Stimulated Raman Excited Fluorescence Spectroscopy of VisibleDyesHanqing Xiong, Naixin Qian,† Yupeng Miao,† Zhilun Zhao, and Wei Min*
Department of Chemistry, Columbia University, New York, New York 10027, United States
*S Supporting Information
ABSTRACT: Fluorescence spectroscopy and Raman spectroscopy are two major classesof spectroscopy methods in physical chemistry. Very recently, stimulated Raman excitedfluorescence (SREF) has been demonstrated (Xiong, H.; et al. Nature Photonics, 2019, 13,412−417) as a new hybrid spectroscopy that combines the vibrational specificity ofRaman spectroscopy with the superb sensitivity of fluorescence spectroscopy (down tothe single-molecule level). However, this proof-of-concept study was limited by both thetunability of the commercial laser source and the availability of the excitable molecules inthe near-infrared. As a result, the generality of SREF spectroscopy remains unaddressed,and the understanding of the critical electronic preresonance condition is lacking. In thiswork, we built a modified excitation source to explore SREF spectroscopy in the visibleregion. Harnessing a large palette of red dyes, we have systematically studied SREFspectroscopy on a dozen different cases with a fine spectral interval of several nanometers.The results not only establish the generality of SREF spectroscopy for a wide range ofmolecules but also reveal a tight window of proper electronic preresonance for thestimulated Raman pumping process. Our theoretical modeling and further experiments on newly synthesized dyes also supportthe obtained insights, which would be valuable in designing and optimizing future SREF experiments for single-moleculevibrational spectroscopy and supermultiplex vibrational imaging.
The exquisite chemical specificity of Raman spectroscopyprovides rich structure and dynamics information.1
However, Raman scattering is intrinsically weak, with a crosssection of 10−30 cm−2 for typical chemical bonds.2 Althoughseveral enhancement mechanisms (such as coherent Ramanscattering and electronic resonance Raman) have beenextensively exploited to amplify the Raman scattering crosssections,3−11 it is still difficult to achieve all-far-field (withoutplasmonic resonance2,12−17) Raman spectroscopy at theultimate single-molecule level. For example, electronic pre-resonance stimulated Raman scattering microscopy, which hascombined coherent Raman scattering and electronic resonanceRaman together under an optical microscope, is still limited toabout 50 molecules.18
Another important school of strategies to enhance the Ramansensitivity is to couple vibrational information to more sensitiveoptical observables. Notably, the pioneering work of Laubereauet al. has employed fluorescence detection with time-resolvedinfrared spectroscopy to study vibrational relaxation dynam-ics.19−21 Along this line of fluorescence-encoded infraredspectroscopy, Whaley-Mayda et. al have coupled this methodwith modern confocal microscopy and reported an impressivesensitivity at the nanomolar level.22 Along the Raman direction,Wright proposed early in the 1980s that the fluorophore can befirst pumped through stimulated Raman scattering (SRS) to avibrational excited state, which can then be brought up to afluorescence excited state by a second excitation23 (Figure 1a,which is related to but distinct from electronic resonancecoherent anti-Stokes Raman scattering (CARS)24). This
approach would have both the sharp lines characteristic ofRaman spectroscopy and an increased sensitivity characteristicof fluorescence spectroscopy. In light of the revolutionarysuccess of single-molecule fluorescence spectroscopy in thesubsequent decades,25−27 Winterhalder et al. were alsooptimistic and proposed in 2011 that this method might offerthe single-molecule sensitivity of vibrational spectroscopy atoptical far-field.28 However, the first experimental attempt byWright’s group in 1983 was hindered by the overwhelming two-photon fluorescence background from perylene dye.29 To thebest of our knowledge, no further experimental studies havebeen published about this type of spectroscopy since 1983.Very recently, our group has revisited this topic and reported
stimulated Raman excited fluorescence (SREF) spectroscopyand imaging (Figure 1a).30 By leveraging the joint advantage ofchemical specificity and detection sensitivity, we have achievedsingle-molecule Raman sensitivity in an all-far-field manner forthe first time and demonstrated multicolor SREF imaging ofisotopologues to break the color barrier of fluorescencemicroscopy. We noted that the stimulated Raman pumping inthe previous proposals and attempts was operated in thenonresonant condition. As a result, SREF can be easily buried byother competing process such as two-photon excited fluo-rescence, which was observed in both the 1983 study29 and our
Received: May 6, 2019Accepted: June 11, 2019Published: June 11, 2019
Letter
pubs.acs.org/JPCLCite This: J. Phys. Chem. Lett. 2019, 10, 3563−3570
own experiment on the coumarin dye. Hence, we decided toharness electronic resonance to promote the stimulated Ramanpumping process. However, we found in our pilot studies thatbringing stimulated Raman pumping to rigorous electronicresonance gave us an overwhelming background largely from theanti-Stokes fluorescence background. We reasoned that thisshould be due to the fact that the linear absorption cross section(which determines anti-Stokes fluorescence background)increases much faster than the resonance Raman cross sectionas the pump excitation approaches rigorous electronicresonance.31 Therefore, we detuned the stimulated Ramanpumping empirically to some extent to electronic preresonanceand successfully achieved vibrational specificity and nanomolarsensitivity on two dyes (nitrile mode of Rh800 and C=C skeletalmode of ATTO 740).Despite the excitement and promise, there are two critical
(and also inter-related) limitations in this proof-of-conceptstudy. First, the experiment is based on a commercial opticalparametric oscillator (OPO) system, which outputs a fixed beamat 1031.2 nm and a tunable pump beam operated in the near-IRregion (i.e., the electronic resonance cannot be studiedindependent of the vibrational resonance). There are limitedchoices of bright fluorophores in the near-IR range. As a result,only two dyes have been demonstrated.30 The generality ofSREF spectroscopy remains unestablished beyond these twodyes. Second, the apparent “rare” success of SREF suggests thatit is critical to achieve a proper electronic preresonance for thestimulated Raman pumping process: too much electronicresonance will make anti-Stokes fluorescence dominate SREF,and too little electronic resonance will make SREF too weak.Thus, the logical next step is to study the effect of electronicpreresonance condition in a systematic way in order toquantitatively characterize and understand this effect. Thisunderstanding is rather important as it will contribute toestablishing the generality of SREF spectroscopy as well asproviding a practical guide for future design and application.Again, this task is difficult to perform with the commercial OPOsystem and the relative lack of dyes in this near-IR range.
In the present work, to address the two critical limitationsrelated to SREF spectroscopy in our proof-of-concept paper, wewill build a modified source to open up a new excitation rangenear 600 nm for SREF, which will allow us to test the generalityof SREF spectroscopy on a number of visible dyes and to studythe effect of electronic resonance conditions in a systematic way.While a two-beam free tunable laser system might be moreflexible to gain independent access to electronic resonance andvibrational resonance, such a laser system is rather difficult andexpensive to obtain in the lab. Alternatively, we will adopt asimple approach by frequency doubling the idler beam from thecommercial OPO laser source and then recombining it with theOPO signal beam as a new pair of pump and Stokes beams(Figure 1, detail in Experimental Methods) to open up a newSREF excitation range near 600 nm. Benefiting from a largevariety of commercial fluorescence dyes in this visible range, thegenerality of SREF can be sufficiently tested. Moreover, we willselect a rich list of dyes with slightly different (a few nanometerinterval) absorption peak (λabs) in this range, which providesdifferent electronic resonance conditions for systematicevaluation.Our new excitation source is based on an optical modification
of the previous commercial OPO system (picoEmerald S, APE),which outputs the signal beam (λsignal, tunable from 790 to 960nm) and the idler beam (λidler), with their wavelengths followinga relationship of 1/λsignal + 1/λidler = 1/515.6 nm. Hence, bytuning of λsignal, λidler will cover the wavelength range of 1484.4nm to 1113.8 nm with the same pulse shape and laser line width(2 ps, 0.6 nm fwhm bandwidth).We then frequency doubled theidler beam by second harmonic generation (SHG) with aperiodically poled lithium niobate (PPLN) crystal (Covesion,MSHG1420-0.5-5) (Figure 1b,c). We then used the SHG(λidler/2) of the idler as the new pump beam (ωp) and the signalλsignal as the new Stokes beam (ωs). When tuning λsignal from 805to 825 nmwith the PPLN temperature matched simultaneously,the λidler/2 will sweep from 717.1 to 687.4 nm. As such, theenergy difference between this new pair covers the Raman shiftbetween 1523 and 2426 cm−1 (Figure 1b). The crystal length (5
Figure 1.New stimulated Raman excited fluorescence (SREF) laser microscopy system opening excitation band near 600 nm. (a) Energy diagram forSREF process. (b) Wavelength of SHG of OPO idler (red) and corresponding resonance Raman shift (blue) as a function of OPO signal wavelengthwhen OPO signal is set as the Stoke beam and the SHG is set as the pump beam. (c) System setup for SREF excitation band around 600 nm. PPLN,periodically poled lithium niobate; QWP, quarter-wave plate; DM, dichroic mirror; PBS, polarization beam splitter; BS, beam splitter; SPCM, single-photon counting module; DAC, digital-to-analog converter; ADC, analog-to-digital converter; EOM, electro-optical modulator; APD, avalanchedphotodiode; PD, photodiode.
The Journal of Physical Chemistry Letters Letter
DOI: 10.1021/acs.jpclett.9b01289J. Phys. Chem. Lett. 2019, 10, 3563−3570
mm) is chosen to be short enough to ensure neglectable groupvelocity mismatch, which well maintains the ∼2 ps pulse widthfor efficient SREF excitation30 (Figure S1a).With this new excitation source, the total excitation energy
((ωp−ωs) +ωp) for a typical C=C skeletal mode (∼1650 cm−1)of fluorescent dyes reaches ∼638 nm. Therefore, the C=Cskeletal mode with dye absorption peak (λabs) around 638 nmcan be potentially excited. Different from the previous near-IRregion which lacks bright fluorophores, many commercialfluorophores with high photostability and large quantum yield,such as Alexa 610, ATTORho14, Nile Blue A, Alexa 633, ATTO633, Alexa 647 etc., can be found with absorption peak around638 nm (likely because of the famous He−Ne laser line at 633nm). To test the generality of SREF spectroscopy and to studythe critical electronic preresonance condition systematically, weselect seven popular fluorescent dyes with λabs gradually shiftingfrom 621 to 662 nm. With the additional help of solvatochromiceffect in different solvents,32 we are able to generate 11 differentλabs within this range with a step size of several nanometers(Table 1). Because the total SREF excitation energy ((ωp − ωs)+ ωp) of the C=C mode is fixed around 638 nm, these 11 λabsfrom 621 to 662 nm serve as a “sweep” to study the effect of theelectronic resonance condition in a systematic way. As shown inTable 1, we can divide these 11 cases into three categoriesdepending on the energetic relationship between λabs and ((ωp− ωs) + ωp).We started from the high-energy side of the absorption peaks.
ATTO 610 NHS ester in phosphate buffered saline (PBS, pH7.4) is the model dye in this category. As shown in Figure 2, theSRS spectrum clearly shows a Raman peak around 1638 cm−1 for
its C=C skeletal mode. Note that the electronic resonance isdetuned substantially so that the total SREF excitation energy((ωp − ωs) + ωp) is lying below the ensemble 0−0 transitionenergy (i.e., the cross point between the absorption spectrumand corresponding emission spectrum). As a result of thisenergetic gap, the pure SREF signal is observed to be very weakand barely detectable above the background (Figure 2b,c). Wereason that only a very small portion of molecules in the solutioncan be pumped to an electronic excited state by SREF (likelybecause of inhomogeneous broadening). Therefore, the 0−0transition energy can be set as the “red” side limit for the totalSREF excitation energy. This limit would become moreapparent after considering the complete “sweep” of the 11cases shown below.There are eight different cases in the second category in which
the total SREF excitation energy slightly exceeds that of the 0−0transition line and lies in the vicinity of the dye absorption peak(λabs). Remarkably, robust SREF peaks were observed againstthe antistokes fluorescence background in all eight cases (Figure3). For several of these dyes such as ATTORho14 and Alexa 633(Figure 3f,g), high-quality SREF spectra can be recorded withsolution even below 50 nM (which corresponds to only a fewmolecules on average within our tight laser focal volume). Wethus attribute this superb sensitivity to the nearly perfect matchbetween ((ωp − ωs) + ωp) and λabs. Moreover, SREF spectracontaining double vibrational peaks were recorded for ATTORho14 in both PBS and DMSO solutions (Figure 3c,f), whichshowcases the accurate reflection of SREF on the vibrationaldimension. This is an importanct technical advance and
Table 1. Spectral Properties of Commercial Red SREF Dyes Investigated in This Study
ATTO 610* a 621 1644 638.1 500 undetectableAlexa 633^ a 630 1651 637.6 100 2.5ATTO 610* b 631 1644 638.1 500 3.0ATTO Rho14* a 632 1652 and 1680 637.5 and 635.7 <2000 7.5 and 2.1Nile Blue A c 635 1652 637.5 500 7.1ATTO 633* a 635 1640 638.4 500 3.1ATTO Rho14* b 638 1652 and 1680 637.5 and 635.7 50 3.0 and 1.5Alexa 633^ b 640 1651 637.6 10 3.0ATTO 633* b 642 1640 638.4 500 1.6Alexa 647* a 654 1602 640.8 500 undetectableJF 646* d 662 1616 639.9 500 undetectable
a^ for carboxy linker; * for NHS linker. ba for PBS (pH 7.4); b for DMSO; c for ethanol; d for 0.1% TFA in ethanol. cSNR, signal-to-noise ratio.Data shown in Figures 2−4.
Figure 2. SREF excitation with the total SREF excitation energy ((ωp − ωs) + ωp) below the 0−0 transition line. (a) SREF excitation and signalcollection diagrams for ATTO 610 (NHS ester) in PBS (pH 7.4). Purple curves and blue curves show the absorption and emission spectra; the darkred, light red, and dashed blue lines show the corresponding wavelength positions of Stokes beam (ωs), pump beam (ωp), and total excitation energy((ωp − ωs) + ωp), respectively; the blue band shows the fluorescence collection band of the filter set (FF01-661/20, Semrock). All laser lines andCARS lines ((ωp − ωs) + ωp) are avoided. (b and c) SRS spectrum and the SREF spectrum of ATTO 610 (NHS ester), respectively. Correspondingconcentrations and solvents are labeled in each panel. The SRS spectrum was measured under 2.5 mW pump power and 13mW Stokes power, and theSREF spectrum was measured under 6 mW pump power and 13 mW Stokes power.
The Journal of Physical Chemistry Letters Letter
DOI: 10.1021/acs.jpclett.9b01289J. Phys. Chem. Lett. 2019, 10, 3563−3570
Figure 3. SREF excitation with the total SREF excitation energy ((ωp − ωs) +ωp) within the proper-electronic preresonance region. The left columnshows the SREF excitation and signal collection diagrams for Alexa 633 (Carboxy) in PBS (pH 7.4), ATTO 610 (NHS ester) in DMSO, ATTORho14(NHS ester) in PBS (pH 7.4), Nile Blue A in ethanol, ATTO 633 (NHS ester) in PBS (pH 7.4), ATTO Rho14 (NHS ester) in DMSO, Alexa 633(Carboxy) in DMSO, and ATTO 633 (NHS ester) in DMSO. In these panels, purple curves and blue curves show the absorption and emission spectrain corresponding solvents, respectively. The dark red, light red, and dashed blue lines show the corresponding wavelength positions of Stokes beam(ωs), pump beam (ωp), and total excitation energy ((ωp−ωs) +ωp), respectively; the blue bands show the fluorescence collection band of the filter set(FF01-661/20, Semrock). All laser lines and CARS lines ((ωp − ωs) + ωp) are avoided. The center column shows the SRS spectra of correspondingdyes. All SRS spectra are measured under 2.5 mW pump power and 13 mW Stokes power. The right column shows the SREF spectra of correspondingdyes. All SREF spectra were measured under 6 mW pump power and 13 mW Stokes power. Concentrations and solvents are marked in thecorresponding panels. The concentration of the right column of panel c is unknown because of the strong absorption of ATTORho14 (initially 2 μM inPBS) on the coverslip and spacer, which results in obvious decreasing of the concentration.
The Journal of Physical Chemistry Letters Letter
DOI: 10.1021/acs.jpclett.9b01289J. Phys. Chem. Lett. 2019, 10, 3563−3570
generalization, because our proof-of-principle study recordedonly a single or isolated vibrational peak in the SREF spectrum.In the third and final category, we have two more cases in
which the total SREF excitation energy exceeds that of the 0−0transition line by 20 nm (approximately exceeding the dyeabsorption peak by 10 nm). As shown in Figure 4, although SRSspectra display the Raman peaks around 1600 cm−1 from theC=C skeletal mode for Alexa 647 in PBS and JF646 in 0.1% TFAethanol, their vibrational features are largely buried by the anti-Stokes fluorescence background in the corresponding SREFspectra. Under this condition, the molecules are said to be overelectronic preresonance and the SREF strategy for vibrational
detection fails, which sets the “blue” side limit for the total SREFexcitation energy.This systematic SREF spectroscopy study on the C=C skeletal
mode of a total of 11 cases distributed in three differentcategories has allowed us to gain quantitative insight into thegenerality and extendibility of SREF. We could draw anexperimental conclusion that the “proper electronic pre-resonance” condition for successful SREF is within a ∼20 nmrange between the absorption peak of fluorescence dye and thetotal SREF excitation energy: 0−0 transition energy sets the“red” limit for the total SREF excitation energy, and the 20 nmabove the 0−0 transition energy sets the “blue” limit. We nowhave strong experimental data to show that, when under such
Figure 4. SREF excitation of two dyes with over electronic preresonance. (a and d) SREF excitation and signal collection diagrams for Alexa 647 (NHSester) and JF646 (NHS ester), respectively. In these panels, purple curves and blue curves show the absorption and emission spectra in thecorresponding solvents, respectively. The dark red, light red, and dashed blue lines show the corresponding wavelength positions of Stokes beam (ωs),pump beam (ωp), and total excitation energy ((ωp − ωs) + ωp), respectively; the blue bands show the fluorescence collection band of the filter set(FF01-661/20, Semrock). All laser lines and CARS lines ((ωp−ωs) +ωp) are avoided. (b and e) The corresponding SRS spectra. All SRS spectra aremeasured under 2.5 mWpump power and 13mWStokes power. (c and f) SREF spectra of corresponding dyes. All SREF spectra were measured under6 mW pump power and 13 mW Stokes power. Concentrations and solvents are marked in the corresponding panels.
Figure 5. SREF excitation of nitrile mode on newly synthesized fluorophores. (a and e) Newly synthesized dyes with electronic-transition-couplednitrile mode. (b and f) Energy diagrams for nitrile mode SREF excitation. The purple curves and blue curves show the absorption and emission spectrain the corresponding solvents, respectively. The dark red, light red, and dashed blue lines show the corresponding wavelength positions of Stokes beam(ωs), pump beam (ωp), and total excitation energy ((ωp − ωs) + ωp), respectively. The blue bands show the fluorescence collection band of the filterset (FF01-661/20, Semrock). (c and g) SRS spectra of the corresponding dyes. (d and h) SREF spectra of corresponding dyes. All SRL and SREFspectra were measured under 2.5 mW pump power and 13 mW Stokes power. Concentrations and solvents are marked in the corresponding panels.
The Journal of Physical Chemistry Letters Letter
DOI: 10.1021/acs.jpclett.9b01289J. Phys. Chem. Lett. 2019, 10, 3563−3570
electronic preresonance condition, SREF would be a generalphenomenon for a large class of fluorophores with highsensitivity, as supported by all eight cases shown in Figure 3.Once outside this range, the SREF signal could either be tooweak or be buried in anti-Stokes fluorescence background, asshown in Figures 2 and 4.To further test if our experimental insight obtained above is
extendable to other electronic coupled Raman modes, we havesynthesized two new fluorophores33,34 (Figure 5a,e; synthesisdetails are in the Supporting Information) with nitrile groupinstalled on their conjugation systems. For both of thesefluorophores, strong electronic coupled nitrile modes areobserved with Raman resonance around 2200 cm−1 (Figure5c,g). For fluorophore A, which is designed with λabs = 635 nm inPBS (pH 7.4) (Figure 5 a), the total SREF excitation energy fornitrile mode reaches 600.4 nm, meaning the SREF excitation isfar beyond the blue side limit of proper electronic preresonance(Figure 5b). Consistent with our insight obtained above, anoverwhelming anti-Stokes fluorescence background was ob-served, and no sharp vibrational feature can be found on thefluorescence excitation spectrum (Figure 5c,d). In contrast, forfluorophore B, which is designed with λabs = 595 nm (Figure 5e),the total SREF excitation energy of nitrile mode (resonance at2250 cm−1) reaches 599.6 nm, which makes it well within theproper electronic preresonance (Figure 5 f). Indeed, an obviousSREF peak was detected above the anti-Stokes fluorescencebackground at the exact Raman resonance of the nitrile mode(Figure 5g,h). Therefore, our newly synthesized dyes ondifferent Raman modes also support the obtained insights.Note that the fluorophore A experiences a strong quenchingeffect in many solvents (including in PBS, pH 7.4), mainlybecause of the ionization dynamics that led to the failure of thepush−pull electronic conjugation system. Together with the lowfluorescence collection efficiency of the emission filter (Figure5f, blue band), only amoderate detection sensitivity of∼700 nMis achieved. Further engineering of less-quenchable fluorophoreswill significantly improve the sensitivity up to the single-molecule level.30
Next, we seek a theoretical explanation for our experimentalinsight obtained above by modeling the detuning trend of SREFsignal and anti-Stokes fluorescence background (Figure 6,details in the Supporting Information, Figures S2−S4). In ourexperiments, the anti-Stokes fluorescence background can bemodeled by Boltzmann statistics35,36 (Figure S4). On the otherhand, the modeling of the SREF signal will depend on whetherthe total excitation energy ((ωp − ωs) + ωp) is above the 0−0transition line. If so, the SREF signal is mainly determined by theSRS pumping rate, which can be modeled by the Albrecht A-term,9,37 as our pump pulse can easily saturate the transitionsfrom ground-state vibrational-excited states to the firstelectronic excited state.30 If not, the SREF transition rate willbe further modulated by the profile of the absorption tail (FigureS3). Figure 6 shows the evolving trend of the ratio between theSREF signal and anti-Stokes fluorescence background, if thecompetition between different processes can be ignored.Obviously, when the total excitation energy ((ωp − ωs) + ωp)is above the 0−0 transition line, the anti-Stokes fluorescenceincreases much faster than the SREF. As a result, when the totalexcitation energy is above the 0−0 transition line by∼20 nm, theratio between SREF and anti-Stokes fluorescence decreases bymore than two times. Note that this ratio can be furtherdecreased because of competition between different processes,such as ground-state depletion, etc. Hence, this trend can explain
our observed “blue” limit (Figure 4). When the total excitationenergy ((ωp − ωs) + ωp) is below the 0−0 transition line, theabsolute SREF signal drops very fast (Figure S3 b), which canexplain our observed “red” limit for successful SREF detection.Besides offering theoretical support to the blue and red limits forthe resonance condition of the total excitation energy, thissimple model also suggests that the SREF to anti-Stokesfluorescence ratio would reach the maximum near the 0−0transition line, which is indeed observed in the SREF spectra ofATTO 633 and ATTORho14 in PBS and DMSO (panels c, f, e,and h of Figure 3).In summary, through constructing a new excitation source
and exploring a large palette of popular dyes, we have establishedthe generality of SREF spectroscopy on a wide variety of visibledyes with nanomolar-level sensitivity. This has put the validity ofSREF spectroscopy on firm experimental ground, which is asignificant result given that only two dyes were successfullydetected since the initial proposal back in 1980. Moreover, bysystematically studying how the SREF performance depends onthe electronic resonance, we have revealed the experimental rulefor the critical electronic preresonance condition. Specially, thetotal excitation energy ((ωp−ωs) +ωp) should lie within the 20nm range around the absorption peak λabs of fluorophores abovethe 0−0 transition energy. Our theoretical modeling (Figure 6)also lends support to this insight. On one hand, such a relativelynarrow range might appear stringent and thus partly explain whyexperimental progress on this line has been slow during the pastthree decades.29 On the other hand, the successful SREFdetection of all 8 cases falling within the proper electronicpreresonance window strongly supports the generality androbustness of SREF spectroscopy (Table 1). Furthermore, as wehave demonstrated here for the novel nitrile-containingfluorophores (Figure 5), the insights obtained here can serveas a valuable guide to design SREF-optimized dyes for advancedapplication, such as supermultiplexed biomedical imag-ing.18,31,38−40 Beside optimized laser excitation configuration,successful single-molecule SREF spectroscopy would requirehigh quantum yield and good photostability of fluorophores inthe environment, which would be a topic for further study.
Figure 6. Theoretical modeling of the SREF signal and anti-Stokesfluorescence background as a function of pump detuning rate. Assumethe ensemble 0−0 transition energy of the molecule is fixed at 638 nm.Yellow line for anti-Stokes fluorescence; red curve for SREF. The bluecurve is the ratio between SREF and anti-Stokes fluorescence. Reddashed lines show the position of 713 nm, while the total excitationenergy ((ωp − ωs) + ωp) equals the 0−0 transition line, which wedefined as the “red” limit for “proper” electronic preresonance.
The Journal of Physical Chemistry Letters Letter
DOI: 10.1021/acs.jpclett.9b01289J. Phys. Chem. Lett. 2019, 10, 3563−3570
The OPO system we used (APE picoEmerald S) has an idlerbeam output up to 350 mW and a signal beam output up to 800mW, both with 0.6 nm HWFM bandwidth, 80 MHz repetitionrate, and∼2 ps pulse width. We weakly focus (focal length f = 75mm) the idler beam (∼1 mm beam diameter) into a 5 mmPPLN crystal (Covesion, MSHG1420-0.5-5) for SHG gen-eration. A home-built software based on LabVIEW is used tosynchronize the OPO tuning and the temperature of the oven(Covesion, PV10 andOC2) that hosts the crystal. A power up to120 mW can be achieved across the whole tuning range of thecrystal. The benefit of using PPLN for frequency doubling is thatthe output beam shares exactly the same polarization andpropagation direction as the input idler beam, which ensures thespatial overlap between pump beam and Stokes beam during thelaser tuning process. A feedback power control loop based on aphotodiode (Thorlabs, PDA36A) and a half-wave platemounted on a motorized rotation stage (Thorlabs, PRM1Z8)has been built to precisely adjust the power within 2%fluctuation range during laser tuning for highly accurateexcitation spectrum recording (Figures 1c and S1). Then, theSHG of the idler was set as the pump beam, and the OPO signalwas set as the Stokes beam. They were expanded, collimated,and sent to a home-built galvanometer-based (GVSM002,Thorlabs) two-dimensional laser scanning microscopy instru-ment. The scan lens (Thorlabs, SL50-CLS2) and tube lens(Thorlabs, TL200-CLS2) enable an additional 4× beamexpansion. Finally, the back pupil of the objective (UPLSAPO,1.2 N.A., Olympus) was overfilled by both beams to approachdiffraction-limited focus. A delay line on SHG of idler was usedto control the delay between the pump beam and the Stokesbeam. An electro-optic modulator (Thorlabs, EO-PM-R-20-C1)was used on the Stokes beam for all SRL measurements. Thelaser itself provide a 20 MHz driving signal; it was amplified by apower amplifier (Mini-Circuits, ZHL-1-2W+) before being sentto the modulator to achieve a modulation depth more than 90%.For all SREF measurements, a short-pass dichroic mirror(Chroma, T690spxxr) was used to reflect the pump and Stokesbeams but pass the backward fluorescence, and two bandpassfilters (FF01−661/20, Semrock) were used to totally blockpump and Stokes laser lines and CARS lines. A 75 mm doubletwas used to relay the fluorescence emitted in the objective focusto a 100 μm diameter small area avalanched photodiode(SPCM-NIR-14-FC, 70 cps dark counts, Excelitas) operating insingle-photon-counting mode to form serious confocaldetection. For all the SRL and SREF signal acquisition, sampleswere prepared by sandwich solutions with a standard 1mm thickglass slide and 0.17 mm coverslip, separated by 0.12 mm thickimaging spacers (20 mm diameter, Sigma, GBL654006). Fornanomolar-concentration solutions, to avoid dye absorption onthe glass interface, both slides and coverslips were cleaned inPiranha solution41 (H2SO4:H2O2 solution = 3:1 v/v) at 90 °Covernight and further rinsed by 30 min of ultrasonic cleaning indeionized water more than 4 times. Data points were allcollected with 1 ms dwell time, while the laser focus was drivento scan in the solution to avoid obvious photobleaching at asingle point. Each data point on the spectra represents the meanvalue of 200 independent measurements; the correspondingerror bar represents 95% confidence intervals of themean values.All data collection and laser-scan control were achieved by a NIcard (PCI-6259, NI) driven by our home-written LabVIEWprogram. The details about the detection of the SRL signal can
be found in ref 30. For all the commercial dyes, they are directlyused without further purification. For the dyes synthesized by us,details about the synthesis and characterization can be found inthe Supporting Information (Synthesis of Fluorophores A andB).
■ ASSOCIATED CONTENT
*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.jpclett.9b01289.
Synthesis of fluorophores A and B, theoretical modelingof SREF and anti-Stoke fluorescence background, andFigures S1−S4 (PDF)
ORCIDWei Min: 0000-0003-2570-3557Author Contributions†N.Q. and Y.M. contributed equally to this work.
NotesThe authors declare no competing financial interest.
■ ACKNOWLEDGMENTSW.M. acknowledges support from National Institutes of Health(NIH) R01 Grant (GM128214) and R01 Grant (GM132860).
■ REFERENCES(1) Herzberg, G. Infrared and Raman spectra of polyatomic molecules;D. Van Nostrand Company: New York, 1945; Vol. 2.(2) Nie, S.; Emory, S. R. Probing single molecules and singlenanoparticles by surface-enhanced Raman scattering. Science 1997, 275,1102−1106.(3) Min, W.; Freudiger, C. W.; Lu, S.; Xie, X. S. Coherent nonlinearoptical imaging: beyond fluorescence microscopy. Annu. Rev. Phys.Chem. 2011, 62, 507−530.(4) Carey, P. Biochemical applications of Raman and resonance Ramanspectroscopes; Elsevier, 2012.(5) Kukura, P.; McCamant, D. W.; Mathies, R. A. Femtosecondstimulated Raman spectroscopy.Annu. Rev. Phys. Chem. 2007, 58, 461−488.(6) Shim, S.; Stuart, C. M.; Mathies, R. A. Resonance Raman cross-sections and vibronic analysis of Rhodamine 6G from broadbandstimulated Raman spectroscopy. ChemPhysChem 2008, 9, 697−699.(7) Spiro, T. G.; Strekas, T. C. Resonance Raman spectra ofhemoglobin and cytochrome c: inverse polarization and vibronicscattering. Proc. Natl. Acad. Sci. U. S. A. 1972, 69, 2622−2626.(8) Johnson, B. B.; Peticolas, W. L. The resonant Raman effect. Annu.Rev. Phys. Chem. 1976, 27, 465−521.(9) Asher, S. A. UV resonance Raman studies of molecular structureand dynamics: applications in physical and biophysical chemistry.Annu.Rev. Phys. Chem. 1988, 39, 537−588.(10) Efremov, E. V.; Ariese, F.; Gooijer, C. Achievements in resonanceRaman spectroscopy: Review of a technique with a distinct analyticalchemistry potential. Anal. Chim. Acta 2008, 606, 119−134.(11) Prince, R. C.; Frontiera, R. R.; Potma, E. O. Stimulated Ramanscattering: from bulk to nano. Chem. Rev. 2017, 117, 5070−5094.(12) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.;Dasari, R. R.; Feld, M. S. Single molecule detection using surface-enhanced Raman scattering (SERS). Phys. Rev. Lett. 1997, 78, 1667−1670.
The Journal of Physical Chemistry Letters Letter
DOI: 10.1021/acs.jpclett.9b01289J. Phys. Chem. Lett. 2019, 10, 3563−3570
(13) Zhang,W.; Yeo, B. S.; Schmid, T.; Zenobi, R. Single molecule tip-enhanced Raman spectroscopy with silver tips. J. Phys. Chem. C 2007,111, 1733−1738.(14) Le Ru, E. C.; Etchegoin, P. G. Single-molecule surface-enhancedRaman spectroscopy. Annu. Rev. Phys. Chem. 2012, 63, 65−87.(15) Zhang, Y.; Zhen, Y.-R.; Neumann, O.; Day, J. K.; Nordlander, P.;Halas, N. J. Coherent anti-Stokes Raman scattering with single-molecule sensitivity using a plasmonic Fano resonance. Nat. Commun.2014, 5, 4424.(16) Yampolsky, S.; Fishman, D. A.; Dey, S.; Hulkko, E.; Banik, M.;Potma, E. O.; Apkarian, V. A. Seeing a single molecule vibrate throughtime-resolved coherent anti-Stokes Raman scattering. Nat. Photonics2014, 8, 650−656.(17) Zrimsek, A.; Chiang, N.; Mattei, M.; Zaleski, S.; McAnally, M.;Chapman, C.; Henry, A.; Schatz, G.; Van Duyne, R. Single-moleculechemistry with surface-and tip-enhanced Raman spectroscopy. Chem.Rev. 2017, 117, 7583−7613.(18) Wei, L.; Chen, Z.; Shi, L.; Long, R.; Anzalone, A. V.; Zhang, L.;Hu, F.; Yuste, R.; Cornish, V. W.; Min, W. Super-multiplex vibrationalimaging. Nature 2017, 544, 465−470.(19) Laubereau, A.; Seilmeier, A.; Kaiser, W. A new technique tomeasure ultrashort vibrational relaxation times in liquid systems. Chem.Phys. Lett. 1975, 36, 232−237.(20) Seilmeier, A.; Kaiser, W.; Laubereau, A.; Fischer, S. A novelspectroscopy using ultrafast two-pulse excitation of large polyatomicmolecules. Chem. Phys. Lett. 1978, 58, 225−229.(21) Hubner, H.-J.; Worner, M.; Kaiser, W.; Seilmeier, A. Subpico-second vibrational relaxation of skeletal modes in polyatomicmolecules. Chem. Phys. Lett. 1991, 182, 315−320.(22) Whaley-Mayda, L.; Penwell, S. B.; Tokmakoff, A. Fluorescenceencoded infrared spectroscopy: ultrafast vibrational spectroscopy onsmall ensembles of molecules in solution. J. Phys. Chem. Lett. 2019, 10,1967.(23) Wright, J. C. Double resonance excitation of fluorescence in thecondensed phase-an alternative to infrared, Raman, and fluorescencespectroscopy. Appl. Spectrosc. 1980, 34, 151−157.(24) Min, W.; Lu, S.; Holtom, G. R.; Xie, X. S. Triple-resonancecoherent anti-Stokes Raman scattering microspectroscopy. ChemPhy-sChem 2009, 10, 344−347.(25) Orrit, M.; Bernard, J. Single pentacene molecules detected byfluorescence excitation in a p-terphenyl crystal. Phys. Rev. Lett. 1990, 65,2716.(26) Nie, S.; Chiu, D. T.; Zare, R. N. Probing individual moleculeswith confocal fluorescence microscopy. Science 1994, 266, 1018−1018.(27) Moerner, W.; Orrit, M. Illuminating single molecules incondensed matter. Science 1999, 283, 1670−1676.(28) Winterhalder, M.; Zumbusch, A.; Lippitz, M.; Orrit, M. Towardfar-field vibrational spectroscopy of single molecules at roomtemperature. J. Phys. Chem. B 2011, 115 (18), 5425−5430.(29) Lee, S.; Nguyen, D.; Wright, J. Double resonance excitation offluorescence by stimulated Raman scattering. Appl. Spectrosc. 1983, 37,472−474.(30) Xiong, H.; Shi, L.; Wei, L.; Shen, Y.; Long, R.; Zhao, Z.; Min, W.Stimulated Raman excited fluorescence spectroscopy and imaging.Nat.Photonics 2019, 13, 412−417.(31) Wei, L.; Min, W. Electronic preresonance stimulated Ramanscattering microscopy. J. Phys. Chem. Lett. 2018, 9, 4294−4301.(32) Reichardt, C. Solvatochromic dyes as solvent polarity indicators.Chem. Rev. 1994, 94, 2319−2358.(33) Shi, J.; Zhang, X.; Neckers, D. C. Xanthenes: fluorone derivatives.1. J. Org. Chem. 1992, 57, 4418−4421.(34) Shi, J.; Zhang, X.-P.; Neckers, D. C. Xanthenes: flouronederivatives II. Tetrahedron Lett. 1993, 34, 6013−6016.(35) Erickson, L. On anti-Stokes luminescence from Rhodamine 6Gin ethanol solutions. J. Lumin. 1972, 5, 1−13.(36) Zander, C.; Drexhage, K. H. Cooling of a dye solution by anti-Stokes fluorescence. Advances in photochemistry 2007, 20, 59−78.
(37) Albrecht, A.; Hutley, M. On the dependence of vibrationalRaman intensity on the wavelength of incident light. J. Chem. Phys.1971, 55, 4438−4443.(38) Shi, L.; Xiong, H.; Shen, Y.; Long, R.; Wei, L.; Min, W. Electronicresonant stimulated raman scattering micro-spectroscopy. J. Phys.Chem. B 2018, 122, 9218−9224.(39) Hu, F.; Zeng, C.; Long, R.; Miao, Y.; Wei, L.; Xu, Q.; Min, W.Supermultiplexed optical imaging and barcoding with engineeredpolyynes. Nat. Methods 2018, 15, 194.(40) Hu, F.; Brucks, S. D.; Lambert, T. H.; Campos, L. M.; Min, W.Stimulated Raman scattering of polymer nanoparticles for multiplexedlive-cell imaging. Chem. Commun. 2017, 53, 6187−6190.(41) Liu, X.; Wu, Z.; Nie, H.; Liu, Z.; He, Y.; Yeung, E. Single DNAmolecules as probes for interrogating silica surfaces after variouschemical treatments. Anal. Chim. Acta 2007, 602, 229−235.
The Journal of Physical Chemistry Letters Letter
DOI: 10.1021/acs.jpclett.9b01289J. Phys. Chem. Lett. 2019, 10, 3563−3570
