Fluorescence-Free Single-Molecule Microscopy via Electronic Resonance Stimulated Raman Scattering

Fluorescence-Free Single-Molecule Microscopy via Electronic Resonance Stimulated Raman Scattering | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Fluorescence-Free Single-Molecule Microscopy via Electronic Resonance Stimulated Raman Scattering Sang-Hee Shim, Sumin Oh, Yunji Eom, Ha Yeon Kim, Ayushi Tripathi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5650021/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Feb, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Vibrational spectroscopy is a powerful tool for spectral imaging of biological samples, thanks to its narrow bandwidth (10 cm⁻¹) compared to fluorescence. Single-molecule vibrational spectroscopy has been demonstrated with near-field amplification as in surface-enhanced Raman spectroscopy or fluorescence detection as in stimulated Raman excited fluorescence and bond-selective fluorescence-detected infrared-excited spectro-microscopy. However, these methods involve complex sample preparation or produce high backgrounds, limiting their practicality. In response to these issues, we developed electronic resonance stimulated Raman scattering (ER-SRS) to achieve single-molecule sensitivity in far-field vibrational microscopy without relying on fluorescence detection. ER-SRS has encountered difficulties due to large electronic backgrounds. To overcome this, we employed Raman-amplified nonfluorescent molecular probe (RANMP) alongside our synchronously pumped, independently tunable double optical parametric oscillators for effective optimization of the signal-to-background ratio. The optimization of probe and light source allowed us to successfully detect ER-SRS signal from single particles in solution and from single molecules embedded in polymer matrix. ER-SRS combined with RANMP, offering single-molecule sensitivity without the aid of fluorescence detection, will open new avenues in biological and chemical fields, particularly in multiplexed imaging. Physical sciences/Chemistry/Physical chemistry/Optical spectroscopy/Raman spectroscopy Physical sciences/Optics and photonics/Optical physics/Nonlinear optics Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Fluorescence microscopy has revolutionized biology with its unmatched molecular specificity and single-molecule sensitivity 1-4 . Single-molecule fluorescence microscopy has unveiled the individual trajectories of biological molecules, revealing the molecular mechanisms 5,6 . Fluorescence detection has driven advancements in cutting-edge technologies such as massively parallel DNA sequencing 7 and super-resolution optical microscopy 8-11 . In recent years, fluorescence microscopy evolved to highly multiplexed imaging, opening new fields of spatial genomics, transcriptomics and proteomics 6,12,13 . However, fluorescence-based multiplexed imaging relies on iterative cycles of labeling, imaging and washing for each molecular target due to the broad bandwidth (1,500 cm -1 ) of excitation and emission that limit its ability to spectrally distinguish different molecules 14 . In contrast, vibrational transitions, such as Raman scattering and infrared (IR) absorption, have a much narrower bandwidth (10 cm -1 ) compared to fluorescence, making them highly suitable for simultaneously differentiating multiple substances. However, the intrinsic low intensity of Raman signals necessitates extensive signal amplification. Surface-enhanced Raman spectroscopy (SERS) enhances the Raman signal by over 10 7 using plasmonic nanoparticles, enabling single-molecule detection 15-17 . However, the preparation of SERS substrates can be complicated and requires precise control of nanoparticle fabrication and probe positioning for consistent results 18,19 . Electronic pre-resonance stimulated Raman scattering (EPR-SRS, Fig. 1A) enhances Raman sensitivity by up to 10 11 compared to spontaneous Raman without near-field effects 20 . However, it still requires fluorescence detection, such as stimulated Raman excited fluorescence 21 (SREF, Supplementary Fig. 1A), for single-molecule sensitivity. Efficient SREF fluorophores must fulfill multiple requirements: (1) conjugation of vibrational modes to a chromophore, (2) large Raman cross-section, (3) high near-infrared (NIR) absorbance, and (4) high fluorescence quantum yields 22 . However, high quantum yields lead to high background including anti-Stokes fluorescence 23 from thermally excited populations, limiting the signal-to-background (S/B) ratios and practicality. Bond-selective fluorescence-detected infrared-excited spectro-microscopy 24 (BonFIRE) also demonstrated single-molecule sensitivity (Supplementary Fig. 1A). However, BonFIRE suffered from anti-Stokes fluorescence and IR-induced photothermal background, requiring high-frequency modulation for background substraction 24 . Our research aims to leverage electronic resonance stimulated Raman scattering (ER-SRS) to amplify Raman sensitivity without the aid of near-field effects or fluorescence detection (Fig. 1A). First demonstrated in 2018, ER-SRS has never demonstrated single-molecule sensitivity due to large electronic background and lack of suitable probes and light sources 25 . In this regard, our strategy combines nonfluorescent, highly conjugated Raman-active probe and frequency optimization for two-beam ER-SRS by using our unique independently tunable double optical parametric oscillator (OPO) laser system (Fig. 1B and Supplementary Fig. 1B), enabling single-molecule detection. Results ER-SRS and EPR-SRS of Rhodamine 800 fluorophore Rhodamine 800 (Rho800) serves as a key probe for EPR-SRS and SREF 20 , 21 , 23 . Utilizing our independently tunable double OPO system, we analyzed the ER-SRS and EPR-SRS spectra of Rho800 under various pump/Stokes wavelength combinations (Figs. 1 C-E). The selected wavelengths meet these conditions: (1) \(\:{{\omega\:}}_{\text{p}}-{{\omega\:}}_{\text{s}}=\:{{\omega\:}}_{\text{v}}\:\) ; (2) \(\:{{\omega\:}}_{\text{p}}-{{\omega\:}}_{\text{a}\text{b}\text{s},\text{m}\text{a}\text{x}}\) < 700 cm - 1 for ER-SRS 25 ; (3) 1,400 cm - 1 < \(\:{{\omega\:}}_{\text{p}}-{{\omega\:}}_{\text{a}\text{b}\text{s},\text{m}\text{a}\text{x}}\) < 4,200 cm - 1 for EPR-SRS 20 , where \(\:{{\omega\:}}_{\text{p}}\) and \(\:{{\omega\:}}_{\text{s}}\) are pump and Stokes frequencies; \(\:{\omega\:}_{\text{a}\text{b}\text{s},\text{m}\text{a}\text{x}}\) and \(\:{{\omega\:}}_{\text{v}}\) are the frequencies at the absorption max and Raman shift of the probe, respectively. For Rho800, \(\:\:{{\omega\:}}_{\text{v}}\) =2,229.6 cm - 1 (Supplementary Fig. 2A). Using our synchronized, yet independently tunable double OPO system with a tuning rage of 660–960 nm, we optimized pump/Stokes combinations to maximize the S/B ratio. The pump wavelength was tuned around the absorption max (λ max = 700 nm) while adjusting the Stokes wavelength to achieve vibrational resonance \(\:{\:({\omega\:}}_{\text{p}}-{{\omega\:}}_{\text{s}}=\:{{\omega\:}}_{\text{v}})\) . When the pump matched λ max (black line, Fig. 1 C), an inverted Raman peak was observed (black spectrum, Fig. 1 D), consistent with prior findings for SiR845 fluorophore 25 . At λ pump = 716 nm, within Rho800’s resonance region, the signal increased over 60-fold compared to the pre-resonant condition at (pump, Stokes) = (838.4 nm, 1,031.2 nm) (Supplementary Fig. 2B), as previously demonstrated with conventional OPO 20 . However, significant background at λ pump = 716 nm led to a low S/B ratio. Deviating λ pump from λ max by > 50 nm significantly improved the S/B ratio (Fig. 1 E). For highly fluorescent probe like Rho800, ER-SRS failed to enhance the S/B ratio due to strong background from fluorescence and pump-probe processes 20 , illustrating the limitations of ER-SRS with conventional fluorophores as resonant Raman probes. ER-SRS of Raman-amplified nonfluorescent molecular probe (RANMP) We developed Raman-amplified nonfluorescent molecular probes (RANMPs) with (1) high far-red to NIR absorption for electronic resonance to the light source, (2) Raman-active groups conjugated to the chromophore for large Raman cross-section and electronic resonance, and (3) low fluorescence quantum yields to reduce background (Fig. 2 A). The series–RANMP, RANMP-4F, RANMP-4Cl, and RANMP-T 26 –is based on an electron-rich core, bis(alkylsulfanyl)methylene-substituted 4,9-dihydro-s-indaceno[1,2-b:5,6-b’]dithiophene (IDT), and the electron-withdrawing terminal group 2-(3-oxo-2,3-dihydro-1H-inden-1-ylidene)malononitrile (INCN) containing Raman-active CN moieties. These molecules were synthesized following a previously reported procedure 26 (Supplementary Fig. 3). The final compounds were obtained by coupling a formylated IDT core with INCN derivatives via a Knoevenagel condensation reaction using pyridine as a base, in approximately 80% yields for all compounds. The effective conjugation was adjusted by controlling intramolecular charge transfer (ICT) interactions in each molecule. This was achieved by incorporating halogen substituents into INCN group (RANMP-4F and RANMP-4Cl) or introducing a thiophene spacer between the core and INCN terminal moieties (RANMP-T), thereby finely modulating the absorption max. The ICT effect allows λ max to fall within the pump tuning range of 660–960 nm, facilitating resonance conditions (Supplementary Fig. 4A). In RANMP-T, the addition of a thiophene linker between the donor (D) and acceptor (A) units increases the effective conjugation length, resulting in a Raman peak shift of the CN groups (Supplementary Fig. 4B). From quantum chemical calculations, we found that the donor unit (IDT core) and the acceptor unit (INCN terminal groups) of the RANMP series are responsible mainly for the HOMO and LUMO, respectively (Supplementary Fig. 5). The natural transition orbitals (HONTO and LUNTO) represent the ICT character from the donor to the acceptor unit upon S 0 → S 1 transition (Supplementary Fig. 6). These A-D-A type molecules have large Raman cross-sections due to the four CN groups in both terminals conjugated to the IDT core and INCN terminal group. The calculated Raman intensity of the CN vibrational mode in tetracyanoethylene (TCNE) is significantly lower—by a factor of 10 to 30—compared to those of the RANMP series, indicating that the influence of the conjugation on the Raman cross-section (Supplementary Fig. 7C and Supplementary Table 1). In addition, the Raman peak of the CN vibrational mode in the RANMP series depends on the molecular structures. The calculated Raman intensities of the CN vibrational modes increase in the order of RANMP, RANMP-4F, RANMP-4Cl, and RANMP-T (Supplementary Fig. 7C), which is consistent with the experimental results (Supplementary Fig. 4). The calculated Raman frequency is red-shifted in the order of RANMP-4F, RANMP-4Cl, RANMP, and RANMP-T, which correlates with the CN bond length of the RANMP series (Supplementary Fig. 7D). The RANMP series exhibit photoluminescence undetectable with our spectrophotometer, significantly improving the Raman S/B ratio. To understand the low photoluminescence of the RANMP series, the intersystem crossing rate constants ( k ISC ) and radiative decay rate constants ( k r ) of the RANMP series and Rho800 were calculated (Supplementary Note 1) 27 . As shown in Table SN2 of Supplementary Note 1, the k ISC / k r ratio of the RANMP series (ranging from 10 2 to 10 3 ) is significantly higher than that of Rho800 (7.3). Therefore, compared with Rho800, the RANMP series exhibit much faster ISC to the triplet state and more efficient quenching of the fluorescence of the S 1 state, which explains the low photoluminescence. Note that fluorescence quenching by rapid ISC is a key strategy to substantially reduce fluorescence background in ER-SRS. As shown in Eq. (S1) in Supplementary Note 1, the ISC rate constant increases with a large spin-orbit coupling (SOC) constant and a small energy difference between singlet and triplet states (Δ E ST ). SOC can be increased by introducing heavy atoms (e.g., S, Se, Br) into organic compounds, a concept widely employed in designing photosensitizers for photodynamic therapy 28 , 29 . For the RANMP series, the SOC constants are found to be large due to sulfur atoms (a heavy atom) (Supplementary Table SN2 in Supplementary Note 1). To optimize ER-SRS for the RANMP series, we measured the modulation transfer signal of RANMP-T film at (pump, Stokes) = (749 nm, 897.9 nm) by scanning the time delay between pump and Stokes pulses (Fig. 2 B). When Stokes preceded pump (blue line, Fig. 2 C), the intensity fits to a Gaussian peak centered at time zero, consistent with SRS convolved with a picosecond pulse. When pump precedes Stokes (red line, Fig. 2 C), the signal decayed exponentially with a 4.26 ps lifetime, which is on the order of that of stimulated emission. This asymmetrical time delay dependence indicates pump-dominated background. Also, the signal exhibited nonlinear pump power dependence as in the previous literature 25 (Fig. 2 D). The Stokes power dependence of ER-SRS signal was largely linear below a saturation level (Supplementary Fig. 8). Using a reduced pump power (500 µW) and a Stokes power below saturation (10 mW), we obtained ER-SRS spectra of RANMP, RANMP-4F, RANMP-4Cl and RANMP-T films (Fig. 2 E, Supplementary Figs. 8–9). The double OPO system enabled flexible pump and Stokes tuning to optimize the S/B ratio. Under rigorous resonance, the overwhelming background signal resulted in inverted Raman peaks, whereas positive peaks appeared slightly off resonance (Supplementary Fig. 9). At the optimized conditions, ER-SRS signals showed 200-400x amplification compared to EPR-SRS of Rho800 [(pump, Stokes) = (838.4 nm, 1,031.2 nm)] (Fig. 2 F). These results align with the theoretical predictions (10-100x amplification), given the four CN groups per RANMP molecule versus one CN in Rho800 25 . Single-particle imaging of RANMP Pdots Previously, we demonstrated that chromophores aggregated into polymer dot (Pdot) forms exhibit high resonance Raman scattering with suppressed fluorescence due to aggregation-induced quenching 30 . Likewise, we expected ER-SRS signals in RANMP-based Pdots to benefit from probe concentration and further fluorescence suppression. RANMP-4Cl and RANMP-T, showing the highest ER-SRS amplification (Fig. 2 F), were fabricated into Pdots (Fig. 3 A, Supplementary Fig. 10). Using independent wavelength tuning, we optimized the ER-SRS S/B ratios, achieving maxima at λ max + 37 nm for RANMP-T and λ max + 20 nm for RANMP-4Cl (Fig. 3 C, Supplementary Fig. 11C). Under the optimum ER-SRS conditions, we achieved dual-color single-particle imaging using the narrow linewidth of Raman scattering (Figs. 3 D-G). In a mixture of RANMP-T and RANMP-4Cl Pdots, indistinguishable particles in the brightfield image became spectrally distinct at 2,218.4 cm − 1 (RANMP-T) and 2,235.4 cm − 1 (RANMP-4Cl) (Fig. 3 D). At the optimal condition for RANMP-T [(pump, Stokes) = (744.4 nm, 891.6 nm)], RANMP-4Cl particles were nearly invisible, and vice versa for RANMP-4Cl [ (pump, Stokes) = (760.2 nm, 915.8 nm)]. Off-resonance condition [(pump, Stokes) = (760.7 nm, 915.8 nm)] rendered all particles invisible. The Full Width at Half Maximum (FWHM) of the cross-sectional profile of the particle image was 361 nm for RANMP-4Cl and 356 nm for RANMP-T, closely matching the diffraction-limited resolution of 324 nm and particle size (Figs. 3 F-G, Supplementary Fig. 10B). Each 5 µm x 5 µm field of view (FOV) was acquired in ~ 62 seconds, enabling two-color imaging under 3 minutes without noticeable bleaching. The imaging speed, limited by the sample piezo stage, could be improved with a Galvano scanner for beam scanning. The 17 cm − 1 Raman shift difference between particles (Fig. 3 E) allowed clear spectral distinction for successful two-color single-particle imaging. Single-molecule imaging of individual molecules embedded in film To detect single molecule at room temperature, we optimized ER-SRS for poly(methyl methacrylate) (PMMA) samples sparsely embedded with RANMP molecules. Surpassing the EPR-SRS detection limit of 250 nM 20 , we detected signals at 1 nM for RANMP-T (Figs. 4 A-B), equivalent to ~ 3 molecules in the laser focal volume. To minimize bleaching, we employed a polyvinyl alcohol (PVA) 31 layer above and below the PMMA (Supplementary Figs. 12A-B) and applied a minimal pump power of 300 µW at optimized wavelengths (Supplementary Fig. 12C). Single-molecule imaging under these conditions (Figs. 4 C-E) showed abrupt photobleaching events in the time trace, confirming their single-molecule nature (Fig. 4 D). Orange and blue arrows in Fig. 4 D correspond to photobleaching of M1 and M2 molecules in Fig. 4 C. ER-SRS specificity was verified by collecting single-molecule images at two temporal overlaps of pump and Stokes pulses (Fig. 4 E). A molecular signal was observed when two pulses arrived concurrently (left “0 ps”, Fig. 4 E). When the Stokes pulse preceded the pump only by 1 ps (+ 1 ps, Fig. 4 E), the signal sharply attenuated, consistent with the bulk time-dependent measurements in Fig. 2 C. Reverting the delay to 0 ps restored the signal (right 0 ps, Fig. 4 E), confirming that the signal reduction was due to the short vibrational lifetime of nitrile groups (< 1 ps) 32 . To confirm vibrational resonance, we analyzed signal variations with pump-Stokes frequency differences (Fig. 4 F). Signals appeared at the CN stretch mode (2,218.2 cm⁻¹) but vanished when the pump wavelength tuned by 1 nm to a frequency difference of 2,236.3 cm⁻¹. Restoring resonance at 2,218.2 cm⁻¹ recovered the signal, confirming the loss was due to off-resonance, not photobleaching. The abrupt photobleaching as well as sensitivity to temporal overlap and frequency difference validate the near-diffraction-limited features in Figs. 4 C-F as single-molecule ER-SRS images. This is the first demonstration of far-field single-molecule detection via resonance Raman without relying on fluorescence or plasmonic enhancement. Discussion In this study, we successfully demonstrated single-molecule ER-SRS by optimizing both light sources and probes. The previous ER-SRS demonstration 25 faced challenges in parameter optimization due to conventional SRS light source providing only a single tunable pump beam and a fixed Stokes beam (usually at 1,031.2 nm). In contrast, using our independently tunable double OPO laser system, we optimized the wavelength combination for two-beam ER-SRS to maximize the S/B ratio for Raman-active molecules with CN-moieties absorbing NIR light. To further enhance the signal and suppress the background, we designed a series of four novel Raman-active nonfluorescent molecules (RANMP, RANMP-4F, RANMP-4Cl and RANMP-T) and pre-analyzed their optical properties using DFT and time-dependent (TD) DFT calculations, thereby facilitating effective ER-SRS signal enhancement. As a result, we experimentally realized over 100-fold signal amplification, achieving the first-ever detection of single molecules via Raman scattering without fluorescence detection or near-field enhancement. The fluorescence-free nature of ER-SRS opens new doors for extending single-molecule vibrational microscopy to tissue and animal imaging. While fluorescence enables SREF and BonFIRE to achieve single-molecule sensitivity, it introduces significant background noise: SREF signals are accompanied by anti-Stokes fluorescence from thermally excited populations, and BonFIRE is affected by both anti-Stokes fluorescence and photothermal-induced fluorescence change. These background signals necessitate confocal detection for imaging single cell layers in SREF and BonFIRE, and additional strategies to suppress out-of-focus signal and background from thick samples for deep-tissue imaging. In contrast, the nonlinear nature of ER-SRS inherently provides optical sectioning, offering significant advantages for in vivo imaging, as extensively demonstrated in SRS imaging 33 , 34 . ER-SRS also facilitates the development of probes for single-molecule vibrational microspectroscopy. In SREF, achieving an optimal S/B requires stringent electronic pre-resonance conditions, limiting the selection of fluorophores. While BonFIRE provides greater flexibility in dye selection, it is constrained by the tuning range of OPO and relies on NIR fluorophores, which often exhibit low photoluminescence quantum yields (PLQYs) —for instance, Rho800 has a PLQY of 17%. Shifting absorption and emission into the NIR wavelengths typically employ strong donor-acceptor conjugation to induce intramolecular charge transfer (ICT). However, this approach often leads to strong ICT quenching, making it challenging to develop highly fluorescent NIR dyes. By contrast, designing nonfluorescent NIR absorbing molecules is more feasible. The A-D-A molecular design motif used in this study effectively extends conjugation to meet the resonance condition for ER-SRS, where strong ICT interaction suppresses PL emission, optimizing the S/B ratio 35 – 37 . Photobleaching, a common challenge in fluorescence microscopy, also occurs in ER-SRS due to the use of electronic resonance. To mitigate this, single-molecule images in Fig. 4 were obtained using a protective PVA layer above and below the probe-containing PMMA layer. When only a single PVA layer was applied, ER-SRS signals from single molecules were still detectable but photobleaching occurred more rapidly (Supplementary Figs. 12D-E). While fast transfer to the triplet state of RANMP series can promote photobleaching, the photobleaching lifetime of ER-SRS signals from a RANMP-T film was significantly longer than that of EPR-SRS signal from a Rho800 film (Supplementary Fig. 13). Our attempts to measure the triplet-state lifetime of RANMP-T film were unsuccessful, as no phosphorescence signal was detectable (data not shown), suggesting an extremely short triplet-state lifetime. This implies that the triplet state of RANMP, likely in the NIR region, is highly susceptible to thermal quenching. Efficient triplet-state quenching may therefore contribute to the slower photobleaching observed in the ER-SRS signal of RANMP. To further suppress triplet-mediated radical formation leading to photobleaching, strategies used in fluorescence imaging—such as adding reducing agents, antioxidants and triplet state quenchers—could be adapted for ER-SRS 38 . ER-SRS with RANMP achieved single-molecule detection from conventional film and solution samples using standard optical microscopes, highlighting a strong potential for advanced biological imaging using existing labeling methods and instruments. The RANMP in this study are organic small molecules, a widely used class of biological probes. For ER-SRS multiplexed imaging, the primary requirement is to synthesize more probes with nonfluorescent, NIR-absorbing conjugated molecules with Raman-active moieties such as nitrile, alkynes and azido groups. Raman shifts of these probes can be tuned through various strategies, including incorporating isotopes into vibrating groups 39 , extending polyene structures 40 and exploiting various core structures of conjugated molecules 41 . In this study, the thiophene spacer inserted between the core and INCN terminal moieties induced a blue shift in the CN vibrational frequency sufficient for two-color imaging (Fig. 3 ). This introduces a new approach for synthesizing multiplex Raman probes. The PSMA-encapsulated Pdot in this study also offers a convenient, general route for boosting the S/B of ER-SRS probes and for efficient targeting in biological samples by using the carboxylic groups on the particle surface. We anticipate that ER-SRS, combined with RANMP, will serve as a powerful tool for multiplexed imaging of individual molecules or nanoparticles within complex biological environments. Methods Independently tunable double OPO system The ER-SRS microscopy setup is described in Fig. 1 B and Supplementary Fig. 1B. Two OPOs are pumped by a common, powerful IR fiber laser with a frequency doubling unit (Angewandte Physik & Elektronik GmbH, Emerald Engine HP). The Emerald Engine generates an output pulse train at 1,031.2 nm with a 2 ps pulse width and an 80 MHz repetition rate. The frequency-doubled beam at 516 nm is used to synchronously seed two OPOs, referred to as the high power (HP) OPO and the low power (LP) OPO. The tunable pump beam (660–960 nm, ~ 2 ps) from the HP OPO is modulated at 10 MHz by an electro-optic modulator (EOM) to achieve shot-noise limited detection sensitivity. The tunable Stokes beam (660–960 nm, ~ 2 ps) is pumped from the LP OPO. ER-SRS microscopy As in Fig. 1 B and Supplementary Fig. 1B, the independently tuned pump and Stokes beams are combined at the dichroic mirror and then directed into a home-built sample-scanning microscope body (Cerna system, Thorlabs). To confirm the spatial overlap of the two beams, two position sensors were each installed on the near and far sides of the dichroic mirror. The temporal overlap of pump and Stokes pulses is precisely defined using an optical delay line, installed in the pump path, and verified with the sum-frequency generation signal of a BBO crystal. The sample is placed on a piezo-stage (PINano XY Piezo System, P-545.3C8S; PI) for X and Y axis scanning. All beams are focused on the sample using a 60X objective lens with NA 1.2 (UplanSApo; Olympus). A condenser lens with NA 1.4 (oil immersion, D-CUO; Nikon) is used to collect the transmitted beams. The output beams pass through two band-pass filters and a dichroic mirror, which transmits the Stokes beam but blocks the pump, measured as the stimulated Raman gain (SRG) signal. Different band-pass filters and dichroic mirrors are used for each wavelength combination (Supplementary Table 2). The transmitted Stokes beam is collected through a large area photodiode and amplified by a lock-in amplifier (APE GmbH). The signal is sent to a computer in which a home-built LabVIEW program records the ER-SRS intensity at each pixel. We measured SR gain due to the peak sensitivity of the Si photodiode around 960 nm. Polymer dots (Pdots) fabrication The nanoprecipitation method was employed to form Pdots (Supplementary Fig. 10). 100 µL of RANMP-4Cl (or RANMP-T) solution in THF (1 mg/mL) was mixed with 400 µL of poly(styrene-co-maleic anhydride) (PSMA, M n ≈ 1600 Da, ~ 1.3:1 styrene anhydride mol ratio) solution in THF (1 mg/mL). Subsequently, 4.5 mL of THF was added. The resulting THF solution was quickly transferred into 10 mL of deionized water under sonication. The water-THF mixture was then heated to 80°C to facilitate the evaporation of THF and achieve nanoparticle formation until the volume was reduced to 2 mL under N 2 bubbling. The concentrated Pdot solution was filtered through a 0.2 µM syringe filter and stored in a brown-colored vial. To verify if the Pdot nanoparticles are produced at a concentration similar to the theoretical concentration of the Raman probe, a certain amount of RANMP-T Pdot nanoparticle solution was taken and quantified through an absorption coefficient calibration curve (Supplementary Fig. 14). Due to losses occurring during bubbling when evaporating THF, the experimental concentration has an error of approximately ~ 10% compared to the theoretical concentration. Imaging sample preparation Single-particle sample A 6% w/w agarose gel was prepared by dissolving the appropriate amount of agarose powder (A4018, Sigma) in a 25 mM MES buffer. The mixture was heated to 95°C to ensure complete dissolution. Subsequently, a glass slide was heated on a hot plate to 95°C. Then, 10 µL of the Pdot solution and 10 µL of the agarose gel were sequentially added on top of the slide. A cover glass was carefully placed over the mixture and gently pressed to achieve full coverage. Two-color Pdot sample The same procedure was performed to prepare the single particle imaging sample. Before adding the agarose gel, 5 µL of the Pdot solution of RANMP-4Cl and 5 µL of the Pdot solution of RANMP-T were sequentially added. Single-molecule sample A glass substrate was washed in acetone, isopropyl alcohol and deionized water (DI) water for 15 minutes then UV-ozone plasma treatment was performed for 10 min. A 5% w/w poly(vinyl alcohol) (PVA)/ DI water solution was spin-coated onto a glass substrate at 1000 rpm for 120 seconds. Using a 1% w/w poly(methyl methacrylate) (PMMA)/CF solution, the desired concentration of RANMP-T solution was prepared to form a single-molecule embedded polymer thin film layer. This solution was then spin-coated onto the PVA layer at 1500 rpm for 120 seconds. Afterward, the 5% w/w PVA layer was spin-coated onto the PMMA/RANMP-T layer at 1000 rpm for 120 seconds. Material characterization The 1 H spectra were measured by a Bruker Advance III system operating at 500 MHz. UV-Vis-NIR spectra were measured by a JASCO V-770 spectrophotometer. The hydrodynamic diameters of Pdots were measured in aqueous solution using Zetasizer Nano S90 (Malvern Panalytical). Spontaneous Raman spectra was obtained by HORIBA LabRAM HR Evolution Raman spectrometer (Japan). The laser beam, 532 nm, was focused on the samples at the microscope stage by a 100x/0.90 NA objective lens (Plan N, Olympus, Japan). The laser conditions were kept consistent with 1% power, a 5 s acquisition time and 5 accumulations. Synthesis RANMP, RANMP-4F, RANM − 4Cl, and RANMP-T were synthesized following a previously reported procedure 26 (Supplementary Figs. 3, 15–18). RANMP 1 H NMR (500 MHz, Chloroform- d ): δ 8.92 (s, 2H), 8.89 (s, 2H), 8.64 (s, 2H), 8.59–8.54 (m, 2H), 7.89–7.82 (m, 2H), 7.72–7.65 (m, 4H), 3.14 (m, 8H), 1.80–1.68 (m, 4H), 1.56–1.13 (m, 64H), 0.96–0.71 (m, 24H). RANMP-4F 1 H NMR (500 MHz, Chloroform- d ): δ 9.10–9.05 (m, 2H), 8.92 (s, 2H), 8.72 (s, 2H), 8.57–8.41 (m, 2H) 7.64 (t, J = 7.4 Hz, 2H), 3.21–3.09 (m, 8H), 1.79–1.70 (m, 4H), 1.53–1.11 (m, 64H), 0.93–0.72 (m, 24H). RANMP-4Cl 1 H NMR (500 MHz, Chloroform- d ): δ 9.11 (s, 2H), 8.92 (s, 2H), 8.72 (s, 2H), 8.53–8.43 (m, 2H), 7.64 (t, J = 7.4 Hz, 2H), 3.15 (m, J = 6.7 Hz, 8H), 1.81–1.66 (m, 4H), 1.52–1.15 (m, 64H), 0.91–0.72 (m, 24H). RANMP-T 1 H NMR (500 MHz, Chloroform- d ): δ 8.93 (s, 2H), 8.80 (s, 2H), 8.69 (d, J = 7.0 Hz, 2H), 8.47 (s, 2H), 7.94–7.90 (m, 2H), 7.78–7.68 (m, 6H), 3.15–3.09 (m, 8H), 2.93 (t, 3H), 1.85–1.68 (m, 4H), 1.52–1.14 (m, 88H), 0.91–0.73 (m, 30H). Quantum chemical calculations Density functional theory (DFT) and time-dependent (TD) DFT methods (B3LYP/6-31G(d,p)) were used to obtain the optimized molecular structures in S 0 , frontier molecular orbitals (HOMO and LUMO), and natural transition orbitals (HONTO and LUNTO) of the molecules in chloroform (Supplementary Figs. 5–6). The optimized structures in S 1 of molecules were obtained by using the TD-DFT method. Additionally, Raman spectra of molecules were calculated and analyzed to understand the dependence of Raman peak (CN vibrational mode) on the molecular structures (Supplementary Fig. 7). The intersystem crossing rate constants ( k ISC ) and radiative decay rate constants ( k r ) of molecules were calculated as described in Supplementary Note 1. The vertical electronic transition energies (singlet and triplet states) and spin-orbit coupling (SOC) constants were obtained using the Tamm-Dancoff approximation (TDA)-DFT method (B3LYP/6-31G(d,p)) implemented in ORCA. Declarations Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Code Availability The codes that support the plots and data analysis within this paper are available from the corresponding author upon reasonable request. Acknowledgements This work was supported by Samsung Science and Technology Foundation SSTF-BA2201-07 for S.-H.S. S.P. and H.Y.W acknowledge the financial support from the National Research Foundation of Korea (NRF 2019R1A6A1A11044070). Author Information Authors and Affiliations Department of Chemistry, Korea University, Seoul, Korea Sumin Oh, Yunji Eom, Ha Yeon Kim, Ayushi Tripathi, Sungnam Park, Han Young Woo, Sang-Hee Shim Contributions H.Y.W and S.-H.S conceived and designed the research. S.O. built the ER-SRS set-up and performed all the SRS spectroscopy and microscopy experiments. Y.E. and A.T. prepared polymer dots and film samples. S.O. and Y.E. collected and analyzed the data. H.Y.W and S.-H.S supervised the experiments. S.P. supervised theoretical and quantum chemical calculations and analyzed the results. H.Y.K performed theoretical and quantum chemical calculations. The manuscript was written by S.O. and S.-H.S. with input from all authors. Corresponding Authors Correspondence and requests for materials should be addressed to Han Young Woo. Correspondence and requests for data should be addressed to Sang-Hee Shim. Correspondence and requests for theoretical results should be addressed to Sungnam Park. Ethics declarations Competing interests The authors declare no competing interests. References Ha, T. et al. Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc. Natl Acad. Sci. USA 93, 6264–6268 (1996). Yildiz, A. et al. Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization. Science 300, 2061–2065 (2003). Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006). Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006). Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods 5, 155–157 (2008). Bintu, B. et al. Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science 362, 6413 (2018). Metzker, M. et al. Sequencing technologies — the next generation. Nat Rev Genet 11, 31–46 (2010). Sigal, Y.M. et al. Visualizing and discovering cellular structures with super-resolution microscopy. Science 361, 880–887 (2018). Balzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606–612 (2017). Weisenburger, S. et al. Cryogenic optical localization provides 3D protein structure data with Angstrom resolution. Nat. Methods 14, 141–144 (2017). Reinhardt, S.C.M. et al. Ångström-resolution fluorescence microscopy. Nature 617, 711–716 (2023). Chen, K.H. et al. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015). Moffitt, J.R. et al. The emerging landscape of spatial profiling technologies. Nat Rev Genet 23, 741–759 (2022). Lakowicz, J. R. Principles of Fluorescence Spectroscopy 3rd edn (Springer, 2011). Nie, S. et al.“Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering”. Science 275, 1102–1106 (1997). Langer, J. et al. Present and Future of Surface-Enhanced Raman Scattering. ACS Nano 14, 28 – 117 (2020). Laing, S. et al. Multiplex in vitro detection using SERS. Chem. Soc. Rev. 45, 1901–1918 (2016). Jimenez De Aberasturi, D. et al. Surface Enhanced Raman Scattering Encoded Gold Nanostars for Multiplexed Cell Discrimination. Chem. Mater. 28, 6779–6790 (2016). Kang, H. et al. Near-Infrared SERS Nanoprobes with Plasmonic Au/Ag Hollow-Shell Assemblies for in Vivo Multiplex Detection. Adv. Funct. Mater. 23, 3719–3727 (2013). Wei, L. et al. Super-multiplex vibrational imaging. Nature 544, 465–470 (2017). Xiong, H. et al. Stimulated Raman excited fluorescence spectroscopy and imaging. Nat. Photon. 13, 412–417 (2019). Xiong, H. et al. Stimulated Raman excited fluorescence spectroscopy of visible dyes. J. Phys. Chem. Lett. 10, 3563–3570 (2019). Xiong, H. et al. Combining the best of two worlds: stimulated Raman excited fluorescence. J. Chem. Phys. 153, 210901 (2020). Wang, H. et al. Bond-selective fluorescence imaging with single-molecule sensitivity. Nat. Photon. 17, 846–855 (2023). Shi, L. et al. Electronic Resonant Stimulated Raman Scattering Micro-Spectroscopy. J. Phys. Chem. B 122, 9218–9224 (2018). Cho, S. J. et al. A-D-A Type Semiconducting Small Molecules with Bis(alkylsulfanyl)methylene Substituents and Control of Charge Polarity for Organic Field-Effect Transistors. ACS Appl. Mater. Interfaces 12, 41842–41851 (2020). Cho, E. et al. Impact of secondary donor units on the excited-state properties and thermally activated delayed fluorescence (TADF) efficiency of pentacarbazole-benzonitrile emitters. J. Chem. Phys. 153, 144708 (2020). Nguyen, V. -N. et al. An Emerging Molecular Design Approach to Heavy-Atom-Free Photosensitizers for Enhanced Photodynamic Therapy under Hypoxia. J. Am. Chem. Soc. 141, 16243–16248 (2019). Nguyen, V. -N. et al. Design and synthesis of efficient heavy-atom-free photosensitizers for photodynamic therapy of cancer. Chem. Commun. 56, 11489–11492 (2020). Jeong, J.-E. et al. Resonant Raman-Active Polymer Dot Barcodes for Multiplex Cell Mapping. ACS Nano 17, 4800–4812 (2023). Kwon, J. et al. Bleaching-Resistant Super-Resolution Fluorescence Microscopy. Adv. Sci. 9, 2101817 (2022). Kocheril, P.A. et al. Nitrile Vibrational Lifetimes as Probes of Local Electric Fields. J. Phys. Chem. Lett. 15, 5306–5314 (2024). Lu, F.-K. et al. Label-free DNA imaging in vivo with stimulated Raman scattering microscopy. Proc. Natl Acad. Sci. USA 112, 11624– 11629 (2015). Fu, D. et al. In vivo metabolic fingerprinting of neutral lipids with hyperspectral stimulated Raman scattering microscopy. J. Am. Chem. Soc. 136, 8820–8828 (2014). Santra, D.C. et al. Charge-Transfer-Induced Fluorescence Quenching of Anthracene Derivatives and Selective Detection of Picric Acid. Chem. Eur. J. 22, 2012–2019 (2016). Bu, L. et al. An AIE and ICT based NIR florescent probe for cysteine and homocysteine. Dyes Pigm. 136, 724–731 (2017). Bednarik, S. et al. Tuning Electron-Accepting Properties of Phthalocyanines for Charge Transfer Processes. Inorganic Chemistry 63, 8799–8806 (2024). Cordes, T. et al. Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy. Phys. Chem. Chem. Phys. 13, 6699–6709 (2011). Chen, Z., Paley, D. W., Wei, L., Weisman, A. L., Friesner, R. A., Nuckolls, C., Min, W. Multicolor Live-Cell Chemical Imaging by Isotopically Edited Alkyne Vibrational Palette. J. Am. Chem. Soc. 136, 8027–8033 (2014). Hu, F. et al. Supermultiplexed optical imaging and barcoding with engineered polyynes. Nat Methods 15, 194–200 (2018). Miao, Y. et al. 9-Cyanopyronin probe palette for super-multiplexed vibrational imaging. Nat Commun 12, 4518 (2021). Additional Declarations There is NO Competing Interest. Supplementary Files 241216SI.docx Supplementary information Cite Share Download PDF Status: Published Journal Publication published 13 Feb, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5650021","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":399746914,"identity":"22954ec0-ec5e-4b63-bc51-ef411c166df2","order_by":0,"name":"Sang-Hee Shim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYJACZhBhwMzA+LABLnaAOC3MhiRqYWBgkyRKi3z72cOvCyps7M3ZudMqZ1Tck+NvYH74geHMPZxaDM7kpVnPOJOWuLOZd9vNDWeKjSUOsBlLMNwoxq2FIcfMmLftcILBYaCWh20JiQ0HGMwYGD4k4HZY/xugln//7UFaCoFa6ucfYP+GVwvDjRzjx7wNBxg3ALUwbmxLSDA4wAO05QZuLQY33pgx8xxLTgRq2Sw540yC4cbDPMUSCWfwOSzH+DNPjZ29wfmzGz/2VCTIyx1v3/jhwzE8DgNGhwQqHxRNeDUAlXzALz8KRsEoGAUjHgAAhAlYQzIr1SMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-9964-7231","institution":"Korea University","correspondingAuthor":true,"prefix":"","firstName":"Sang-Hee","middleName":"","lastName":"Shim","suffix":""},{"id":399746915,"identity":"f399ab72-28c0-4afc-b264-3acc16056f9b","order_by":1,"name":"Sumin Oh","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Sumin","middleName":"","lastName":"Oh","suffix":""},{"id":399746916,"identity":"228affbe-2457-4629-a2e7-ea151307604e","order_by":2,"name":"Yunji Eom","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Yunji","middleName":"","lastName":"Eom","suffix":""},{"id":399746917,"identity":"211b8612-b565-4e73-8ca5-32caf2e8704d","order_by":3,"name":"Ha Yeon Kim","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Ha","middleName":"Yeon","lastName":"Kim","suffix":""},{"id":399746918,"identity":"aa5ebd84-1e15-44b4-bfea-0a9f47ad57b0","order_by":4,"name":"Ayushi Tripathi","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Ayushi","middleName":"","lastName":"Tripathi","suffix":""},{"id":399746919,"identity":"ddb6c37b-f6ff-440d-8c22-d1bc2b19a9cd","order_by":5,"name":"Sungnam Park","email":"","orcid":"https://orcid.org/0000-0001-6288-4620","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Sungnam","middleName":"","lastName":"Park","suffix":""},{"id":399746920,"identity":"7bdbc6f7-5aad-4c3e-846b-3f541fd3c038","order_by":6,"name":"Han Young Woo","email":"","orcid":"https://orcid.org/0000-0001-5650-7482","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"Young","lastName":"Woo","suffix":""}],"badges":[],"createdAt":"2024-12-16 03:05:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5650021/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5650021/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-026-69348-6","type":"published","date":"2026-02-13T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75517741,"identity":"1ca5cebb-3689-4bcd-ab82-60293a502218","added_by":"auto","created_at":"2025-02-05 11:41:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":198731,"visible":true,"origin":"","legend":"\u003cp\u003eIndependently tunable ER-SRS microscope and its application to Rho800 fluorophore. (A) Energy diagram of two-beam EPR-SRS (left) and ER-SRS (right) processes, where the frequency difference between the pump and Stokes beams is resonant to the vibrational transition. In ER-SRS (right), the pump beam is rigorously resonant to the electronic transition while in EPR-SRS (left), the pump beam is slightly off-resonant but still close to the electronic transition. (B) Two-beam ER-SRS instrument with synchronously pumped double OPO system with two independently tunable OPOs whose signal beams are each used as the pump and Stokes beams. (C) Absorption and emission spectra of Rho800 and the wavelength combinations of pump and Stokes used for collecting ER-SRS spectra. (D) ER-SRS spectra with various combinations of the pump and Stokes wavelengths indicated in C. SR gain signal is plotted against the frequency differences between the pump and Stokes beams. Powers are set as 5 mW for pump and 5 mW for Stokes. (E) Signal and background intensities in the ER-SRS spectra in D plotted against the pump wavelengths.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5650021/v1/7a5a677055575bbcbf69c3c3.png"},{"id":75517738,"identity":"c7a6b6e0-ec02-44af-86d7-a00f668282dc","added_by":"auto","created_at":"2025-02-05 11:41:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":231803,"visible":true,"origin":"","legend":"\u003cp\u003eRANMP for ER-SRS (A) Molecular structures of the RANMP series in which four nitrile groups conjugated to the highly conjugated structures with near-infrared absorption and extremely low fluorescence quantum yield: RANMP, RANMP-4F, RANMP-4Cl, and RANMP-T. (B) Sample geometry of chromophore film for SRS detection in transillumination geometry. (C) Time trace of ER-SRS signal of RANMP-T. Blue curve is Gaussian fitting to the trace when the Stokes pulse precedes the pump. Red line is exponential fitting to the trace when pump pulse precedes Stokes. (D) Pump power dependence of ER-SRS signal. (E) ER-SRS spectra of the four RANMPs at the optimal combination of pump and Stokes wavelengths. (F) Summary of ER-SRS spectra in E in terms of the absorption max (l\u003csub\u003emax\u003c/sub\u003e), wavelength difference of the pump wavelength from the absorption max (l\u003csub\u003emax\u003c/sub\u003e - l\u003csub\u003epump\u003c/sub\u003e) at the optimal condition and signal enhancement in comparison to the EPR-SRS signal of Rho800 with fixed Stokes wavelength at 1,031.2 nm as in previous studies.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5650021/v1/9773ff2465a56aa2d585cb46.png"},{"id":75518429,"identity":"58c53355-348b-4cfd-b622-25b2eaec2a12","added_by":"auto","created_at":"2025-02-05 11:49:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":255868,"visible":true,"origin":"","legend":"\u003cp\u003eER-SRS of Pdots encapsulating RANMP-4Cl and RANMP-T. (A) Schematic of Pdot structure. (B) ER-SRS spectra of RANMP-T under various wavelength combinations of pump and Stokes for optimizing the S/B ratio. Powers are set as 1 and 8 mW for pump and Stokes beam, respectively. (C) Signal and background intensities in the ER-SRS spectra in B plotted against the pump wavelengths. (D) Two-color single-particle images of RANMP-T and RANMP-4Cl. Far left: brightfield image shows four particles with no way to identify the probe. Middle left: ER-SRS image under optimal condition for RANMP-T. Middle right: ER-SRS image under the optimal condition for RANMP-4Cl. Far right: Off-resonance condition in which pump-Stokes frequency difference is in between the vibrational frequencies of the probe. Powers are set as 1.2 and 16.8 mW for pump and Stokes beams, respectively. (E) Spontaneous spectra of RANMP-T and RANMP-4Cl Pdots. (F-G) Cross-sectional profiles of the ER-SRS images of RANMP-T (F) and RANMP-4Cl (G) along the dotted lines in D and their fitting to Gaussian (lines).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5650021/v1/aad9871d027d402b7d347725.png"},{"id":75517756,"identity":"1571fad7-7f7b-460f-b4d5-571c9f71133c","added_by":"auto","created_at":"2025-02-05 11:41:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":124333,"visible":true,"origin":"","legend":"\u003cp\u003eSingle-molecule ER-SRS imaging. (A) ER-SRS spectrum of 1 nM RANMP-T embedded in PMMA film. (B) SR gain intensity in various concentrations of RANMP-T. (C) Two single-molecule images (M1 and M2) embedded in PMMA sandwiched by PVA films for photobleaching prevention. (D) Time traces of single-molecule signals in C. Single-step bleaching events (arrows) indicate single-molecule nature of the imaged objects. (E) Single-molecule time delay dependence. Measurements were performed in the sequence of 0 ps, +1 ps, and 0 ps. The recovery of the molecular signal upon returning to 0 ps verifies that the vanishment of signal at +1 ps is not due to photobleaching. Powers are set as 300 µW and 4 mW for pump and Stokes beams, respectively. (F) Single-molecule frequency difference dependence. Measurements were performed in the sequence of 2218.2 cm\u003csup\u003e-1\u003c/sup\u003e, 2236.3 cm\u003csup\u003e-1\u003c/sup\u003e and 2218.2 cm\u003csup\u003e-1\u003c/sup\u003e. Powers are set as 300 µW and 4 mW for pump and Stokes beams, respectively. Scale bars: 500 nm. Exposure times: 1 ms.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5650021/v1/0bed758e26cd0291f3f3d048.png"},{"id":105357103,"identity":"1392ea66-8cd4-4495-9425-6412e6bfbc46","added_by":"auto","created_at":"2026-03-25 07:06:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1420941,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5650021/v1/ae834443-7dea-4e9d-8faf-270d02995f35.pdf"},{"id":75520414,"identity":"b187e7b2-992a-46ae-b97d-d66db6d764cf","added_by":"auto","created_at":"2025-02-05 12:05:56","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4525315,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"241216SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-5650021/v1/197d591dd893f36254f081a2.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003eFluorescence-Free Single-Molecule Microscopy via Electronic Resonance Stimulated Raman Scattering\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFluorescence microscopy has revolutionized biology with its unmatched molecular specificity and single-molecule sensitivity\u003csup\u003e1-4\u003c/sup\u003e. Single-molecule fluorescence microscopy has unveiled the individual trajectories of biological molecules, revealing the molecular mechanisms\u003csup\u003e5,6\u003c/sup\u003e.\u0026nbsp;Fluorescence detection has driven advancements in cutting-edge technologies such as massively parallel DNA sequencing\u003csup\u003e7\u003c/sup\u003e and super-resolution optical microscopy\u003csup\u003e8-11\u003c/sup\u003e. In recent years, fluorescence microscopy evolved to highly multiplexed imaging, opening new fields of spatial genomics, transcriptomics and proteomics\u003csup\u003e6,12,13\u003c/sup\u003e. However, fluorescence-based multiplexed imaging relies on iterative cycles of labeling, imaging and washing for each molecular target due to the broad bandwidth (1,500 cm\u003csup\u003e-1\u003c/sup\u003e) of excitation and emission that limit its ability to spectrally distinguish different molecules\u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn contrast, vibrational transitions, such as Raman scattering and infrared (IR) absorption, have a much narrower bandwidth (10 cm\u003csup\u003e-1\u003c/sup\u003e) compared to fluorescence, making them highly suitable for simultaneously differentiating multiple substances. However, the intrinsic low intensity of Raman signals necessitates extensive signal amplification. Surface-enhanced Raman spectroscopy (SERS) enhances the Raman signal by over 10\u003csup\u003e7\u003c/sup\u003e using plasmonic nanoparticles, enabling single-molecule detection\u003csup\u003e15-17\u003c/sup\u003e. However, the preparation of SERS substrates can be complicated and requires precise control of nanoparticle fabrication and probe positioning\u0026nbsp;for\u0026nbsp;consistent results\u003csup\u003e18,19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eElectronic pre-resonance stimulated Raman scattering (EPR-SRS, Fig. 1A) enhances Raman sensitivity by up to 10\u003csup\u003e11\u003c/sup\u003e compared to spontaneous Raman without near-field effects\u003csup\u003e20\u003c/sup\u003e. However, it still requires fluorescence detection, such as stimulated Raman excited fluorescence\u003csup\u003e21\u003c/sup\u003e (SREF, Supplementary Fig. 1A), for single-molecule sensitivity. Efficient SREF fluorophores must fulfill multiple requirements: (1) conjugation of vibrational modes to a chromophore, (2) large Raman cross-section, (3) high near-infrared (NIR) absorbance, and (4) high fluorescence quantum yields\u003csup\u003e22\u003c/sup\u003e. However, high quantum yields lead to high background including anti-Stokes fluorescence\u003csup\u003e23\u003c/sup\u003e from thermally excited populations, limiting the signal-to-background (S/B) ratios and practicality. Bond-selective fluorescence-detected infrared-excited spectro-microscopy\u003csup\u003e24\u003c/sup\u003e (BonFIRE) also demonstrated single-molecule sensitivity (Supplementary Fig. 1A). However, BonFIRE suffered from anti-Stokes fluorescence and IR-induced photothermal background, requiring high-frequency modulation for background substraction\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur research aims to leverage electronic resonance stimulated Raman scattering (ER-SRS) to amplify Raman sensitivity without the aid of near-field effects or fluorescence detection (Fig. 1A). First demonstrated in 2018, ER-SRS has never demonstrated single-molecule sensitivity due to large electronic background and lack of suitable probes and light sources\u003csup\u003e25\u003c/sup\u003e. In this regard, our strategy combines nonfluorescent, highly conjugated Raman-active probe and frequency optimization for two-beam ER-SRS by using our unique independently tunable double optical parametric oscillator (OPO) laser system (Fig. 1B and Supplementary Fig. 1B), enabling single-molecule detection.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eER-SRS and EPR-SRS of Rhodamine 800 fluorophore\u003c/h2\u003e \u003cp\u003eRhodamine 800 (Rho800) serves as a key probe for EPR-SRS and SREF\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Utilizing our independently tunable double OPO system, we analyzed the ER-SRS and EPR-SRS spectra of Rho800 under various pump/Stokes wavelength combinations (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E). The selected wavelengths meet these conditions: (1) \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{{\\omega\\:}}_{\\text{p}}-{{\\omega\\:}}_{\\text{s}}=\\:{{\\omega\\:}}_{\\text{v}}\\:\\)\u003c/span\u003e\u003c/span\u003e; (2)\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{{\\omega\\:}}_{\\text{p}}-{{\\omega\\:}}_{\\text{a}\\text{b}\\text{s},\\text{m}\\text{a}\\text{x}}\\)\u003c/span\u003e\u003c/span\u003e\u0026lt; 700 cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e for ER-SRS\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e; (3) 1,400 cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e \u0026lt; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{{\\omega\\:}}_{\\text{p}}-{{\\omega\\:}}_{\\text{a}\\text{b}\\text{s},\\text{m}\\text{a}\\text{x}}\\)\u003c/span\u003e\u003c/span\u003e \u0026lt; 4,200 cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e for EPR-SRS\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, where \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{{\\omega\\:}}_{\\text{p}}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{{\\omega\\:}}_{\\text{s}}\\)\u003c/span\u003e\u003c/span\u003e are pump and Stokes frequencies; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\omega\\:}_{\\text{a}\\text{b}\\text{s},\\text{m}\\text{a}\\text{x}}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{{\\omega\\:}}_{\\text{v}}\\)\u003c/span\u003e\u003c/span\u003e are the frequencies at the absorption max and Raman shift of the probe, respectively. For Rho800,\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:{{\\omega\\:}}_{\\text{v}}\\)\u003c/span\u003e\u003c/span\u003e=2,229.6 cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;2A). Using our synchronized, yet independently tunable double OPO system with a tuning rage of 660\u0026ndash;960 nm, we optimized pump/Stokes combinations to maximize the S/B ratio. The pump wavelength was tuned around the absorption max (λ\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;700 nm) while adjusting the Stokes wavelength to achieve vibrational resonance\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\:({\\omega\\:}}_{\\text{p}}-{{\\omega\\:}}_{\\text{s}}=\\:{{\\omega\\:}}_{\\text{v}})\\)\u003c/span\u003e\u003c/span\u003e. When the pump matched λ\u003csub\u003emax\u003c/sub\u003e (black line, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), an inverted Raman peak was observed (black spectrum, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), consistent with prior findings for SiR845 fluorophore\u003csup\u003e25\u003c/sup\u003e. At λ\u003csub\u003epump\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;716 nm, within Rho800\u0026rsquo;s resonance region, the signal increased over 60-fold compared to the pre-resonant condition at (pump, Stokes) = (838.4 nm, 1,031.2 nm) (Supplementary Fig.\u0026nbsp;2B), as previously demonstrated with conventional OPO\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, significant background at λ\u003csub\u003epump\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;716 nm led to a low S/B ratio. Deviating λ\u003csub\u003epump\u003c/sub\u003e from λ\u003csub\u003emax\u003c/sub\u003e by \u0026gt;\u0026thinsp;50 nm significantly improved the S/B ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). For highly fluorescent probe like Rho800, ER-SRS failed to enhance the S/B ratio due to strong background from fluorescence and pump-probe processes\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, illustrating the limitations of ER-SRS with conventional fluorophores as resonant Raman probes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eER-SRS of Raman-amplified nonfluorescent molecular probe (RANMP)\u003c/h2\u003e \u003cp\u003eWe developed Raman-amplified nonfluorescent molecular probes (RANMPs) with (1) high far-red to NIR absorption for electronic resonance to the light source, (2) Raman-active groups conjugated to the chromophore for large Raman cross-section and electronic resonance, and (3) low fluorescence quantum yields to reduce background (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The series\u0026ndash;RANMP, RANMP-4F, RANMP-4Cl, and RANMP-T\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e\u0026ndash;is based on an electron-rich core, bis(alkylsulfanyl)methylene-substituted 4,9-dihydro-s-indaceno[1,2-b:5,6-b\u0026rsquo;]dithiophene (IDT), and the electron-withdrawing terminal group 2-(3-oxo-2,3-dihydro-1H-inden-1-ylidene)malononitrile (INCN) containing Raman-active CN moieties. These molecules were synthesized following a previously reported procedure\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;3). The final compounds were obtained by coupling a formylated IDT core with INCN derivatives via a Knoevenagel condensation reaction using pyridine as a base, in approximately 80% yields for all compounds. The effective conjugation was adjusted by controlling intramolecular charge transfer (ICT) interactions in each molecule. This was achieved by incorporating halogen substituents into INCN group (RANMP-4F and RANMP-4Cl) or introducing a thiophene spacer between the core and INCN terminal moieties (RANMP-T), thereby finely modulating the absorption max. The ICT effect allows λ\u003csub\u003emax\u003c/sub\u003e to fall within the pump tuning range of 660\u0026ndash;960 nm, facilitating resonance conditions (Supplementary Fig.\u0026nbsp;4A). In RANMP-T, the addition of a thiophene linker between the donor (D) and acceptor (A) units increases the effective conjugation length, resulting in a Raman peak shift of the CN groups (Supplementary Fig.\u0026nbsp;4B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom quantum chemical calculations, we found that the donor unit (IDT core) and the acceptor unit (INCN terminal groups) of the RANMP series are responsible mainly for the HOMO and LUMO, respectively (Supplementary Fig.\u0026nbsp;5). The natural transition orbitals (HONTO and LUNTO) represent the ICT character from the donor to the acceptor unit upon S\u003csub\u003e0\u003c/sub\u003e \u0026rarr; S\u003csub\u003e1\u003c/sub\u003e transition (Supplementary Fig.\u0026nbsp;6). These A-D-A type molecules have large Raman cross-sections due to the four CN groups in both terminals conjugated to the IDT core and INCN terminal group. The calculated Raman intensity of the CN vibrational mode in tetracyanoethylene (TCNE) is significantly lower\u0026mdash;by a factor of 10 to 30\u0026mdash;compared to those of the RANMP series, indicating that the influence of the conjugation on the Raman cross-section (Supplementary Fig.\u0026nbsp;7C and Supplementary Table\u0026nbsp;1). In addition, the Raman peak of the CN vibrational mode in the RANMP series depends on the molecular structures. The calculated Raman intensities of the CN vibrational modes increase in the order of RANMP, RANMP-4F, RANMP-4Cl, and RANMP-T (Supplementary Fig.\u0026nbsp;7C), which is consistent with the experimental results (Supplementary Fig.\u0026nbsp;4). The calculated Raman frequency is red-shifted in the order of RANMP-4F, RANMP-4Cl, RANMP, and RANMP-T, which correlates with the CN bond length of the RANMP series (Supplementary Fig.\u0026nbsp;7D). The RANMP series exhibit photoluminescence undetectable with our spectrophotometer, significantly improving the Raman S/B ratio. To understand the low photoluminescence of the RANMP series, the intersystem crossing rate constants (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eISC\u003c/sub\u003e) and radiative decay rate constants (\u003cem\u003ek\u003c/em\u003e\u003csub\u003er\u003c/sub\u003e) of the RANMP series and Rho800 were calculated (Supplementary Note 1)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. As shown in Table SN2 of Supplementary Note 1, the \u003cem\u003ek\u003c/em\u003e\u003csub\u003eISC\u003c/sub\u003e / \u003cem\u003ek\u003c/em\u003e\u003csub\u003er\u003c/sub\u003e ratio of the RANMP series (ranging from 10\u003csup\u003e2\u003c/sup\u003e to 10\u003csup\u003e3\u003c/sup\u003e) is significantly higher than that of Rho800 (7.3). Therefore, compared with Rho800, the RANMP series exhibit much faster ISC to the triplet state and more efficient quenching of the fluorescence of the S\u003csub\u003e1\u003c/sub\u003e state, which explains the low photoluminescence. Note that fluorescence quenching by rapid ISC is a key strategy to substantially reduce fluorescence background in ER-SRS. As shown in Eq. (S1) in Supplementary Note 1, the ISC rate constant increases with a large spin-orbit coupling (SOC) constant and a small energy difference between singlet and triplet states (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e). SOC can be increased by introducing heavy atoms (e.g., S, Se, Br) into organic compounds, a concept widely employed in designing photosensitizers for photodynamic therapy\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. For the RANMP series, the SOC constants are found to be large due to sulfur atoms (a heavy atom) (Supplementary Table SN2 in Supplementary Note 1).\u003c/p\u003e \u003cp\u003eTo optimize ER-SRS for the RANMP series, we measured the modulation transfer signal of RANMP-T film at (pump, Stokes) = (749 nm, 897.9 nm) by scanning the time delay between pump and Stokes pulses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). When Stokes preceded pump (blue line, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), the intensity fits to a Gaussian peak centered at time zero, consistent with SRS convolved with a picosecond pulse. When pump precedes Stokes (red line, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), the signal decayed exponentially with a 4.26 ps lifetime, which is on the order of that of stimulated emission. This asymmetrical time delay dependence indicates pump-dominated background. Also, the signal exhibited nonlinear pump power dependence as in the previous literature\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The Stokes power dependence of ER-SRS signal was largely linear below a saturation level (Supplementary Fig.\u0026nbsp;8).\u003c/p\u003e \u003cp\u003eUsing a reduced pump power (500 \u0026micro;W) and a Stokes power below saturation (10 mW), we obtained ER-SRS spectra of RANMP, RANMP-4F, RANMP-4Cl and RANMP-T films (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, Supplementary Figs.\u0026nbsp;8\u0026ndash;9). The double OPO system enabled flexible pump and Stokes tuning to optimize the S/B ratio. Under rigorous resonance, the overwhelming background signal resulted in inverted Raman peaks, whereas positive peaks appeared slightly off resonance (Supplementary Fig.\u0026nbsp;9). At the optimized conditions, ER-SRS signals showed 200-400x amplification compared to EPR-SRS of Rho800 [(pump, Stokes) = (838.4 nm, 1,031.2 nm)] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results align with the theoretical predictions (10-100x amplification), given the four CN groups per RANMP molecule versus one CN in Rho800\u003csup\u003e25\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSingle-particle imaging of RANMP Pdots\u003c/h3\u003e\n\u003cp\u003ePreviously, we demonstrated that chromophores aggregated into polymer dot (Pdot) forms exhibit high resonance Raman scattering with suppressed fluorescence due to aggregation-induced quenching\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Likewise, we expected ER-SRS signals in RANMP-based Pdots to benefit from probe concentration and further fluorescence suppression. RANMP-4Cl and RANMP-T, showing the highest ER-SRS amplification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), were fabricated into Pdots (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;10). Using independent wavelength tuning, we optimized the ER-SRS S/B ratios, achieving maxima at λ\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;37 nm for RANMP-T and λ\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;20 nm for RANMP-4Cl (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Supplementary Fig.\u0026nbsp;11C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnder the optimum ER-SRS conditions, we achieved dual-color single-particle imaging using the narrow linewidth of Raman scattering (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-G). In a mixture of RANMP-T and RANMP-4Cl Pdots, indistinguishable particles in the brightfield image became spectrally distinct at 2,218.4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (RANMP-T) and 2,235.4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (RANMP-4Cl) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). At the optimal condition for RANMP-T [(pump, Stokes) = (744.4 nm, 891.6 nm)], RANMP-4Cl particles were nearly invisible, and vice versa for RANMP-4Cl [ (pump, Stokes) = (760.2 nm, 915.8 nm)]. Off-resonance condition [(pump, Stokes) = (760.7 nm, 915.8 nm)] rendered all particles invisible. The Full Width at Half Maximum (FWHM) of the cross-sectional profile of the particle image was 361 nm for RANMP-4Cl and 356 nm for RANMP-T, closely matching the diffraction-limited resolution of 324 nm and particle size (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G, Supplementary Fig.\u0026nbsp;10B). Each 5 \u0026micro;m x 5 \u0026micro;m field of view (FOV) was acquired in ~\u0026thinsp;62 seconds, enabling two-color imaging under 3 minutes without noticeable bleaching. The imaging speed, limited by the sample piezo stage, could be improved with a Galvano scanner for beam scanning. The 17 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Raman shift difference between particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) allowed clear spectral distinction for successful two-color single-particle imaging.\u003c/p\u003e\n\u003ch3\u003eSingle-molecule imaging of individual molecules embedded in film\u003c/h3\u003e\n\u003cp\u003eTo detect single molecule at room temperature, we optimized ER-SRS for poly(methyl methacrylate) (PMMA) samples sparsely embedded with RANMP molecules. Surpassing the EPR-SRS detection limit of 250 nM\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, we detected signals at 1 nM for RANMP-T (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B), equivalent to ~\u0026thinsp;3 molecules in the laser focal volume. To minimize bleaching, we employed a polyvinyl alcohol (PVA)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e layer above and below the PMMA (Supplementary Figs.\u0026nbsp;12A-B) and applied a minimal pump power of 300 \u0026micro;W at optimized wavelengths (Supplementary Fig.\u0026nbsp;12C). Single-molecule imaging under these conditions (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E) showed abrupt photobleaching events in the time trace, confirming their single-molecule nature (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Orange and blue arrows in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD correspond to photobleaching of M1 and M2 molecules in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC. ER-SRS specificity was verified by collecting single-molecule images at two temporal overlaps of pump and Stokes pulses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). A molecular signal was observed when two pulses arrived concurrently (left \u0026ldquo;0 ps\u0026rdquo;, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). When the Stokes pulse preceded the pump only by 1 ps (+\u0026thinsp;1 ps, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), the signal sharply attenuated, consistent with the bulk time-dependent measurements in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC. Reverting the delay to 0 ps restored the signal (right 0 ps, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), confirming that the signal reduction was due to the short vibrational lifetime of nitrile groups (\u0026lt;\u0026thinsp;1 ps)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. To confirm vibrational resonance, we analyzed signal variations with pump-Stokes frequency differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Signals appeared at the CN stretch mode (2,218.2 cm⁻\u0026sup1;) but vanished when the pump wavelength tuned by 1 nm to a frequency difference of 2,236.3 cm⁻\u0026sup1;. Restoring resonance at 2,218.2 cm⁻\u0026sup1; recovered the signal, confirming the loss was due to off-resonance, not photobleaching. The abrupt photobleaching as well as sensitivity to temporal overlap and frequency difference validate the near-diffraction-limited features in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-F as single-molecule ER-SRS images. This is the first demonstration of far-field single-molecule detection via resonance Raman without relying on fluorescence or plasmonic enhancement.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we successfully demonstrated single-molecule ER-SRS by optimizing both light sources and probes. The previous ER-SRS demonstration\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e faced challenges in parameter optimization due to conventional SRS light source providing only a single tunable pump beam and a fixed Stokes beam (usually at 1,031.2 nm). In contrast, using our independently tunable double OPO laser system, we optimized the wavelength combination for two-beam ER-SRS to maximize the S/B ratio for Raman-active molecules with CN-moieties absorbing NIR light. To further enhance the signal and suppress the background, we designed a series of four novel Raman-active nonfluorescent molecules (RANMP, RANMP-4F, RANMP-4Cl and RANMP-T) and pre-analyzed their optical properties using DFT and time-dependent (TD) DFT calculations, thereby facilitating effective ER-SRS signal enhancement. As a result, we experimentally realized over 100-fold signal amplification, achieving the first-ever detection of single molecules via Raman scattering without fluorescence detection or near-field enhancement.\u003c/p\u003e \u003cp\u003eThe fluorescence-free nature of ER-SRS opens new doors for extending single-molecule vibrational microscopy to tissue and animal imaging. While fluorescence enables SREF and BonFIRE to achieve single-molecule sensitivity, it introduces significant background noise: SREF signals are accompanied by anti-Stokes fluorescence from thermally excited populations, and BonFIRE is affected by both anti-Stokes fluorescence and photothermal-induced fluorescence change. These background signals necessitate confocal detection for imaging single cell layers in SREF and BonFIRE, and additional strategies to suppress out-of-focus signal and background from thick samples for deep-tissue imaging. In contrast, the nonlinear nature of ER-SRS inherently provides optical sectioning, offering significant advantages for \u003cem\u003ein vivo\u003c/em\u003e imaging, as extensively demonstrated in SRS imaging\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eER-SRS also facilitates the development of probes for single-molecule vibrational microspectroscopy. In SREF, achieving an optimal S/B requires stringent electronic pre-resonance conditions, limiting the selection of fluorophores. While BonFIRE provides greater flexibility in dye selection, it is constrained by the tuning range of OPO and relies on NIR fluorophores, which often exhibit low photoluminescence quantum yields (PLQYs) \u0026mdash;for instance, Rho800 has a PLQY of 17%. Shifting absorption and emission into the NIR wavelengths typically employ strong donor-acceptor conjugation to induce intramolecular charge transfer (ICT). However, this approach often leads to strong ICT quenching, making it challenging to develop highly fluorescent NIR dyes. By contrast, designing nonfluorescent NIR absorbing molecules is more feasible. The A-D-A molecular design motif used in this study effectively extends conjugation to meet the resonance condition for ER-SRS, where strong ICT interaction suppresses PL emission, optimizing the S/B ratio\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePhotobleaching, a common challenge in fluorescence microscopy, also occurs in ER-SRS due to the use of electronic resonance. To mitigate this, single-molecule images in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e were obtained using a protective PVA layer above and below the probe-containing PMMA layer. When only a single PVA layer was applied, ER-SRS signals from single molecules were still detectable but photobleaching occurred more rapidly (Supplementary Figs.\u0026nbsp;12D-E). While fast transfer to the triplet state of RANMP series can promote photobleaching, the photobleaching lifetime of ER-SRS signals from a RANMP-T film was significantly longer than that of EPR-SRS signal from a Rho800 film (Supplementary Fig.\u0026nbsp;13). Our attempts to measure the triplet-state lifetime of RANMP-T film were unsuccessful, as no phosphorescence signal was detectable (data not shown), suggesting an extremely short triplet-state lifetime. This implies that the triplet state of RANMP, likely in the NIR region, is highly susceptible to thermal quenching. Efficient triplet-state quenching may therefore contribute to the slower photobleaching observed in the ER-SRS signal of RANMP. To further suppress triplet-mediated radical formation leading to photobleaching, strategies used in fluorescence imaging\u0026mdash;such as adding reducing agents, antioxidants and triplet state quenchers\u0026mdash;could be adapted for ER-SRS\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eER-SRS with RANMP achieved single-molecule detection from conventional film and solution samples using standard optical microscopes, highlighting a strong potential for advanced biological imaging using existing labeling methods and instruments. The RANMP in this study are organic small molecules, a widely used class of biological probes. For ER-SRS multiplexed imaging, the primary requirement is to synthesize more probes with nonfluorescent, NIR-absorbing conjugated molecules with Raman-active moieties such as nitrile, alkynes and azido groups. Raman shifts of these probes can be tuned through various strategies, including incorporating isotopes into vibrating groups\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, extending polyene structures\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and exploiting various core structures of conjugated molecules\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In this study, the thiophene spacer inserted between the core and INCN terminal moieties induced a blue shift in the CN vibrational frequency sufficient for two-color imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This introduces a new approach for synthesizing multiplex Raman probes. The PSMA-encapsulated Pdot in this study also offers a convenient, general route for boosting the S/B of ER-SRS probes and for efficient targeting in biological samples by using the carboxylic groups on the particle surface. We anticipate that ER-SRS, combined with RANMP, will serve as a powerful tool for multiplexed imaging of individual molecules or nanoparticles within complex biological environments.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIndependently tunable double OPO system\u003c/h2\u003e \u003cp\u003eThe ER-SRS microscopy setup is described in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Supplementary Fig.\u0026nbsp;1B. Two OPOs are pumped by a common, powerful IR fiber laser with a frequency doubling unit (Angewandte Physik \u0026amp; Elektronik GmbH, Emerald Engine HP). The Emerald Engine generates an output pulse train at 1,031.2 nm with a 2 ps pulse width and an 80 MHz repetition rate. The frequency-doubled beam at 516 nm is used to synchronously seed two OPOs, referred to as the high power (HP) OPO and the low power (LP) OPO. The tunable pump beam (660\u0026ndash;960 nm, ~\u0026thinsp;2 ps) from the HP OPO is modulated at 10 MHz by an electro-optic modulator (EOM) to achieve shot-noise limited detection sensitivity. The tunable Stokes beam (660\u0026ndash;960 nm, ~\u0026thinsp;2 ps) is pumped from the LP OPO.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eER-SRS microscopy\u003c/h3\u003e\n\u003cp\u003eAs in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Supplementary Fig.\u0026nbsp;1B, the independently tuned pump and Stokes beams are combined at the dichroic mirror and then directed into a home-built sample-scanning microscope body (Cerna system, Thorlabs). To confirm the spatial overlap of the two beams, two position sensors were each installed on the near and far sides of the dichroic mirror. The temporal overlap of pump and Stokes pulses is precisely defined using an optical delay line, installed in the pump path, and verified with the sum-frequency generation signal of a BBO crystal. The sample is placed on a piezo-stage (PINano XY Piezo System, P-545.3C8S; PI) for X and Y axis scanning. All beams are focused on the sample using a 60X objective lens with NA 1.2 (UplanSApo; Olympus). A condenser lens with NA 1.4 (oil immersion, D-CUO; Nikon) is used to collect the transmitted beams. The output beams pass through two band-pass filters and a dichroic mirror, which transmits the Stokes beam but blocks the pump, measured as the stimulated Raman gain (SRG) signal. Different band-pass filters and dichroic mirrors are used for each wavelength combination (Supplementary Table\u0026nbsp;2). The transmitted Stokes beam is collected through a large area photodiode and amplified by a lock-in amplifier (APE GmbH). The signal is sent to a computer in which a home-built LabVIEW program records the ER-SRS intensity at each pixel. We measured SR gain due to the peak sensitivity of the Si photodiode around 960 nm.\u003c/p\u003e\n\u003ch3\u003ePolymer dots (Pdots) fabrication\u003c/h3\u003e\n\u003cp\u003eThe nanoprecipitation method was employed to form Pdots (Supplementary Fig.\u0026nbsp;10). 100 \u0026micro;L of RANMP-4Cl (or RANMP-T) solution in THF (1 mg/mL) was mixed with 400 \u0026micro;L of poly(styrene-co-maleic anhydride) (PSMA, M\u003csub\u003en\u003c/sub\u003e \u0026asymp; 1600 Da, ~\u0026thinsp;1.3:1 styrene anhydride mol ratio) solution in THF (1 mg/mL). Subsequently, 4.5 mL of THF was added. The resulting THF solution was quickly transferred into 10 mL of deionized water under sonication. The water-THF mixture was then heated to 80\u0026deg;C to facilitate the evaporation of THF and achieve nanoparticle formation until the volume was reduced to 2 mL under N\u003csub\u003e2\u003c/sub\u003e bubbling. The concentrated Pdot solution was filtered through a 0.2 \u0026micro;M syringe filter and stored in a brown-colored vial.\u003c/p\u003e \u003cp\u003eTo verify if the Pdot nanoparticles are produced at a concentration similar to the theoretical concentration of the Raman probe, a certain amount of RANMP-T Pdot nanoparticle solution was taken and quantified through an absorption coefficient calibration curve (Supplementary Fig.\u0026nbsp;14). Due to losses occurring during bubbling when evaporating THF, the experimental concentration has an error of approximately\u0026thinsp;~\u0026thinsp;10% compared to the theoretical concentration.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImaging sample preparation\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eSingle-particle sample\u003c/strong\u003e \u003cp\u003eA 6% w/w agarose gel was prepared by dissolving the appropriate amount of agarose powder (A4018, Sigma) in a 25 mM MES buffer. The mixture was heated to 95\u0026deg;C to ensure complete dissolution. Subsequently, a glass slide was heated on a hot plate to 95\u0026deg;C. Then, 10 \u0026micro;L of the Pdot solution and 10 \u0026micro;L of the agarose gel were sequentially added on top of the slide. A cover glass was carefully placed over the mixture and gently pressed to achieve full coverage.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTwo-color Pdot sample\u003c/strong\u003e \u003cp\u003eThe same procedure was performed to prepare the single particle imaging sample. Before adding the agarose gel, 5 \u0026micro;L of the Pdot solution of RANMP-4Cl and 5 \u0026micro;L of the Pdot solution of RANMP-T were sequentially added.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSingle-molecule sample\u003c/strong\u003e \u003cp\u003eA glass substrate was washed in acetone, isopropyl alcohol and deionized water (DI) water for 15 minutes then UV-ozone plasma treatment was performed for 10 min. A 5% w/w poly(vinyl alcohol) (PVA)/ DI water solution was spin-coated onto a glass substrate at 1000 rpm for 120 seconds. Using a 1% w/w poly(methyl methacrylate) (PMMA)/CF solution, the desired concentration of RANMP-T solution was prepared to form a single-molecule embedded polymer thin film layer. This solution was then spin-coated onto the PVA layer at 1500 rpm for 120 seconds. Afterward, the 5% w/w PVA layer was spin-coated onto the PMMA/RANMP-T layer at 1000 rpm for 120 seconds.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMaterial characterization\u003c/h2\u003e \u003cp\u003eThe \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH spectra were measured by a Bruker Advance III system operating at 500 MHz. UV-Vis-NIR spectra were measured by a JASCO V-770 spectrophotometer. The hydrodynamic diameters of Pdots were measured in aqueous solution using Zetasizer Nano S90 (Malvern Panalytical). Spontaneous Raman spectra was obtained by HORIBA LabRAM HR Evolution Raman spectrometer (Japan). The laser beam, 532 nm, was focused on the samples at the microscope stage by a 100x/0.90 NA objective lens (Plan N, Olympus, Japan). The laser conditions were kept consistent with 1% power, a 5 s acquisition time and 5 accumulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis\u003c/h2\u003e \u003cp\u003eRANMP, RANMP-4F, RANM \u0026minus;\u0026thinsp;4Cl, and RANMP-T were synthesized following a previously reported procedure\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e (Supplementary Figs.\u0026nbsp;3, 15\u0026ndash;18).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRANMP\u003c/b\u003e \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, Chloroform-\u003cem\u003ed\u003c/em\u003e): δ 8.92 (s, 2H), 8.89 (s, 2H), 8.64 (s, 2H), 8.59\u0026ndash;8.54 (m, 2H), 7.89\u0026ndash;7.82 (m, 2H), 7.72\u0026ndash;7.65 (m, 4H), 3.14 (m, 8H), 1.80\u0026ndash;1.68 (m, 4H), 1.56\u0026ndash;1.13 (m, 64H), 0.96\u0026ndash;0.71 (m, 24H). \u003cb\u003eRANMP-4F\u003c/b\u003e \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, Chloroform-\u003cem\u003ed\u003c/em\u003e): δ 9.10\u0026ndash;9.05 (m, 2H), 8.92 (s, 2H), 8.72 (s, 2H), 8.57\u0026ndash;8.41 (m, 2H) 7.64 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 2H), 3.21\u0026ndash;3.09 (m, 8H), 1.79\u0026ndash;1.70 (m, 4H), 1.53\u0026ndash;1.11 (m, 64H), 0.93\u0026ndash;0.72 (m, 24H). \u003cb\u003eRANMP-4Cl\u003c/b\u003e \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, Chloroform-\u003cem\u003ed\u003c/em\u003e): δ 9.11 (s, 2H), 8.92 (s, 2H), 8.72 (s, 2H), 8.53\u0026ndash;8.43 (m, 2H), 7.64 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 2H), 3.15 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.7 Hz, 8H), 1.81\u0026ndash;1.66 (m, 4H), 1.52\u0026ndash;1.15 (m, 64H), 0.91\u0026ndash;0.72 (m, 24H). \u003cb\u003eRANMP-T\u003c/b\u003e \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, Chloroform-\u003cem\u003ed\u003c/em\u003e): δ 8.93 (s, 2H), 8.80 (s, 2H), 8.69 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.0 Hz, 2H), 8.47 (s, 2H), 7.94\u0026ndash;7.90 (m, 2H), 7.78\u0026ndash;7.68 (m, 6H), 3.15\u0026ndash;3.09 (m, 8H), 2.93 (t, 3H), 1.85\u0026ndash;1.68 (m, 4H), 1.52\u0026ndash;1.14 (m, 88H), 0.91\u0026ndash;0.73 (m, 30H).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eQuantum chemical calculations\u003c/h2\u003e \u003cp\u003eDensity functional theory (DFT) and time-dependent (TD) DFT methods (B3LYP/6-31G(d,p)) were used to obtain the optimized molecular structures in S\u003csub\u003e0\u003c/sub\u003e, frontier molecular orbitals (HOMO and LUMO), and natural transition orbitals (HONTO and LUNTO) of the molecules in chloroform (Supplementary Figs.\u0026nbsp;5\u0026ndash;6). The optimized structures in S\u003csub\u003e1\u003c/sub\u003e of molecules were obtained by using the TD-DFT method. Additionally, Raman spectra of molecules were calculated and analyzed to understand the dependence of Raman peak (CN vibrational mode) on the molecular structures (Supplementary Fig.\u0026nbsp;7). The intersystem crossing rate constants (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eISC\u003c/sub\u003e) and radiative decay rate constants (\u003cem\u003ek\u003c/em\u003e\u003csub\u003er\u003c/sub\u003e) of molecules were calculated as described in Supplementary Note 1. The vertical electronic transition energies (singlet and triplet states) and spin-orbit coupling (SOC) constants were obtained using the Tamm-Dancoff approximation (TDA)-DFT method (B3LYP/6-31G(d,p)) implemented in ORCA.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe codes that support the plots and data analysis within this paper are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Samsung Science and Technology Foundation SSTF-BA2201-07 for S.-H.S.\u0026nbsp;S.P.\u0026nbsp;and H.Y.W acknowledge the financial support from the National Research\u003c/p\u003e\n\u003cp\u003eFoundation of Korea (NRF 2019R1A6A1A11044070).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003eDepartment of Chemistry, Korea University, Seoul, Korea\u003c/p\u003e\n\u003cp\u003eSumin Oh, Yunji Eom, Ha Yeon Kim, Ayushi Tripathi, Sungnam Park, Han Young Woo, Sang-Hee Shim\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.Y.W and S.-H.S conceived and designed the research. S.O. built the ER-SRS set-up and performed all the SRS spectroscopy and microscopy experiments. Y.E. and A.T. prepared polymer dots and film samples. S.O. and Y.E. collected and analyzed the data. H.Y.W and S.-H.S supervised the experiments. S.P. supervised theoretical and quantum chemical calculations and analyzed the results. H.Y.K performed theoretical and quantum chemical calculations. The manuscript was written by S.O. and S.-H.S. with input from all authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence and requests for materials should be addressed to Han Young Woo. Correspondence and requests for data should be addressed to Sang-Hee Shim.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCorrespondence and requests for theoretical results should be addressed to Sungnam Park.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHa, T. et al. Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. \u003cem\u003eProc. Natl Acad. Sci. USA\u003c/em\u003e 93, 6264\u0026ndash;6268 (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYildiz, A. et al. Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization. Science 300, 2061\u0026ndash;2065 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBetzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642\u0026ndash;1645 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRust, M. J., Bates, M. \u0026amp; Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793\u0026ndash;795 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods 5, 155\u0026ndash;157 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBintu, B. et al. Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science 362, 6413 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMetzker, M. et al. Sequencing technologies \u0026mdash; the next generation. Nat Rev Genet 11, 31\u0026ndash;46 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSigal, Y.M. et al. Visualizing and discovering cellular structures with super-resolution microscopy. Science 361, 880\u0026ndash;887 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606\u0026ndash;612 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeisenburger, S. et al. Cryogenic optical localization provides 3D protein structure data with Angstrom resolution. Nat. Methods 14, 141\u0026ndash;144 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReinhardt, S.C.M. et al. \u0026Aring;ngstr\u0026ouml;m-resolution fluorescence microscopy. Nature 617, 711\u0026ndash;716 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, K.H. et al. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoffitt, J.R. et al. The emerging landscape of spatial profiling technologies. Nat Rev Genet 23, 741\u0026ndash;759 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLakowicz, J. R. \u003cem\u003ePrinciples of Fluorescence Spectroscopy\u003c/em\u003e 3rd edn (Springer, 2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNie, S. et al.\u0026ldquo;Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering\u0026rdquo;. \u003cem\u003eScience\u003c/em\u003e 275, 1102\u0026ndash;1106 (1997). Langer, J. et al. Present and Future of Surface-Enhanced Raman Scattering. \u003cem\u003eACS Nano\u003c/em\u003e 14, 28\u0026thinsp;\u0026ndash;\u0026thinsp;117 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaing, S. et al. Multiplex in vitro detection using SERS. Chem. Soc. Rev. 45, 1901\u0026ndash;1918 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJimenez De Aberasturi, D. et al. Surface Enhanced Raman Scattering Encoded Gold Nanostars for Multiplexed Cell Discrimination. Chem. Mater. 28, 6779\u0026ndash;6790 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang, H. et al. Near-Infrared SERS Nanoprobes with Plasmonic Au/Ag Hollow-Shell Assemblies for in Vivo Multiplex Detection. Adv. Funct. Mater. 23, 3719\u0026ndash;3727 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei, L. et al. Super-multiplex vibrational imaging. Nature 544, 465\u0026ndash;470 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiong, H. et al. Stimulated Raman excited fluorescence spectroscopy and imaging. Nat. Photon. 13, 412\u0026ndash;417 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiong, H. et al. Stimulated Raman excited fluorescence spectroscopy of visible dyes. J. Phys. Chem. Lett. 10, 3563\u0026ndash;3570 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiong, H. et al. Combining the best of two worlds: stimulated Raman excited fluorescence. J. Chem. Phys. 153, 210901 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, H. et al. Bond-selective fluorescence imaging with single-molecule sensitivity. Nat. Photon. 17, 846\u0026ndash;855 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi, L. et al. Electronic Resonant Stimulated Raman Scattering Micro-Spectroscopy. J. Phys. Chem. B 122, 9218\u0026ndash;9224 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCho, S. J. et al. A-D-A Type Semiconducting Small Molecules with Bis(alkylsulfanyl)methylene Substituents and Control of Charge Polarity for Organic Field-Effect Transistors. ACS Appl. Mater. Interfaces 12, 41842\u0026ndash;41851 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCho, E. et al. Impact of secondary donor units on the excited-state properties and thermally activated delayed fluorescence (TADF) efficiency of pentacarbazole-benzonitrile emitters. J. Chem. Phys. 153, 144708 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen, V. -N. et al. An Emerging Molecular Design Approach to Heavy-Atom-Free Photosensitizers for Enhanced Photodynamic Therapy under Hypoxia. J. Am. Chem. Soc. 141, 16243\u0026ndash;16248 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen, V. -N. et al. Design and synthesis of efficient heavy-atom-free photosensitizers for photodynamic therapy of cancer. Chem. Commun. 56, 11489\u0026ndash;11492 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJeong, J.-E. et al. Resonant Raman-Active Polymer Dot Barcodes for Multiplex Cell Mapping. ACS Nano 17, 4800\u0026ndash;4812 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKwon, J. et al. Bleaching-Resistant Super-Resolution Fluorescence Microscopy. Adv. Sci. 9, 2101817 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKocheril, P.A. et al. Nitrile Vibrational Lifetimes as Probes of Local Electric Fields. J. Phys. Chem. Lett. 15, 5306\u0026ndash;5314 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu, F.-K. et al. Label-free DNA imaging in vivo with stimulated Raman scattering microscopy. \u003cem\u003eProc. Natl Acad. Sci. USA\u003c/em\u003e 112, 11624\u0026ndash; 11629 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu, D. et al. In vivo metabolic fingerprinting of neutral lipids with hyperspectral stimulated Raman scattering microscopy. J. Am. Chem. Soc. 136, 8820\u0026ndash;8828 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantra, D.C. et al. Charge-Transfer-Induced Fluorescence Quenching of Anthracene Derivatives and Selective Detection of Picric Acid. Chem. Eur. J. 22, 2012\u0026ndash;2019 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBu, L. et al. An AIE and ICT based NIR florescent probe for cysteine and homocysteine. Dyes Pigm. 136, 724\u0026ndash;731 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBednarik, S. et al. Tuning Electron-Accepting Properties of Phthalocyanines for Charge Transfer Processes. Inorganic Chemistry 63, 8799\u0026ndash;8806 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCordes, T. et al. Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy. Phys. Chem. Chem. Phys. 13, 6699\u0026ndash;6709 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, Z., Paley, D. W., Wei, L., Weisman, A. L., Friesner, R. A., Nuckolls, C., Min, W. Multicolor Live-Cell Chemical Imaging by Isotopically Edited Alkyne Vibrational Palette. J. Am. Chem. Soc. 136, 8027\u0026ndash;8033 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu, F. et al. Supermultiplexed optical imaging and barcoding with engineered polyynes. Nat Methods 15, 194\u0026ndash;200 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiao, Y. et al. 9-Cyanopyronin probe palette for super-multiplexed vibrational imaging. Nat Commun 12, 4518 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5650021/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5650021/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVibrational spectroscopy is a powerful tool for spectral imaging of biological samples, thanks to its narrow bandwidth (10 cm⁻¹) compared to fluorescence. Single-molecule vibrational spectroscopy has been demonstrated with near-field amplification as in surface-enhanced Raman spectroscopy or fluorescence detection as in stimulated Raman excited fluorescence and bond-selective fluorescence-detected infrared-excited spectro-microscopy. However, these methods involve complex sample preparation or produce high backgrounds, limiting their practicality. In response to these issues, we developed electronic resonance stimulated Raman scattering (ER-SRS) to achieve single-molecule sensitivity in far-field vibrational microscopy without relying on fluorescence detection. ER-SRS has encountered difficulties due to large electronic backgrounds. To overcome this, we employed Raman-amplified nonfluorescent molecular probe (RANMP) alongside our synchronously pumped, independently tunable double optical parametric oscillators for effective optimization of the signal-to-background ratio. The optimization of probe and light source allowed us to successfully detect ER-SRS signal from single particles in solution and from single molecules embedded in polymer matrix. ER-SRS combined with RANMP, offering single-molecule sensitivity without the aid of fluorescence detection, will open new avenues in biological and chemical fields, particularly in multiplexed imaging.\u003c/p\u003e","manuscriptTitle":"Fluorescence-Free Single-Molecule Microscopy via Electronic Resonance Stimulated Raman Scattering","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-05 11:41:51","doi":"10.21203/rs.3.rs-5650021/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2fd7135b-9149-44e8-a9cf-a522d0b52c24","owner":[],"postedDate":"February 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":43825641,"name":"Physical sciences/Chemistry/Physical chemistry/Optical spectroscopy/Raman spectroscopy"},{"id":43825642,"name":"Physical sciences/Optics and photonics/Optical physics/Nonlinear optics"}],"tags":[],"updatedAt":"2026-03-25T07:06:37+00:00","versionOfRecord":{"articleIdentity":"rs-5650021","link":"https://doi.org/10.1038/s41467-026-69348-6","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-02-13 05:00:00","publishedOnDateReadable":"February 13th, 2026"},"versionCreatedAt":"2025-02-05 11:41:51","video":"","vorDoi":"10.1038/s41467-026-69348-6","vorDoiUrl":"https://doi.org/10.1038/s41467-026-69348-6","workflowStages":[]},"version":"v1","identity":"rs-5650021","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5650021","identity":"rs-5650021","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}