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Pulse-shaped two-photon excitation of a fluorescent baseanalogue approaches single-molecule sensitivity

Citation for published version:Fisher, RS, Nobis, D, Füchtbauer, AF, Bood, M, Grøtli, M, Wilhelmsson, LM, Jones, AC & Magennis, SW2018, 'Pulse-shaped two-photon excitation of a fluorescent base analogue approaches single-moleculesensitivity', Physical chemistry chemical physics. https://doi.org/10.1039/C8CP05496G

Digital Object Identifier (DOI):10.1039/C8CP05496G

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Pulse-shapedtwo-photonexcitationofafluorescentbaseanalogueapproachessingle-moleculesensitivityRachelS.Fisher,a‡DavidNobis,b,‡AndersF.Füchtbauer,cMattiasBood,dMortenGrøtli,dL.MarcusWilhelmsson,cAnitaC.Jones*aandStevenW.Magennis*b

Fluorescentnucleobaseanalogues(FBAs)havemanydesirablefeaturesincomparisontoextrinsicfluorescentlabels,butthey are yet to find application in ultrasensitive detection. Many of the disadvantages of FBAs arise from their shortexcitation wavelengths (often in the ultraviolet), making two-photon excitation a potentially attractive approach.Pentacyclic adenine (pA) is a recently developed FBA that has an exceptionally high two-photon brightness.We havestudiedthetwo-photon-excitedfluorescencepropertiesofpAandhowtheyareaffectedbyincorporationinDNA.Wefindthat pA is more photostable under two-photon excitation than via resonant absorption. When incorporated in anoligonucleotide, pA has a high two-photon cross section and emission quantum yield, varying with sequence context,resultinginthehighestreportedbrightnessforsuchaprobe.Theuseofatwo-photonmicroscopewithultrafastexcitationandpulseshapinghasallowedthedetectionofpA-containingoligonucleotidesinsolutionwithalimitofdetectionof~5molecules,demonstratingthatpracticalsingle-moleculedetectionofFBAsisnowwithinreach.

IntroductionFluorescence spectroscopy and imaging are powerfultechniques for studying the structure, dynamics andinteractions of nucleic acids. As the naturally occurringnucleic acids are virtually non-emissive, they require afluorescent label. The most common approach is tocovalently attach external dyes by long linkers, but theseare generally insensitive to local base interactions. Analternative is the use of a fluorescent analogue of one ofthenaturalnucleobases,whichcanreplacethenucleobasewhilemaintaininghydrogenbondingandleavingtheoverallconformation of the duplex unaffected.1-3 A range offluorescentnucleobaseanalogues (FBAs) forDNAandRNAhave been developed,1-8 notably the archetypal baseanalogue2-aminopurine(2-AP).9 Although ensemble measurements of FBAs are veryuseful tools, single-molecule spectroscopy could provideunprecedented insight into the mechanism of processessuchasDNAmethylationattheindividualbase level,10but

this has proved challenging for nucleobase analogues forseveral reasons. Firstly, despite good emission quantumyields (f), especially as monomeric units, the absorptioncoefficients of nucleobase analogues (ɛ) are much lowerthan for extrinsic dyes, limiting their brightness (ɛf).Secondly, nucleobase analogues absorb at shortwavelength, often in the UV, and are prone tophotobleaching. Thirdly, light penetration in biologicalmedia is poorer at short wavelength due to increasedabsorption and scattering. The optimised emission signalfor single-molecule detection, given as count rate permolecule (CPM), for free 2-AP in water (unquenched andnot incorporated in anoligonucleotide)withUVexcitationwasfoundtobe~2kHz,11withasignal-to-backgroundratio(SBR) per molecule of 0.3. This CPM is very low incomparison with typical extrinsic organic labels (30-100kHz). Similarly, the CPMof a guanine analogue, 3-MI,wasreported as 4 kHz with a SBR of ~ 5 following UVexcitation.12 It has been reported that oligonucleotideslabelled with 2-AP or pyrrolo-cytosine (Py-C) could bedetectedatthesingle-moleculelevelinnucleicacidsviaUVexcitation on a surface.13 Although the authors did notreporttheCPMof2-APandPy-C, it iswellknownthatthefluorescenceof2-APisquenchedbetween10and100-folddue to base stacking interactions.9 In short, it seemsunlikely that UV excitation of such bases will lead to arobust method of single-molecule detection of nucleicacids. A way to overcome these problems could be to usemultiphotonexcitation,thesimultaneousabsorptionoftwoor more longer wavelength photons.14 In addition to

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allowing deeper penetration into tissue in the near-IR,multiphoton excitation allows 3D localization of lightabsorption, without exciting out-of-focus chromophores;out-of-focus photobleaching is therefore also reduced.Furthermore, changes in the optical selection rules formultiphoton excitation can result in the more efficientpopulationoftheexcitedstates,orcompletelynewexcitedstates being accessed.14 Other possible advantages ofmultiphoton excitation include reduced photobleaching inthe excitation focus, a reduction of backgroundfluorescence from optical components, greater spectralseparation of excitation and emission bands, and thesimultaneousexcitationofmultiplefluorophores.15 Therehavebeen fewstudiesofmultiphotonexcitationof nucleobase analogs.16-20 The guanineanalogue, 6-MI (6-methyl isoxanthopterin), is reported tohavea two-photon(2P)crosssection (σ2)of2.5GMunits (Goeppert-Mayer,1GM = 10−50 cm4 s photon−1),16 while 6MAP (4-amino-6-methyl-8-(2-deoxy-b-D-ribofuranosyl)-7(8H)-pteridone), anadenineanalog,hasacrosssectionof3.4GM.17The2Pandthree-photon excitation of 2-AP (~0.2 GM) was reportedrecently, along with the 2P excitation of the cytosineanalogue tC in an oligonucleotide (1.5 GM).18 Althoughphotobleachingwasreducedfor2Pexcitation,thestabilitywas not high enough to perform fluorescence correlationspectroscopy (FCS).18 In a very recent study,Mikhaylov etal. assessed the potential utility of several establishedisomorphic FBAs formultiphotonmicroscopy.19 As well asreporting2Pcrosssections, theyusedapreviouslyderivedrelationshipbetweenthe2Pcrosssectionandthespeedofmultiphoton imaging21 to estimate the maximum framerate that would be achievable, in a generic scanningmultiphoton microscope. The best-performing FBA in thiscontextwasfoundtobe6-MI,withanestimatedframerateof200framespersecond.21

Chart 1 a) Structure of pentacyclic adenine (pA), as the free base (left) and theriboside(right).b)pA-containingdeoxyoligonucleotides(10mers).SequencesdifferonlybytheneighbouringbasesofpA,andarenamedaccordingly.

The potential for major improvements in 2P fluorescencebrightnessviasystematicmodificationswashighlightedinastudy of a series of structurally-related nucleosides.20 The1P and 2P cross sections and spectra in this report alsoagree well with subsequent theoretical calculations,22offering the prospect of the rational design ofphotophysicalproperties.Nevertheless,until recentlyFBAshave either possessed a low two-photon cross section, alow quantum yield, an absorbance maximum outside thepractical rangeof two-photonexcitation,oracombinationofallthree. Apromisingnewdevelopmentistherecentreportthatpentacyclic adenine, pA, a bright fluorescent adenineanalogue(Chart1a)canbeexcitedvia2Pexcitationwitharelativelyhighcrosssectionof6.6GM.23The1PexcitationmaximumofpA is391nmwithaquantumyieldof0.66 inwater. This value is on a parwith 2-AP and surpasses thebrightnessofmanyotherFBAs.Unlike2-AP(andmostotherfluorescent analogues), pA retains a relatively highquantum yield in oligonucleotides, in several sequencecontexts.ThecrosssectionofpAinanoligonucleotide(GA,Chart1b)was reportedas3GM,whichwasencouraginglyhigh.Thiswasonlythesecondexampleofthe2PdetectionofanFBAinDNA.18 Inthisworkwereportacomparativeanalysisofthe1Pand2PphotophysicsofpAas thenucleobase, theribosideand incorporated in oligonucleotides (Chart 1). WedemonstratethatpAhasanenhancedphotostabilityunder2P excitation.We then report the use of a 2Pmicroscopewithpulse-shaped,ultrafastlaserexcitationtoestablishthelimits of detection for DNA containing pA. FCS allowed adetermination of the CPM as 0.5 kHz per molecule. Byoptimizingthebroadbandlaserpulse,asfewas5moleculescouldbedetected.Weconcludewithadiscussionofsomeof thenextdevelopments thatcould result in theultimatesingle-moleculelevelofsensitivity.

ExperimentalMaterials

The synthesis and characterization of the pA nucleobaseandpA-modifiedoligodeoxynucleotideshasbeen reportedrecently;23 the pA riboside24 was used instead of thenucleobase for photophysical measurements in aqueoussolution,duetoitshighersolubility.For ensemble 1P and 2P spectroscopy, the free pAnucleobase was dissolved in ethanol (>99.9%, FisherScientific) and the pA riboside was dissolved in eitherethanolorphosphatebuffer(12.5mMphosphate,100mMNaCl, pH 7.5 (Sigma-Aldrich)). pA-containing modifiedoligonucleotides were also dissolved in phosphate bufferand were annealed by combining each modified single

a

bGA 5´-d(CGCAGpAATCG)-3´GG 5´-d(CGCAGpAGTCG)-3´AT 5´-d(CGCAApATTCG)-3´CC 5´-d(CGCACpACTCG)-3´TT 5´-d(CGCATpATTCG)-3´

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strandwith10%excessof its complementary strandatRTfollowedbyheatingto95°Candleavingtocoolovernight.Solution concentrationswere ~10–6Mwith absorbance of~0.05atthelong-wavelengthmaximum. For 2P microscopy, a pA-modified oligonucleotide wasdissolved in buffer containing 20mM Tris (Sigma-Aldrich),150 mM NaCl (Fluka) at pH 7.4. Solutions were filteredthrough activated charcoal to remove fluorescentimpurities.Ultrapurewaterwasusedforbufferpreparation(DirectQ3,MerckMillipore).All solventsandbufferswerecheckedforbackgroundfluorescencepriortouse.1PSpectroscopy

Absorption spectra were recorded using a Cary 300 UV-visible spectrophotometer and fluorescence spectra wererecorded using a Jobin Yvon Horiba FluoroMax-3spectrofluorimeterrunningFluorEssenceTMsoftwarev.3.5. Fluorescence lifetimes were determined using time-correlated single photon counting on an EdinburghInstruments spectrometer equipped with TCC900 photoncounting electronics. A mode-locked Ti:sapphire laser(Coherent Mira pumped by Coherent Verdi), producingpulsesofduration~150 fsata repetition rateof76MHz,wasusedastheexcitationsource.Thepulserepetitionratewas reduced to 4.75 MHz using a pulse picker (Coherent9200) and the light was frequency doubled using aCoherent 5-050 harmonic generator. Fluorescence decaycurves were recorded over 50 ns, 4096 channels andcollected to a total of 10000 counts in the peak channel.Decayswerefittedbybyiterativere-convolution,assumingamulti-exponentialfunction,givenineq1.

𝐼 𝑡 = 𝐴%exp−𝑡𝜏%

(1),

%-.

whereIisthefluorescenceintensityasafunctionoftime,t,(normalised to the intensity at t=0); τi is the fluorescencelifetimeof the ithdecaycomponentandAi is the fractionalamplitude (A-factor) of that component. (The fractionalamplitude depends on the radiative lifetime of thatemittingspeciesandthefractionoftheemittingpopulationthatitconstitutes).Decayswerecollectedatthreeemissionwavelengths and were analysed globally, with τi as thecommon parameters, using Edinburgh InstrumentssoftwareFAST. Theaverage lifetime,<t>,of theemittingpopulation isrelatedtotheaveragequantumyield,<f>determinedfromsteady-stateintensitymeasurements,asgivenbyeq2.

<τ> =𝐴%𝜏%,

%-.

𝐴%,%-.

=<ϕ><kr>

(2)

where <kr> is average radiative lifetime of the emittingpopulation.The fraction of the steady-state emission intensity due toeachspeciesi,SSi,isgivenbyeq3.

𝑆𝑆% =𝐴%𝜏%𝐴%𝜏%,

%-.(3)

2PSpectroscopy

SeeFigureS1 foraschematicdiagramof theexperimentalsystem. A mode-locked Ti:sapphire laser (see above) wasused as the excitation source.A variable reflectiveneutraldensity filter was used to attenuate the excitation beam,which then passed through a dichroic mirror (SemrockBrightlineFF735-Di02)andwas focusedbya20´objective(Olympus)intothesamplesolution,whichwascontainedina 1 cm path-length cuvette. Fluorescence emission wascollectedbythesameobjective,reflectedfromthedichroicmirror, passed through a shortpass filter (SemrockBrightlineFF01-720/SP-25)anddetectedbyafibre-coupledspectrometer(OceanOpticsUSB2000+),withanacquisitiontimeof2s.Measurementsweremadeatbetweentwentyand thirty different incident powers. Three spectra werecollectedateachpowerandanaveragetaken.TheincidentpowerwasmeasuredusingaCoherentFieldMasterpowermeter. Two-photoncrosssectionsweremeasuredrelativetoanumberofdifferentstandards(seeSIformoredetails).Thetwo-photon cross sections have an estimated accuracy of10% due to uncertainty in the cross sections of thestandards and errors in the measurement of the spectralthroughput,absorptionspectraandemissionspectra.

Fluorescencelifetimemeasurementsusing2Pexcitationwere recorded as described for 1P excitation, except thatthe pulse-picked fundamental output of the Ti:sapphirelaser at 790 nm was used, and this was focused into thesamplesolutionusinga10´microscopeobjective. 2PMicroscopy

The experimental system is shown in Figure 1. TheexcitationsourcewasabroadbandTi:Sapphirelaserwitharepetitionrateof80MHz(VitaraUBB,Coherent).Thelaserspectrum was centered on 800 nm with a FWHM of 135nm; the compressed pulses from the oscillator had aduration of 15 fs. The pulse shape at the sample wascontrolled via a pulse shaper (MIIPS-Box 640, BiophotonicSolutions Inc.) containing two liquid-crystal spatial light-modulators (SLM) in series (SLM-S640d, Jenoptics). Afterthe pulse shaper the beam entered the invertedmicroscope(IX7,Olympus),whereitwasfocussedontothesample by a 60´ water-immersion objective (UPlanSApo,Olympus). It was thus possible to independently shapeamplitudeandphaseoftheexcitationpulseatthesample.Thepulsecompressionandshapingutilised themethodofmulti-photon intra-pulse interference phase scan (MIIPS)25whereby a reference phase function is applied to thedispersed beam. Through measurement of the second-

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harmonic generation (SHG) from a SHG crystal at thesample position (10 µm BBO, Biophotonic Solutions Inc.),phasedistortionsoftheoriginalbeamcanbemeasuredandcancelled, yielding a transform-limited pulse or a desiredpulseshape.TheSHGspectrumwasmeasuredwithafibre-coupled spectrometer (USB4000, Ocean Optics). Theoptimalcompensationphaseandamplitudemaskwasthendetermined (MIIPS software version 2.0, BiophotonicSolutionsInc.).

Fig.1.Schematicofthe2Pmicroscopysetup.

After pulse shaping, the SHG crystal was replaced by thesample. The solution sampleswere placed on a cover slip(Menzel Gläser, Thermo Scientific) and the solutiontemperature was controlled with an incubator (Live CellInstrument, CU-501). All measurements were recorded at22±1°C.Sample fluorescencewas collectedby the sameobjective and transmitted through a dichroic mirror(Chroma 675dcspxr), split by a polarising cube (PBS) anddetected by two avalanche photodiodes (APD) (MPDPDM50c andMPD $PD-050-CTB). The signal was subsequentlydetectedbyeitheraphoton-countingcard(SPC-132,BeckerandHicklGmbH)formultichannelscalar(MCS)analysisorahardwarecorrelator(ALV-7002,ALVGmbH)forFCS.

Resultsanddiscussion1PPhotophysics

FreepAThe steady-state absorption, excitation and emissionspectra of the pA riboside in a phosphate buffer and inethanol are shown in Figure 2. There are notabledifferences between the spectra in the two solvents. Thestructure apparent in the emission spectrum in ethanol (apeakat405nmandashoulderat425nm)isabsentinthebuffer,andtheStokesshiftislargerintheaqueoussolvent.Theincrease inStokesshift isaresultofablue-shift intheexcitationspectrumanda red-shift inemission, relative tothe respective spectra in ethanol. These effects of solventinteraction indicate that the excited state of pA is morepolar than the ground state. Despite the differences inspectra,thequantumyieldofpAinbothwaterandethanolisreportedtobe0.66.23Comparisonoftheabsorptionandexcitation spectra shows small differences. Both spectrahavetwopeaks,adominantpeakataround390nmanda

secondpeakat370nm.Ineachsolvent,thereisared-shiftinthelongerwavelengthbandintheabsorptionspectrum,comparedwiththeexcitationspectrum.Theintensityratioof the two bands also differs between absorption andexcitationspectra,therelativeintensityofthe390-nmbandis greater in absorption; this is seen to a greater extent inethanol than in phosphate buffer. These observationssuggest the presenceof a specieswith lowquantumyieldthat have significant absorbance but make littlecontribution to the emission. The existence of more thanone species is supported by closer examination of theexcitation spectrum,which reveals a small dependence ofthe intensity ratio of the two excitation bands on theemission wavelength (Figure S2); the relative intensity ofthe 390-nm band increases with increasing emissionwavelength. It appears that both the absorption andemissionspectraofthespecieswithlowquantumyieldareshiftedtolongerwavelength,relativetothoseofthemorestronglyemittingspecies. Further evidence of more than one emitting speciescomes from fluorescence lifetime measurements. In bothphosphatebufferandethanol,thepAribosideexhibitsabi-exponential fluorescencedecay (TableS1)whenexcitedat390 nm. The average lifetimes in phosphate buffer andethanol are very similar at 3.9 ns and 3.8 ns, respectively.Asdiscussedabove,thequantumyield isthesameinbothsolvents, with a value of 0.66.23 The consistency betweenthe average lifetimes and quantum yields implies that theradiative lifetimes of the two emitting species are verysimilar, and hence the A-factors are indicative of thefractional populations. The dominant component has alifetime of 5.97 ns in buffer and 4.82 ns in ethanol andaccounts for themajority of the emitting population (65%and79%,respectively).Ashortlifetimeof0.19nsinbufferand 0.16 ns in ethanol accounts for the remainingpopulation.

Fig.2Absorption(red)andexcitationspectra(black),atemissionwavelength420nm,ofpAribosideina)phosphatebufferandb)ethanol.

From the decay parameters we find that, in bothsolvents, the short-lifetime component contributes lessthan 2% of the steady-state emission intensity, i.e. the

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steady-statespectrumisduealmostentirelytothelonger-lifetime species (Table S1). This is consistent with theobserved discrepancy between the excitation andabsorptionspectra(Figure2).Comparisonoftheabsorptionandexcitationspectrasuggestthattheshort-lifetime(low-quantum-yield) specieshasanabsorptionspectrumthat isslightlyred-shiftedcomparedwiththatofthe long-lifetime(high-quantum-yield)species.Thefractionalpopulations(A-factors) show negligible dependence on emissionwavelengthoverthemeasurementrangeof430to470nm,(TableS2) indicatingthatthetwospecieshaveverysimilaremission spectra over this range, but they may differ atshorterwavelengths. These observations point to the existence of twoground-state species with quantum yields that differ bymore than an order of magnitude. Consideration of thestructureofpA(Chart1a)suggeststhattheonlylikelycauseof this is N-H tautomerism, as illustrated in Figure S3.Tautomerism is a well-known phenomenon in the naturalnucleobases and has also been identified in fluorescentadenineanalogueslike2-aminopurine26andthetC-family.27There is alsoaprecedent for a largedifference inexcited-statelifetimesbetweentautomers.Foradenineinaqueoussolution,thelifetimeofthe7Htautomerisabout40timeslongerthanthatofthemoreabundant(78%)9Htautomer,anditisthustheminortautomerthatisresponsibleforthe(veryweak)fluorescenceofadenine.28 pA-containingoligonucleotidesWhen pA is incorporated into each of the fiveoligonucleotides(Chart1b), itsabsorptionspectrumisred-shiftedcomparedtothatofthefreeriboside inphosphatebuffer,andisverysimilartothatoftheribosideinethanol

(Figure S4). The red-shift ismost likely due to the polaritydifference between local environment in theoligonucleotidesandthebuffer.Thereislittlechangeintheabsorptionspectrumonduplexformation(FigureS4).

Fig.3Normalizedexcitation(dottedlines)andemissionspectra(solidlines)ofpAin single- (black) and double-stranded (red) oligonucleotides with differentcombinations of nearest neighbours. a) AT b) CC c) GA d) GG e) TT

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Fig. 4 Graphical representation of the fluorescence decay parameters (lifetimes and A-factors) and the fractional contribution of each decay component to the steady-stateemission intensity, for pA in (a) single-stranded and (b) double-stranded oligonucleotides, with different combinations of nearest neighbours. Excitationwas at 390 nm andemissionwasdetectedat430nm.

The excitation and emission spectra of the pA-containingoligonucleotides, both single- and double-stranded, areshown in Figure 3. The excitation spectra of the singlestrands,inthewavelengthrange310to410nm,wherepAis excited exclusively, closely resemble the correspondingabsorption spectra (FigureS5). (Atwavelengthsbelow310nm there is large discrepancy between excitation andabsorptionspectraasa resultofabsorptionby thenaturalbases.) The emission spectrum of pA in the single strandsmorecloselyresemblesthatofthefreeribosideinethanolthan that of the riboside in buffer (see Figure S6),consistentwithalesspolar,base-stackedenvironment. Comparisonofthespectraofsingleanddoublestrandsreveals a sequence-dependent effect. For CC, GA and GG,duplex formation has little effect on either excitation oremissionspectra.However,forATandTT,duplexformationresults in a noticeable blue-shift in both the emission andtheexcitation spectrum, and a sharpeningof thebands inbothspectra. Time-resolved fluorescence measurements, exciting at390nm,revealthepresenceofmultipleemittingspeciesinbothsingleanddoublestrands;inmostcasesthreelifetimecomponentsareobserved.Thedecayparameters togetherwith the fractional contribution of each decay componentto the steady-state emission intensity, are summarisedgraphicallyinFigure4.Themulti-exponentialdecaysreflectthe heterogeneity of inter-base interactions in theoligonucleotides; pA exists in a variety of base-stackedenvironments and hence experiences a variety ofquenching rates. The decay parameters are influenced bysequence context and duplex formation. A detailedinterpretation of these results is not relevant to the two-photonstudythatisthemainfocusofthispaperandwillbeaddressed in a future publication.However, it is pertinentto consider the contributions of the different lifetimecomponents to the steady-state emission intensities. Forthree of the single strands, GA, GG and AT, >70% of thesteady-state intensity isdue to thedecay componentwiththelongestlifetime(t3 ~ 7ns),whichalsoconstitutes>70%of the emitting population. In contrast, each of the threedecaycomponentsmakesasubstantialcontributiontotheemission intensity for TT andCC, and, in the latter case, acomponent with a much shorter lifetime (0.8 ns) isdominant.Inspiteofthis,theemissionspectraofTTandCCdo not differ significantly from those of the other singlestrands.This suggests thatallof theemitting specieshavesimilar emission spectra. This is supported by theobservation that, in all cases, the fraction populations (A-factors)areessentiallyindependentofemissionwavelengthbetween410and450nm. Forfourofthedoublestrands,GA,GG,ATandTT,onedecay component (t2 ~ 2 ns) contributes >70% of thesteady-stateintensity.ForCC,twocomponents(t1 =0.5nsand t2 = 1.2 ns) contribute almost equally to the emission

intensity;nevertheless,theemissionspectrumofCCisverysimilar to thoseofGAandGG.As in thecaseof thesinglestrands, the A-factors for all of the double strands arewavelength-independent over the emission range 410-450nm.It isevidentthatinbothsingleanddoublestrandstheemissionspectra show littleevidenceof theheterogeneityof the emitting population. This is partly because thesteady-state intensity tends to be dominated by onespecies, but also because the different emitting speciesappeartohavesimilarspectra. 2PPhotophysics

FreepA

Fig.5EmissionspectraofpAnucleobase inethanolfollowing1Pexcitationat360nm(top)and2Pexcitationat780nm(bottom).

Wenow turn our attention to non-linear excitation of pA,starting with the nucleobase. We recently reported thatfree pA could be excited via 2P-excitation at 780 nm,corresponding to double the wavelength of the 1Pexcitation peak at 390 nm.23 Interestingly, 2P-excitationresults in a different emission spectral profile than thatseenfor1P-excitation(Figure5).Theshoulderat425nminthe 1P-excited emission spectrum becomes the emissionmaximum in the 2P-excited spectrum, with the emissionmaximumat410nminthe1Pspectrumbeingreducedtoashoulderinthe2Pspectrum.Theemissionspectrumshownhere was excited at 360 nm, in order to record thecompleteprofile,butthepartialspectrum(omittingtherededge) recorded for excitation at 390 nm shows the samerelative intensity of the emission maxima, as shown inFigure S7. This confirms that the difference in spectralprofiles between 1P and 2P excitation is not theconsequenceofinequivalentexcitationwavelengths. Spectraldifferencesarealsoapparent inthe1Pand2Pexcitation spectra (Figure 6). The 2P excitation spectrumwas recorded both at a single emission wavelength(corresponding to that used for the 1P spectrum) and fortheintegratedemissionintensityovertheentirespectrum.As canbe seen, these spectra are identical, rulingout any

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effects that might arise from the selective detection ofdifferentemittingspecies.

Fig.6NormalizedfluorescenceexcitationspectraofpAnucleobase inethanol for1Pexcitation (black squares,bottomaxis) and2Pexcitation (red circlesandbluetriangles, topaxis). For the1P spectrum,emission intensitywas recordedat 420nm (bandpass3nm). For the2P spectra,emission intensitywas recordedat420nm(bluetriangles)andintegratedoverthewholespectrum(redcircles).

The2Pexcitationmaximum is red-shiftedbyaround5nmcompared to the one-photonmaximum (the 2P peak is at790 nm,whereas the 1P peak is at 390 nm). This is not ashift of the entire spectrum, but a change in the spectralprofile, since, in the 2P spectrum, the second maximum(370/740 nm) is not red-shifted and is also more intenserelativetotheshort-wavelengthband.Itisnotablethatthe2P excitation spectrum more closely resembles the 1Pabsorption spectrum than the 1P excitation spectrum(Figure2). Theseobservationsareconsistentwiththepresenceoftwotautomerswhoserelative2Pcrosssectionsdifferfromtheir relative one-photon absorption coefficients. This isconfirmed by the results of time-resolved fluorescencemeasurements under 2P excitation (Table S3)which showthe same two lifetime components as those seen for 1Pexcitation, butwith significantly different A-factors. Under2Pexcitation,theshort-lifetime(t1)speciesconstitutes80%oftheemittingpopulation,comparedwithonly21%under1P excitation. Consequently, the contribution of the t1tautomertothesteady-stateemissionintensityisincreasedfrom1%under1Pexcitationto12%(at435nm)under2Pexcitation,resultingintheobservedchangeintheprofileofthe emission spectrum. A greater contribution to theemission from the t1 species also accounts for thediscrepancybetween2Pand1Pexcitationspectra,andtheresemblance of the 2P spectrum to the 1P absorptionspectrum. The emission spectral profile is essentiallyindependentofexcitationwavelength(FigureS8),butthereare small differences in the relative intensities of the twomaxima.Thespectrumexcitedat810nm,ontherededgeof theexcitationspectrum,showsthegreatest intensityofthe longer wavelength band and is likely to be mostcharacteristicofthet1tautomer.

Unlike the 1P absorption coefficient, which isdetermined by the transition dipole moment betweengroundandexcitedstates,the2Pcrosssection,s2,dependson both the transition dipole moment and the change inpermanent dipole moment on excitation (in a two-levelmodelforadipolarchromophore),asexpressedineq4.

σ9 ∝ 𝜇.< 9 ∆𝜇.< 9(4) where 𝜇.< is the transition dipole moment between thegroundstate(S0)andtheexcitedstate(S1),and∆𝜇.<isthedifference between the permanent dipole moments of S1andS0.

29Thedifference inrelative2Pcrosssectionsofthetwo tautomers, compared with their relative 1P crosssectionscan,therefore,beattributedtoagreaterchangeinpermanent dipole moment on excitation for the t1tautomer. We end this section with a caveat regarding thecalculationofthe2Pcrosssectionfromtheexperimentallymeasured parameter, the 2P brightness, σ2f, which is theproduct of the quantum yield and the 2P cross section.Calculationof thecrosssectionassumesthat thequantumyieldisindependentoftheroutetoexcitation.Wheretherearemultiplespeciesinasample,thesteady-statequantumyield isanaverageoverthevariousabsorbingpopulations.Should these populations have different 2P cross sectionsrelative to 1P absorption coefficients, then the averagequantum yield will change accordingly. This applies in thecaseofpA.The2Pbrightnessof5.3GM(at780nm)isthetrue value of the average 2P brightness of the twotautomers and, in the context of fluorescence detection,thisistheparameterofinterest.However,itisnowevidentthat the previously quoted (average) 2P cross section,calculated using the average 1P quantum yield,23 wasconsiderably underestimated. On the basis of the averagelifetimes under 1P and 2P excitation, 3.8 ns and 1.2 ns,respectively (Tables S1 and S3), we now obtain a revisedvalue of 21 GM for the (average) 2P cross section of pA.This is almost three times greater than the highest valuereportedpreviouslyforaFBA,7.6GMfor5-(thiophen-2-yl)-2ʹ-deoxyuridine.20 pA-containingoligonucleotides.For each pA-modified single-stranded DNA (ssDNA) anddouble-stranded DNA (dsDNA, Chart 1), log-log plots ofemission intensity versus laser power show a gradient of~2,confirmingatwo-photonabsorptionprocess(FigureS9).The2P-excitedemissionspectraareshowninFigure7.Inallcases, the spectra are very similar to their 1P-excitedcounterparts (Figure 3), displaying similar effects ofsequence context and duplex formation. However, thereare small differences in emission spectral profile between2P and 1P excitation. These differences, which are muchless pronounced in the oligonucleotides than that seen inthe riboside, can be attributed to small differences in the

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relative 2P and 1P cross sections of themultiple emittingspeciespresent. The measured 2P brightness values together with thederived cross sections of the five pA-modifiedoligonucleotides(ssDNAanddsDNA)arepresentedinTable3.(Anextendedversion(TableS4)alsogivesthepreviouslyreportedvaluesforthe1Pquantumyield,molarabsorptioncoefficient(e390)and1Pbrightness (e390f).

23 Inderivingthecross section values, we again need to be mindful of thepresence of multiple species. The brightness values aresequence-dependent,particularly forthesinglestrands.Asshown in Table S4 and Figure S10, the 2P brightnesscorrelates very closely with the sequence-dependent 1P(average) quantum yield reported previously,23 across theentire set of samples. This implies that there is littlediscrepancybetweentheaveragequantumyieldsunder2Pand 1P excitation. There could be a systematic difference,but this seems unlikely, given the variation in thecompositionoftheemittingpopulationsacrosstherangeofsamplesthatisevidentinthedecayparameters.

Fig.72P-excitedemissionspectraofpAinsingle-(red)anddouble-strandedDNA(black)with different combinations of nearest neighbours. Excitationwas at 780nm.

Table1Two-photonbrightness(σ2f) andcrosssection(σ2)ofpAin ss- and dsDNA with different combinations of nearestneighbours(seeChart1forsequencenamesNN).a

ssDNA dsDNA

σ2f / GM σ2 / GM σ2f / GM σ2 / GM

GAb 1.3 3.0 0.35 2.4

GG 0.65 2.3 0.30 2.4

AT 0.39 2.6 0.46 2.7

CC 0.09 2.5 0.13 3.0

TT 0.06 2.5 0.30 2.7

aExcitationat780nm.Errorinthestandardvaluesofσ2isreportedas8%.bThe2PcrosssectionsforGAwerereportedpreviously.23

The cross sections are similar for pA in ssDNA and dsDNAanddonot showany significant dependenceon sequencecontext. (In view of the possible uncertainty in quantumyieldvaluesunder2Pexcitation,weconsiderthevaluesof2P cross sections for the various oligonucleotides to bewithinerrorofeachother.)However,theyaresubstantiallylower, by about a factor of 2, than the value for the pAnucleobase, 6.6 ± 0.5 GM (at 780 nm), that we reportedpreviously.23Incomparison,the1PabsorptioncoefficientofpA decreases by only about 20% on incorporation inoligonucleotides.23 A decrease in 2P cross section onincorporation in an oligonucleotide has been reported foran intercalating dye,30 but, to our knowledge, thisphenomenon has not been reported previously for a FBA.This effect appears to be analogous to the influence ofmolecular environment on 2P absorption in fluorescentproteins,thathasbeeninvestigatedindetailbyRebaneandcoworkers.31 As shown in their studies, this can beexplainedbytheinfluenceofthelocalelectrostaticfieldonthe difference in permanent electric dipole momentbetweengroundandexcitedstates,∆𝜇.<andhenceonthe2P cross section (see eq 4). Application of an externalelectricfieldresultsinanadditionalinducedcomponentofthedipolemoment,asaresultofthepolarizabilityofthep-electron systemof the chromophore. If thepolarizabilitiesof thegroundandexcited states aredifferent, the changeof induced dipole moment will contribute to the totalchange of dipole moment. For pA in oligonucleotides, itappears that the local electrostatic field acts to decrease∆𝜇.<. If this fieldwere due predominantly to the chargedphosphate groups, this could account for the lack ofdependenceonsequencecontextorduplexformation. 1Pvs2Pphotostability

Inadditiontopossessingahighbrightness,itisessentialformany applications that FBAs have high photostability. Thefluorescence intensity of pA during 1P and 2P excitationwasmeasured as a function of irradiation time,with laserpoweradjusted to give the same initial, absoluteemissionintensity for both excitation regimes. The experimentalarrangement is shown in Figure S1. As shown in Figure 8,overthe35-minutetimescaleinvestigated,the2Pemissionintensity remained constant, whereas the 1P signaldecreased to about a third of its initial value. The 1Pphotobleaching decay is non-exponential, suggesting atime-dependent rate constant that may be due to thedepletionofoxygenconcentrationintheirradiatedvolumeduringthemeasurement,sincethesolutionwasnotstirred.The dependence of photobleaching rate on the excitationregime suggests that the photochemical process does notoccur from the initially excited state, but as a result offurther 1P absorption by the excited singlet state (during

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the initial excitation pulse) and/or a longer-lived tripletstate(duringsubsequentexcitationpulses).

Fig.8ComparisonofthedependenceofemissionintensityoffreepAnucleobaseonirradiationtimefor2Pexcitationat780nm(redsymbols)and1Pexcitationat390 nm (black symbols). Excitation intensities were adjusted to give the sameinitial,absoluteemissionintensityineachcase.2PexcitationusedthefundamentaloutputoftheTi:sapphirelaser,repetitionrate76MHz,averagepower930mW.1Pusedthesecond-harmonic,pulsed-pickedoutputofthesamelaser,repetitionrate4.75MHz,averagepower690μW.

Thehighphotostabilityunder2Pexcitationencouragedustoattemptultrasensitivedetection,asdescribedinthenextsection.

Pulse-shaped2PmicroscopyofpA-containingDNA

The GA single-strand oligonucleotide was identified ashavingthehighest2Pbrightness(Table1)andwasusedforsubsequent measurements. For microscopy, we used abroadbandTi:sapphirelaserastheexcitationsource(Figure1).Inordertogenerateatransform-limitedpulse,orotheruser-definedpulseshapes,at thesample it isnecessary tomeasure and control dispersion in the optical setup. Inparticular, the higher-order dispersion terms becomesignificant as the bandwidth increases (andpulse durationdecreases).32The need for dispersion control can be understood byconsidering2Pexcitationofatwo-levelsystemwithenergydifferenceℏ𝜔<:

𝑃9 ∝ 𝐸𝜔<

2+ 𝛿𝜔 𝐸

𝜔<

2

E

FE

− 𝛿𝜔 exp 𝑖 𝜑𝜔<

2+ 𝛿𝜔

+ 𝜑𝜔<

2− 𝛿𝜔 𝑑𝛿𝜔

9

5

where 𝑃9 is the two-photon transition probability; 𝐸 𝜔 and 𝜑 𝜔 are the spectral amplitude and phase,respectively; and 𝛿𝜔 represents a spectral detuning from

𝜔<.33, 34 The probability of 2P absorption (eq 5) is due to

contributionsfromallpairsofphotonsthatsumtogivethetransition frequency𝜔<. In otherwords, different parts ofthesamepulsecan interferewitheachother.Bychangingthe spectral phase of broadband pulses, this intrapulseinterferencecanbecontrolled.35Weusedthemultiphotonintrapulse interference phase scan (MIIPS) method tomeasure and compensate for dispersion.36 A dual pulseshaperallowedus tocontrol independently thephaseandamplitude. It has been shown previously that using suchshort pulses at the focus of a high numerical apertureobjectivecangivesignificantgainsinsignalin2P-excitationfluorescence microscopy.37,38 This approach can also beextended to wide-field excitation and detection.39 Formolecules,with broad absorption spectra, it is possible totailor the spectral phase40-42 or amplitude38 in a desiredfrequency range for selective excitation of fluorophores.Ourgoalwas tomaximise the ratioof the2P fluorescencetobackgroundsignalbyshapingthelaserpulse. The full spectrum of the laser extends from ~650-950nm (Figure 9). For each pulse shape, the signal andbackgroundwhere recorded as a function of laser power.Intensitywasproportionaltothesquareofthelaserpoweruntilathresholdwasreached,thevalueofwhichdependedon the particular laser spectrum and phase (Figure 10).Firstly,we investigated theeffectof phase shapingon thesignal.Usingthefulllaserspectrum,wefoundthatthebestcontrastwas achieved using transform-limited pulseswithSHG signal centred on 410 nm and pulse width of 8 fs(Figure9).

Fig. 9 The full spectrum of the laser source (blue line). Grey areas indicate thespectralcomponents,blockedfortheoptimalpulseshape.Theinsetgraphshowsthesecondharmonicgeneration (SHG)spectrumfor the full laserspectrum(red)andtheSHGspectrumfromthemaskedlaserspectrum(black).

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Further improvements were achieved by adjusting thecentreandthewidthofthespectrum,removingregionsofthespectrumthatleadpredominantlytobackgroundsignalrather than two-photon absorption by pA. The best SBRratiowasobtainedwithanamplitudemaskcentredat787nmandawidthof182nm(Figure9).ThisincreasedtheSBRfrom7.1 to11.7,with respect to the full spectrum.This isconsistentwiththelong-wavelengthmaximumidentifiedinthe two-photon excitation spectra (Figure 5) andwith theSHG spectrum (Figure 9),which shows that the amplitudeshaping shifts the maximum towards the pA excitationmaximum. Compared to a compressed pulse with noadditional shaping, the SBR ratio increases by almost 65%whenashapedpulse isused.ThepAcountratedecreasesslightly when using the shaped pulse. Therefore, weattribute the increase in the SBR to backgroundsuppression,i.e.moreselectiveexcitationofpA.

Fig. 10 Logarithmic power dependence of the fluorescence intensity of GAoligonucleotide,with no spectralmasking. Dependence is linear,with a slope of1.98 ± 0.04, up to a power of 7.5mW (1.7 TW/cm2). Loss of linearity occurs athigherpowers.Errorbarsrepresentthestandarderrorofthemean(N=4).

The SBR was highest for a laser power of 9.7 mW withphase and amplitude shaping (Figure 9). Having optimizedthe signal, we used FCS to probe the freely-diffusing pA-modifedoligonucleotideandassess the2Pbrightness.Thesetupwas first optimised bymeasuring the 2P-FCS of thedyerhodamine110(FigureS11).ArepresentativeFCScurverecorded for pA in the GA oligonucleotide is shown inFigure 11. Background measurements of the buffer weremade under identical conditions to ensure that thecontribution of fluorescence background was negligible(Figure11,inset).Therewasnoevidenceofphotobleachingduringtheconfocaltransit(stablesignalfor30minutes)at11mW(FigureS12).Furthermore,thenumberofmoleculesmeasured in the focus scales linearly with sampleconcentration (Figure S13). The emission is resolvedaccording to the polarization with respect to the laser

alignment. The collection efficiency for the two detectionchannels are matched, so it is apparent that there is ahigher signal in the parallel channel (Figure S12), which isduetotherestrictedmobilityofthepAinthe10merssDNAand2Pphotoselection.43

Fig. 11 Fluorescence correlation spectroscopy for GA oligonucleotide at 9.8mWwiththeoptimumpulseshape.Thecorrelationcurveisshowninblue.Thefit(redcurve)gaveanaverageof6.7molecules intheexcitationvolumewithadiffusiontimeof81μs.The insetshowsthecorrelationcurve forpurebuffer, recordedatthesameconditions.IB=0.725kHz;S=4.889kHz.Thetemperatureofthesolutionwas22±1°C,thesampleconcentrationwas54nMandtheacquisitiontimewas30minutes.

Thecorrelationcurve𝐺 𝑡 wasfitted(Figure11)toamodelof diffusion through a 3D Gaussian volume (eq 6), takingthe background contribution 𝐼O and the strength of thesignal𝑆intoaccount44:

𝐺 𝑡 =1 − 𝐼O 𝑆 9

8𝑁1

1 + 𝑡 𝜏R

⋅1

1 + 𝑤< 𝑧< 9 𝑡 𝜏R(6)

where 𝑤< and 𝑧< are the waist and the height of theassumedGaussianexcitationvolumeand𝜏R istheaveragediffusion time. The value of 𝑤</𝑧< was taken from thecontrol measurement of rhodamine 110 (Figure S11). Theaverage number ofmolecules in the confocal volumewasdetermined from the fit (4.2-7.1 depending on themeasurement), allowing us to calculate the CPM for theoptimal pulse shape and power as 0.50 ± 0.03 kHz permolecule. Thediffusion time for theGAoligonucleotide inbufferwas89µs±5µs,whereasthatofthefreerhodamine110dyeinwaterwas49µs±5µs(FigureS11). TheFCSmodel(eq5)assumesahomogeneoussample.To examine the possibility of sub-populations of pA stateswithdifferentbrightness,wedilutedthesolutiontothe~10

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pM level and collectedMCS traces (Figure S14). TheMCStraces for sample (Figure S14a) and buffer (Figure S14b),recordedfor30minutes,aresimilarwithslightlymorehigh-intensity bursts apparent for the sample. The burstfrequency histogram (Figure S13c) from theseMCS tracesconfirms thatmost of the bursts are of a similar size anddistributiontothebuffer,asexpectedforameanCPMof~0.50 kHz, with no evidence of any highly-emissivepopulations(e.g.duetoaggregateformation). WeattemptedFCSoftheGAduplex,butthesignalwastoo weak to achieve a reliable correlation curve.Nevertheless, sinceweknowthat the2Pbrightnessof thedsDNA is ca. 3 time lower than for the ssDNA (Table S4),this is further proof that we are detecting pA in ssDNA,ratherthanthefreebaseorsomeotherimpurity.

ConclusionsThe 1P and 2P fluorescence properties of the FBApentacyclic adenine, pA, have been characterised for thenucleobase,nucleosideand ina seriesofoligonucleotides.Time-resolved fluorescence measurements indicate thatthe nucleoside in aqueous solution exists as two emittingspecies with quantum yields that differ by more than anorder of magnitude, and it is likely that these aretautomers.Thetwotautomersdifferintheirrelative2Pand1P cross sections, giving rise to different spectral profiles,both excitation and emission, for 2P and 1P excitationregimes. Consequently, the average (steady-state)fluorescence quantum yield is significantly lower for 2Pexcitationthanfor1Pexcitation. In oligonucleotides, pA exhibits multi-exponentialfluorescence decays indicative of different inter-baseinteractionsarising fromtheconformationalheterogeneityof DNA oligomers. There is little evidence of theheterogeneity of the emitting population in the emissionspectra, partly because the steady-state intensity tends tobe dominated by one species, but also because thedifferentemittingspecieshavesimilarspectra. Althoughthe2PcrosssectionofpA inoligonucleotidesis lower than that of the nucleobase, the 2P brightness ofpA is thehighest reported foraFBA inanoligonucleotide.The 2P brightness depends on sequence context as aconsequence of the sensitivity of the quantum yield tointeractionwithdifferentneighbouringbases.The2Pcrosssectionvaluesaresimilarforsingleanddoublestrandsanddo not show any significant dependence on sequencecontext. The decrease in 2P cross section on incorporation inDNA can be attributed to the influence of the localelectrostatic field on the change in dipole moment onexcitation.This isexpected tobeageneraleffect forFBAsand highlights the importance of measuring 2P crosssectionsinoligonucleotideswhencharacterisingnewFBAs.AlthoughthiseffectresultsinadecreaseincrosssectionforpA, it could, in principle, result in an increase for other

FBAs,dependingontheprojectionofthelocalelectricfieldvectoron∆𝜇.<. Thehigh2PbrightnessofpA,itsphotostabilityunder2Pexcitation, compared with resonance excitation, and thefavourable excitation and emissionwavelengths, led us toexamine itspotential forultrasensitiveanalysis.Usinga2Pmicroscopewith shaped broadband laser pulses,wewereable to detect as few as five pA-modified oligonucleotidemoleculesfreeinsolution. A number of theoretical studies of the 1P and 2Pphotophysics of fluorescent nucleobases have appearedrecently22, 45-48However, the influenceof the local electricfield on the 2P cross section in DNA has not beenaddressed.We hope that the results of the present workwill stimulate theoretical studies of this effect. As designprinciples emerge, structural modifications will beimplemented to systematically improve the 2P propertiesof FBAs. Other gains in signal are likely to come from theuse of strategies to increase the photostability. Forexample, antifade reagentswhich can reduce bleaching athigh powers and increase the brightness are commonlyusedforotherorganicdyes.49Throughsuchdevelopments,we believe that the goal of routine single-moleculedetection of FBA-modified nucleic acids will soon berealised. Although the 2P brightness of pA falls short of thatrequired for single-molecule spectroscopy, it is anexceptionally promising fluorescent probe formultiphotonmicroscopy. In combining high detection sensitivity, highphotostability, optimal wavelength for Ti-sapphire laser-excitation, and an estimated achievable imaging rate of~300 frames per second (under the generic conditionsdefined in reference21),pAcomprehensivelyoutperformsotherFBAs.Weanticipate that theapplicationofpA in2Pmicroscopywill greatly enhance the capability for imagingnucleicacidswithminimalstructuralperturbation.

ConflictsofinterestTherearenoconflictstodeclare.

AcknowledgementsThis work was supported by EPSRC (studentships for RSFandDN) and theUniversityof Edinburgh (RSF). ThisworkwasalsosupportedbytheSwedishFoundationforStrategicResearch (SSF, grants No. ID14-0036 (MG), IS14-0041(LMW) and IRC15-0065 (LMW)) and the Swedish ResearchCouncil (VR, grantNo. 2017-03707 (LMW)).Wewould liketo thankDr.DmitryPestov (Biophotonic Solutions Inc.) fortechnicalsupport.Research data supporting this publication can be found athttp://dx.doi.org/10.7488/ds/2457.

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UltrasensitivedetectionofDNAisachievedviatwo-photonexcitationofafluorescentbaseanalogue.