im mages of U87 cells c expresssing DrBphP P-PCM and MTLn3 cellls expressinng DrSplit inn the presennce of rapamyciin. The LIR L image (right collumn) dem monstrates thhat DrSplitt recoveredd its photoswiitching abiliity in the prresence of raapamycin. Inn addition, DrSplit has lower switcching contrast than t DrBphP P-PCM. Scaale bar, 2 mm m. (c) PA siggnals of MT TLn3 cells exxpressing DrrSplit in the preesence of raapamycin ch hange follow wing the illum mination moodulation. Inn comparisonn, the PA signaals of the co ontrolled MT TLn3 cells without w rapaamycin havee non-detectaable responsses to the illum mination mod dulation. (d) Comparison n of CNRs qquantified frrom the ON--state images and the LIR images. On ne sample was w imaged for f each typpe of cells eexpressing eeither DrSpllit (in presence or absence of rapam mycin) or DrBphP-PCM D M. The datta were averaged overr ten measurem ment cycles on each sam mple; error bars b are s.e.m m. (n = 40), calculated bbased on the pixel values fro om regions of o interests. Rapa is shorrt for rapamyycin. 12
mentary Figu ure 10. Supplem Cytotoxicity of DrSp plit in mamm malian cells.
uorescence iintensity disstribution off live HeLa ((a, b) Flow cyttometry wass used to anaalyze the flu and U87 (c, d) cells expressing both EGFP and mCherrry. EGFP w was co-expressed upstreaam of IRES2 sequence s fro om EGFP-IIRES2-DrPA AS-FRB. mC Cherry wass co-expresssed upstream m of IRES2 seequence from mCherry-IRES2-FKB BP-DrGAF-P PHY. The m mean valuess of EGFP ((a, c) and mCh herry (b, d)) fluorescen nce intensity y are presennted. Black lines correespond to m mocktransfecteed cells, red d lines correspond to distribution d of cells trannsfected witth EGFP-IR RES2DrPAS-F FRB and mCherry-IRE m S2-FKBP-D DrGAF-PHY Y but non-trreated with rapamycin, blue lines correspond to o distributio on of cells transfectedd with EGF FP-IRES2-D DrPAS-FRB and mCherry y-IRES2-FKB BP-DrGAF-PHY and treeated with 100 nM of rapaamycin.
13
Supplementary Figure 11. Validation of the existence of reconstituted DrSplit via near-infrared fluorescence.
MTLn3 cells stably expressing both, EGFP-IRES2-DrPAS-FRB and mCherry-IRES2-FKBPDrGAF-PHY. Constructs were treated with rapamycin. The fluorescence of non-treated (‒ RAPA) and treated (+ RAPA) cells was compared by the fluorescence of EGFP (green; ex 480/40 nm, Em 535/40 nm), mCherry (red; ex 570/30 nm, Em 615/30 nm) and DrSplit (NIR; ex 605/40 nm, Em 640/LP nm). (a) Fluorescence microscopy images of non-treated (top row) and treated (bottom row) cells. (b) Flow cytometry was used to analyze the fluorescence intensity distribution of live MTLn3 cells, that stably expressing DrBphP-PAS downstream of IRES2 sequence with EGFP and DrBphP-GAF-PHY downstream of IRES2 with mCherry. The mean values of EGFP, mCherry and DrSplit fluorescence intensity are presented. Gray curves correspond to non-treated samples, red curves correspond to samples treated with rapamycin. (c) Quantification of the data presented in (a). Error bars, s.e.m. (n=3; transfection experiments). Scale bars, 10 µm. RAPA is short for rapamycin. 14
mentary Figu ure 12. Supplem In vivo RS-SIP-PAC R CT of liver tumors t exprressing DrSpplit, showing photoacouustically dettected PPIs at depth. d
T image of the t mouse liiver on Day 32 after tum mor cells injjection but bbefore rapam mycin (a) PACT injection. The LIR im mage was ov verlaid on th he anatomicaal image, buut no lock-in frequency ssignal was deteected. Scale bar, 5 mm. (b) PACT image of tthe mouse oon Day 34 after tumor cells injection (44 h after rapamycin r in njection). Th he LIR imagge was overllaid on the aanatomical im mage, showing the PPI-ind duced compllementation of DrBphP P-PCM from m DrSplit. (cc) Representtative H&E histological im mage of a harv vested tumo orous liver, w where the tum mor region is bordered bby the green lin ne. Scale bar, 2 mm. (d) Close-up off the black reectangular bbordered porttion of the im mage in (c). Sccale bar, 1 mm. m
15
mentary Figu ure 13. Supplem In vivo fluorescence f imaging off liver tumorrs expressinng DrSplit, cconfirming tthe occurrennce of PPIs.
Fluoresceence imagin ng of tumor growth g and metastasis oof cells exprressing DrSpplit. MTLn3 cells expressin ng DrSplit were w injected d in the mou use liver. Fluuorescence im mages were acquired ~440-44 hours aftter injection of rapamyciin through th he tail vein. The differenntial image w was computeed by subtractin ng the Pfr sttate image frrom the Pr sttate image. ((a) Fluorescence imagess of the mouuse on Day 5 affter the injecction of tum mor cells (~4 40 hours aftter rapamycin injection)), the differeential image (in n color) wass overlaid on n the anatom mical image (in gray). (bb) Fluoresceence image oof the mouse on n Day 32 affter the injection of tum mor cells (~444 hours aft fter rapamyccin injection)), the differentiial image (in n color) was overlaid on the anatomiical image (iin gray). Scaale bar, 5 mm m.
16
mentary Figu ure 14. Supplem Hydrody ynamic liver transfection of mice witth plasmid enncoding DrB BphP-PCM iin vivo.
s and (b)) OFF state images of a mouse liver 24 h afteer injection of DrBphP--PCM (a) ON state plasmid. Due to the overwhelmiing backgrou und signal fr from blood, tthe injected plasmid-indduced expressio on of DrBph hP-PCM is not n visible in i either thee ON state oor OFF statee image. (c)) LIR image ov verlaid on the anatomiical image shows the DrBphP-PC CM signal iin color andd the backgrou und blood sig gnal in gray.. Scale bar, 5 mm.
17
mentary Figu ure 15. Supplem RS-SIP-P PACT of a hydrodynami h ically transfeected mousee liver ex vivvo.
The liverr was exciseed from a mouse m after 24 2 hours of hhydrodynam mic transfection. (a) ON state and (b) OFF O state PA A images off the left lob be of the livver. (c) LIR image of thhe left lobe oof the liver, seleectively showing the sig gnals from th he transfecti on-induced expression oof DrBphP-C CPM. (d) Overllay image off the LIR sig gnal with an n anatomicall image of thhe left lobe of the liver,, with the DrBp phP-PCM sig gnal in colorr and the bacckground bloood signal inn gray. (e–h)) As shown iin (a– e), the sig gnals from th he right lobee of the liverr. Scale bar, 2 mm.
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mentary Figu ure 16. Supplem Ex vivo fluorescence f e imaging off the liver excised e from m a hydrodynnamically trransfected m mouse expressin ng DrSplit.
use was treatted with rap pamycin afteer 24 hours of hydrodynnamic transffection. The liver The mou was excised from thee mouse afteer 44 hours of o rapamyci n treatment.. (a) Pr statee and (b) Pfrr state fluoresceent images of o the liver. (c) ( Differenttial fluoresccent image (P Pr image – P Pfr image) oof the liver. (d) Overlay image of differrential imagee with the Prr state imagee, showing thhe DrBphP--PCM he backgroun nd liver sign nal in gray. S Scale bar, 2 m mm. signal in color and th
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