Fluorescence-free single-molecule microscopy via electronic resonance stimulated Raman scattering

Domain: neuroscience
Published: 2026-02-13
Editorial outcome: minor_revision

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 often require elaborate sample preparation or sometimes generate background signals when unintended processes lead to fluorescence emission. 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 provides single-molecule sensitivity without fluorescence detection, enabling applications in biological and chemical imaging, particularly in multiplexed imaging.

Introduction
Fluorescence microscopy has revolutionized biology with its unmatched molecular specificity and single-molecule sensitivity1–4. Single-molecule fluorescence microscopy has unveiled the individual trajectories of biological molecules, revealing the molecular mechanisms5,6. Fluorescence detection has driven advancements in cutting-edge technologies such as massively parallel DNA sequencing7 and super-resolution optical microscopy8–11. In recent years, fluorescence microscopy evolved to highly multiplexed imaging, opening new fields of spatial genomics, transcriptomics and proteomics6,12,13. However, fluorescence-based multiplexed imaging relies on iterative cycles of labeling, imaging and washing for each molecule target due to the broadbandwidth (1500 cm-1) of excitation and emission that limit its ability to spectrally distinguish different molecules14.

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 107 using plasmonic nanoparticles, enabling single-molecule detection15–17. However, the preparation of SERS substrates can be complicated and requires precise control of nanoparticle fabrication and probe positioning for consistent results18,19.

Electronic pre-resonance stimulated Raman scattering (EPR-SRS, Fig. 1A) enhances Raman sensitivity by up to 1011 compared to spontaneous Raman without near-field effects20. However, it still requires fluorescence detection, such as stimulated Raman excited fluorescence21 (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 yields22. However, high quantum yields lead to high background, including anti-Stokes fluorescence23 from thermally excited populations, limiting the signal-to-background (S/B) ratios and practicality. Bond-selective fluorescence-detected infrared-excited spectro-microscopy24 (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 substraction24.

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 sources25. 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 SREF20,21,23. 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. 1C-E). The selected wavelengths meet these conditions: (1) ωp - ωs = ωv; (2) |ωp - ωabs,max| < 700 cm−1 for ER-SRS25; (3) 1400 cm−1 < |ωp - ωabs,max| < 4200 cm−1 for EPR-SRS20, where ωp and ωs are pump and Stokes frequencies; ωabs,max and ωv are the frequencies at the absorption max and Raman shift of the probe, respectively. For Rho800, ωv = 2229.6 cm−1 (Supplementary Fig. 2A). Using our synchronized, yet independently tunable double OPO system with a tuning range of 660–960 nm, we optimized pump/Stokes combinations to maximize the S/B ratio (peak height/background). The pump wavelength was tuned around the absorption max (λmax = 700 nm) while adjusting the Stokes wavelength to achieve vibrational resonance (ωp - ωs = ωv). When the pump matched λmax (black line, Fig. 1C), an inverted Raman peak was observed (black spectrum, Fig. 1D), consistent with prior findings for SiR845 fluorophore25. At λpump = 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, 1031.2 nm) (Supplementary Fig. 2B), as previously demonstrated with conventional OPO20. However, significant background at λpump = 716 nm led to a low S/B ratio. Deviating λpump from λmax by >50 nm significantly improved the S/B ratio (Fig. 1E). For highly fluorescent probe like Rho800, ER-SRS failed to enhance the S/B ratio due to strong background from fluorescence and pump-probe processes20, 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. 2A). The series–RANMP, RANMP-4F, RANMP-4Cl, and RANMP-T26–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 procedure26 (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 λmax 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 S0 → S1 transition (Supplementary Fig. 6A). 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 exhibits 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 (kISC) and radiative decay rate constants (kr) of the RANMP series and Rho800 were calculated (Supplementary Note 1)27. As shown in Table SN2 of Supplementary Note 1, the kISC/kr ratio of the RANMP series (ranging from 102 to 103) 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 S1 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 (ΔEST). The 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 therapy28,29. For the RANMP series, the SOC constants are found to be large due to sulfur atoms (heavy atom effects) (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. 2B). When Stokes preceded pump (blue line, Fig. 2C), 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. 2C), 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 literature25 (Fig. 2D). 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. 2E, 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, the S/B values ranged from 0.1 to 1.2 (Fig. 2E), comparable to or exceeding those reported for SREF and BonFIRE. Also, ER-SRS signals showed 200-400x amplification compared to EPR-SRS of Rho800 [(pump, Stokes)=(838.4 nm, 1031.2 nm)] (Fig. 2F). ER-SRS spectra of RANMP and RANMP-T solutions in ortho-dichlorobenzene (o-DCB) under optimized conditions yielded S/B ratios of 0.1 and 0.8, respectively. These spectra showed approximately 577-fold and 1152-fold signal amplification compared to EPR-SRS of Rho800 for RANMP and RANMP-T solutions, respectively (Supplementary Figs. 9E–H). These results align with the theoretical predictions (10-100x amplification), given the four CN groups per RANMP molecule versus one CN in Rho80025.

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 quenching30. 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. 2F), were fabricated into Pdots (Fig. 3A, Supplementary Fig. 10). Using independent wavelength tuning, we optimized the ER-SRS S/B ratios, achieving maxima at λmax +37 nm for RANMP-T and λmax +20 nm for RANMP-4Cl (Fig. 3B, C, Supplementary Fig. 11C). Under the optimized conditions, the S/B ratios were 1.2 and 0.9 for RANMP-4Cl and RANMP-T Pdots, respectively. The sharp signal enhancement observed at λmax +77 nm (λpump = 784.4 nm) is attributed to the proximity of the pump wavelength to the second absorption maximum of the RANMP-T Pdot (778.2 nm), enhancing electronic resonance effect.

Under the optimum ER-SRS conditions, we achieved dual-color single-particle imaging using the narrow linewidth of Raman scattering (Fig. 3D-G). In a mixture of RANMP-T and RANMP-4Cl Pdots, indistinguishable particles in the bright field image became spectrally distinct at 2218.4 cm−1 (RANMP-T) and 2235.4 cm−1 (RANMP-4Cl) (Fig. 3D). 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 378 nm and particle size (Fig. 3F, G, Supplementary Fig. 10B). Each 5 µm x 5 µm field of view (FOV) was acquired in ~62 s, enabling two-color imaging under 3 min 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. 3E) 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 nM20, we detected signals at 1 nM for RANMP-T (Figs. 4A and 4B), equivalent to ~6 molecules in the laser focal volume. To minimize bleaching, we employed a polyvinyl alcohol (PVA)31 layer above and below the PMMA (Fig. 4C and Supplementary Fig. 12A) and applied a minimal pump power of 300 µW at optimized wavelengths (Supplementary Fig. 12B). Under these conditions, we performed single-molecule imaging of RANMP-T (1 nM) in a PMMA matrix and successfully obtained single-molecule images and single-step photobleaching traces (Fig. 4D, E and Supplementary Fig. 12E). Orange and blue arrows in Fig. 4D correspond to photobleaching of M1 and M2 molecules, where abrupt photobleaching events in the time trace confirm their single-molecule nature. We then compared traces from a sample with no PVA layer (Supplementary Fig. 12E-G) to those from a PVA-PMMA-PVA configuration (Fig. 4C). Both samples exhibited clear single-step bleaching events and show diverse range of bleaching times (Fig. 4E and Supplementary Fig. 12E). For quantitative analysis, we constructed histograms of photobleaching durations and amplitude (Fig. 4F, G and Supplementary Fig. 12F, G). The distributions of photobleaching time and signal amplitude were well-fitted to single exponential32 and log-normal functions33, respectively, consistent with single-molecule behavior. The extracted time constants (τ) were 42.2 ms for the sample without PVA (n=63) and 410.3 ms for the PVA-PMMA-PVA configuration (n=32), indicating an nearly 10-fold increase in photobleaching lifetime—consistent to previous single-molecule studies that used PVA to suppress photobleaching34.

Single-molecule behaviors were also indicated in multi-step photobleaching event (Supplementary Fig. 12C) obtained at a concentration of 10 nM, 10 times more concentrated than our single step photobleaching results in Fig. 4. The multi step trace is consistent with multiple molecules within a focal spot where discrete emitters bleach in steps—demonstrating the sensitivity of our method to resolve individual bleaching events. Also, supplementary Fig. 12D presents a half-moon photobleaching pattern in an image constructed by raster scanning, which serves as a signature of single-molecule detection.

ER-SRS specificity was verified by collecting single-molecule images at two temporal overlaps of pump and Stokes pulses (Fig. 4H). A molecular signal was observed when two pulses arrived concurrently (left 0 ps, Fig. 4H). When the Stokes pulse preceded the pump only by 1 ps (+1 ps, Fig. 4H), the signal sharply attenuated, consistent with the bulk time-dependent measurements in Fig. 2C. Reverting the delay to 0 ps restored the signal (right 0 ps, Fig. 4H), confirming that the signal reduction was due to the short vibrational lifetime of nitrile groups (<1 ps)35.

To confirm vibrational resonance, we analyzed signal variations with pump-Stokes frequency differences (Fig. 4I). Signals appeared at the CN stretch mode (2218.2 cm⁻¹) but vanished when the pump wavelength tuned by 1 nm to a frequency difference of 2236.3 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 Fig. 4D, H and I as single-molecule ER-SRS images.

Methods
Independently tunable double OPO system
The ER-SRS microscope setup is described in Fig. 1B 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 1031.2 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. 1B 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 (PI Nano 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. Many spectra in this study were obtained by averaging multiple measured spectra (five times in most cases). The spectra in Fig. 1D, Fig. 2E, Fig. 3B, Fig. 3C, Fig. 4B, Figure S9E and Figure S9F are presented with the mean± standard deviation to indicate measurement variance. We did not use any spectral fitting to our spectra except for Fig. 4A, but used spline interpolation without any additional data processing. Figure 4A was the only spectrum that we fitted with a Voigt function50. For single-molecule imaging, we used a 1 ms exposure time per pixel to balance signal strength and imaging speed. This allows for rapid acquisition while minimizing sample drift and photodamage, making it suitable for sensitive or time-limited samples. For photobleaching analysis (Fig. 4E-G and Supplementary Fig. 12E-G), only traces lasting longer than 30 ms were included.

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, Mn ≈ 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 N2 bubbling. The concentrated Pdots 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 completed dissolution. Subsequently, a glass slide was heated on a hot plate to 95°C. Then, 10 µL of the Pdots 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 Pdots sample. The same procedure was performed to prepare the single particle imaging sample. Before adding the agarose gel, 5 µL of the Pdots solution of RANMP-4Cl and 5 µL of the Pdots 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 s. Using a 1% w/w poly(methyl methacrylate) (PMMA)/CF solution, 1 nM 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 s.

Material characterization
The 1H 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 were 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 procedure26 (Supplementary Figs. 3, 15-18). RANMP, RANMP-4F, and RANMP-4Cl were synthesized via Vilsmeier–Haack formylation of a bis(alkylsulfanyl)methylene-substituted IDT core, followed by Knoevenagel condensation with (halogenated) INCN. In contrast, RANMP-T was prepared through stannylation of the bis(alkylsulfanyl)methylene-substituted IDT core, subsequent Stille coupling with an octyl-substituted thiophene spacer, and a final Knoevenagel condensation with INCN.

RANMP 1H NMR (500 MHz, CDCl3): 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 1H NMR (500 MHz, CDCl3): 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 1H NMR (500 MHz, CDCl3): 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 1H NMR (500 MHz, CDCl3): 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 S0, frontier molecular orbitals (HOMO and LUMO), and natural transition orbitals (HONTO and LUNTO) of the molecules in chloroform (Supplementary Figs. 5-6). DFT calculations were performed on simplified structures in which the bulky alkyl chains (R1 and R2 in Fig. 2A) were shortened to methyl groups, retaining only the planar conjugated core. As a result, the calculations may overestimate molecular planarity and packing propensity. The full RANMP molecules contain bulky alkyl chains that can sterically hinder π–π stacking (Supplementary Fig. 6B). The optimized structures in S1 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 (kISC) and radiative decay rate constants (kr) 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.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Report:\nIn this manuscript, S. Oh reports the detection of a single polyatomic molecule based\non the contrast from electronic resonance stimulated Raman scattering microscopy\n(ER-SRS). The key innovation for this work is to use two independently tunable\nOPOs, pumped by the same fundamental laser, to optimize excitation conditions\nwith best achievable signal-to-background ratios (S/B). Through coupling with\nmolecules that feature low fluorescence quantum yield with high inter-system\ncrossing rate, electronic background might be reduced. While the ER-SRS\nspectroscopy was previously reported (J. Phys. Chem. B, 2018) with more than 10-\ntime higher Raman cross-sections compared to the electronic pre-resonance SRS\nmicroscopy (EPR-SRS, 2017, Nature), single molecule detection without\nfluorescence readout has not been been demonstrated. The authors claimed that\nthis new setup, in combination with the selected molecules, enhances sensitivity by\napproximately 100-fold compared to EPR-SRS and achieved the first single-\nmolecule Raman detection without fluorescence detection and plasmon\nenhancement. It is great to see efforts toward this challenging goal.\n\nHowever, the data quality and presentation raise significant concerns for many\nclaims throughout the manuscript, some of which may be overstated. In particular,\nthe evidence for single-molecule detection is unconvincing. It remains very unclear\nwhether the authors were indeed observing single molecules, based on the data\npresented. Given that single-molecule detection is central to this work, much more\nrigorous experiments are required to substantiate this claim.\n\nFurthermore, the application scope seems quite limited here due to high background,\nand the reliance on specific molecular structures, which are prone to aggregation.\nFor broader utility, it is essential to clarify what novel and unique measurements this\ntechnique enables that are not achievable with existing methods. Are the\nmeasurements compatible with biological cell imaging as the authors outline in the\noutlook part? Maybe some preliminary data would be more convincing.\n\nBelow are my major comments:\n1. One of the key advantages of the EPR-SRS microscopy is the achieved high\nS/B while significantly enhancing Raman imaging sensitivity. This capability\nhas the potential to enable a wide range of applications, including highly\nmultiplexed imaging in tissues and live biological cells. In the introduction, the\nauthors strongly criticized about the potential background issues for SREF\nand BonFIRE, both of which are fluorescence-detected vibrational excitation\nschemes. For instance, in the abstract, they indicated that:“However, these\nmethods involve complex sample preparation or produce high backgrounds,\nlimiting their practicality. However, some of these claims are not entirely\naccurate. For example, the authors discussed the limit of NIR dyes for\nBonFIRE. However, possibility of visible (VIS)-absorbing dyes has also been\ndemonstrated. Nonetheless, given the emphasis on background suppression,\none would expect ER-SRS to achieve significantly improved S/B ratios. The\nauthors mentioned in multiple locations that they achieved optimal S/B, yet no\nspecific values are provided. For the best-performing molecule, RANMP-T,\nthe estimated S/B seems to be only around 0.2-0.3. Some other molecules\nare even worse. This background would require even more complicated\ninstrumentation to remove the background, which in turn significantly limits\nthe method’s practicality for general imaging applications.\n\n2. Related to this comment, equally importantly, what is the signal to noise (S/B)\nratio? Here the detector shot noise appears to be less a concern, but\nbackground fluctuation seems to be the dominant limiting factor. All spectra\nshown only have single point measurements. No statistical repeats were\nshown to indicate measurement variance. The severe background\nfluctuations observed in the data appear to significantly constrain S/N and,\nconsequently, the actual detectability of the signal. The authors successfully\nfit the measured curves based on the prior knowledge about the spectral\nshape. However, a key question remains: what would be the typical fitting\nconfidence if no prior information were available? Specifically, in a scenario\nwhere the resonance frequency, linewidth, and other parameters are not\npredetermined—closer to real conditions in unknown environments—how\nrobust would the signal extraction be? Addressing this issue would be crucial\nfor demonstrating the method’s practical applicability. In addition, it is also\nchallenging to evaluate how much measurement fluctuation contributed to the\nanalysis in pure SRG for computing the actual signal sizes.\n\n3. How is the pure SRG signal calculated? In Fig. 3B, the authors illustrated how\nthe peak height was determined. However, looking at Fig. S8A, I would not be\nable to obtain the same conclusion shown in (B). To me, the SRG is close\nbetween 1 mW and 2 mW, and then somehow there is a abrupt jump in the\nbackground (can the authors explain why?) for 5 mW data. The signals\nremain about the same across 5 mW to 15 mW data.\n\n4. Related, the background change seems very random. For example, in Fig.\n3B, +37 nm has ok background, but a decent peak; then +57 nm has a very\nsmall background, also very smaller peak. This is understandable. But at + 77\nnm, both the peak and background restored. Could the authors provide more\ninsights about the variations here?\n\n5. Excitation under the resonance condition would induce more severe\nphotobleaching. The authors showed longer survival time for RANMP-T\ncompared to Rho800 films, most obviously in two PVA layers. If this is the\nrequirement, it would make the general application very challenging. What\nwould the curve look like if there is no PVA layers at all, as in a more general\ncase? Additionally, why is photobleaching fitted as a double-exponential\ndecay? The authors emphasized that the key of achieving ER-SRS is to use\nlow quantum yield molecules with high intersystem crossing rate (ISC). In this\ncase, RANMP series are expected to have 100-1000 fold higher ISC rate\ncompared to Rh800. However, it is known that triplet state would lead to\nmuch quicker photobleaching, which suggest RANMP should fundamentally\nphoto-bleach much faster than Rh800 under ambient conditions.\n\n6. Additionally, how about photo-toxicity? Triplet state would evoke significant\ngeneration of singlet oxygen as the author also mentioned in the application\nof PDT, and further limits the general applicability especially in biological cells\nas an imaging agent.\n\n7. In this regard, are RANMP molecules compatible with water and hence\ncellular environment measurement? The structures of the RANMP should\nmake them highly prone for aggregation, which should be a severe issue in\naqueous environments.\n\n8. Related, in Fig. S4, the authors showed an about 50-100 nm bathochromic\nshift in film compared to the solution, which I assume is in DMSO. The\nsignificant redshift, accompanied by an increased shoulder peak, strongly\nsuggests J-aggregate formation, which is more pronounced in Pdot. The\nauthors reported enhancement fold based on film measurement in Fig. 2F,\nwhich raises the issue of quantification accuracy. Can the authors also show\ncomparison from solutions? Additionally, instead of normalized abs, maybe\nabsolute-intensity abs spectra would be more informative for quantitative\nunderstandings.\n\n9. In this regard, was the wavelength used for solution measurements\nsignificantly different from that in film? How does this change the\ndetectability?\n\n10. The authors corresponded their 1 nM solution detection to ~ 3 molecules?\nBased on their setup parameters (e.g. objective NA=1.2), single molecule\nequivalent should be well above 1 nM.\n\n11. Importantly, in “single-molecule” data, the signals vanished with temporal\ndelay of just 1 ps (Fig. 4E). However, the pulse width here is 2 ps, I would\nexpect delaying the pulse overlap by 1 ps, would only decrease the signal by\nmaybe 30%, rather than causing it to disappear entirely. Similarly, signals are\nnearly absent at 2236 cm-1 (Fig. 4E), whereas Fig. 4A suggests that\napproximately 30% of the signal should still be present. This again raises\nquestions about how true SRG signal is determined. The limited dynamic\nrange also raises a critical issue about confirming the single molecule identity\nin Fig. 4, which was purely based on signal-step photobleaching on ER-SRS\nsignal. While it is a standard procedure in fluorescence, the signal dynamic\nrange there is significantly higher. In addition, due to the aggregation-prone\nproperty of the RANMP, as evidence by absorption spectra and also the\nstructural basis, determining the detected spots are actual singal molecules,\nbut not molecular aggregates, is a key control experiment. More unequivocal\nevidence would be needed to substantiate this importance claim in this paper.\n\n12. Related, in Fig. 3D, in off-resonance channel at 2226 cm-1, both sets of dots\ndisappear. However, based on Fig. 3E, wouldn’t that at least 50% of signal\nremain for Ranmp-4Cl?\n\n13. The temporal profile in Fig. 2C looks very odd too. For true ER-SRS signals,\nthe temporal profile should be more symmetrical. This raises issues about the\nsignal origins. Is this the characterization of signal or background?\n\n14. It is also confusing that the authors first indicated a 4.26 ps lifetime of the\nsignal, but then indicated the “confirming that the signal reduction was due to\nthe 218 short vibrational lifetime of nitrile groups (<1 ps)”. How is the time-\ndelay measurement here probe any vibrational lifetime of nitrile?\n\n15. For the origin of optimal S/B in ER-SRS, the authors claim that the key was\nthe low quantum yield, so low electronic background. However, for ER-SRS\ninvestigations on Rho800, pump wavelengths were kept redder compared to\nthe absorption max of the dye. In RANMP, almost all optimal S/B were\nachieved by using Pump excitation bluer than the absorption maximum. SRS\nis immune to fluorescence – why is low quantum yield the key? Alternatively,\nis the shifted pump excitation the reason for improved S/B? Have the authors\nexplored Rho800 excitation at the blue absorption end?\n\n16. In film measurement, the concentrations also seemed extremely high. 5.8\nmg/ml or 10mg/ml would be estimated to about 5-10 mM in concentration?\nWould this number become even higher when films dry out? Since ER-SRS\noffers high sensitivity, why are such high conc. still needed for measurement?\nIt again raises significant concerns about severe aggregate formation.\n\n17. There is no discussion on acquisition time related to the detection sensitivity,\nespecially for single molecule detection, and also the concentration curve in\nFig. 4B. The only report about time was “5 μm x 5 μm FOV in 62 s”. However,\nthis depends on the pixel numbers. If 10-by-10 pixels, the pixel dwell time is\nabout 620 ms? This is extremely long compared to typical acquisition time\nused in EPR-SRS, SREF and BonFIRE.", "reviewer_2": "How to achieve the ultimate single-molecule sensitivity of Raman spectroscopy in the optical far-field (without near-field surface enhancement) has been a long sought-after goal in physical chemistry. Recent efforts of stimulated Raman excited fluorescence (SREF) have harnessed Raman-encoded fluorescence for this goal but are limited by the shortcoming of fluorescence requirement.\nThis new work shows the fundamental (more basic) stimulated Raman scattering spectroscopy, without the help of fluorescence detection, itself is able to achieve this sensitivity for specially designed molecular systems. Raman-amplified nonfluorescent molecular probe (RANMP) is a clever chemistry design. The quantum calculation agrees with experimental measurement, supporting the mechanism. The spectroscopy and imaging experiments are well presented. While RANMP might need some effort to make (which is comparable to sophisticated fluorescence sensors), I expect its utility in broad sensing and imaging applications. This study sends an important and strong message to the community. It shall inspire many new works in this field.", "reviewer_3": "Oh and coworkers report on enhanced sensitivity in electronically resonant SRS imaging using a new set of designer Raman probes and an experimental system based on two synchronized OPOs. The main finding is the achievemnt (for the first time) of single molecule sensitivity in SRS microscopy without resorting to SERS.\nOverall this manuscript is excellent. The study is detailed, provides all the necessary background and convincing findings. It can be accepted to Nature Communications provided that the authors address the following (rather minor) comments:\n1. With an atomic number of 16, Sulfur is not really a heavy atom.\n2. Integration times (per pixel) for the measurements are stated in several places, but it is important to provide in the discussion details about the practicality of this method in terms of imaging (what can be achieved with pixel dwell times of up to ~1ms) and single molecule spectroscopy (same, but on a time scale of 100s of ms).\n3. Photobleaching is compelling evidence that the experiment detected single molecules but not an ultimate proof. The authors should take note of that."}, "author_response": "Point-by-point Responses to Reviewer's Comments\nManuscript #: NCOMMS-25-04102-T\nTitle: “Fluorescence-free single-molecule microscopy via electronic resonance stimulated Raman\nscattering”\nAuthors: Sumin Oh, Yunji Eom, Ha Yeon Kim, Ayushi Tripathi, Sungnam Park, Han Young Woo,\nSang-Hee Shim\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nReport:\nIn this manuscript, S. Oh reports the detection of a single polyatomic molecule based on\nthe contrast from electronic resonance stimulated Raman scattering microscopy (ER-\nSRS). The key innovation for this work is to use two independently tunable OPOs,\npumped by the same fundamental laser, to optimize excitation conditions with best\nachievable signal-to-background ratios (S/B). Through coupling with molecules that\nfeature low fluorescence quantum yield with high inter-system crossing rate, electronic\nbackground might be reduced. While the ER-SRS spectroscopy was previously reported\n(J. Phys. Chem. B, 2018) with more than 10- time higher Raman cross-sections\ncompared to the electronic pre-resonance SRS microscopy (EPR-SRS, 2017, Nature),\nsingle molecule detection without fluorescence readout has not been been demonstrated.\nThe authors claimed that this new setup, in combination with the selected molecules,\nenhances sensitivity by approximately 100-fold compared to EPR-SRS and achieved the\nfirst single- molecule Raman detection without fluorescence detection and plasmon\nenhancement. It is great to see efforts toward this challenging goal.\nWe thank the reviewer for recognizing the importance of our work.\nHowever, the data quality and presentation raise significant concerns for many claims\nthroughout the manuscript, some of which may be overstated. In particular, the evidence\nfor single-molecule detection is unconvincing. It remains very unclear whether the authors\nwere indeed observing single molecules, based on the data presented. Given that single-\nmolecule detection is central to this work, much more rigorous experiments are required\nto substantiate this claim.\nWe thank the reviewer for raising this important point. We fully acknowledge that single-\nmolecule detection is central to the work.\nUnlike previous single-molecule vibrational fluorescence studies such as SREF and\nBonFIRE—where one-photon fluorescence provides a well-established means of directly\nidentifying individual fluorescent molecules—our probes are nonfluorescent. This\nlimitation compels us to seek other characteristic signatures to support the single-\nmolecule interpretation. Single-step photobleaching is commonly used as a hallmark of\nsingle-molecule detection in nonfluorescent single-molecule studies [A. Gaiduk et al,\nScience 330, 353-356 (2010)]. As Reviewer #3 noted, “photobleaching is compelling\nevidence that the experiment detected single molecules.”\nof single RANMP-T molecules embedded in PMMA for statistical analysis of single-\nmolecule intensities and photobleaching kinetics (Fig. R1). Specifically, we analyzed 63\ntraces from PMMA without any PVA and 32 traces from a PVA-PMMA-PVA configuration,\nin which PVA layers were placed above and below the PMMA sample (Figure R1A). As\nshown in Fig. R1B, both configurations exhibited a broad distribution of photobleaching\ntimes.\nFor quantitative analysis, we constructed histograms of photobleaching durations and\nfitted them using a single-exponential decay model (Fig. R1C), following approaches\nused in many single-molecule fluorescence studies [Belyy, V. et al, Nat. Commun 8,\n13978 (2017); Wennmalm et al, J. Phys. Chem. B 103, 2516-2519 (1999)]. The extracted\ntime constants () were 42.2ms for the sample without PVA (n=63) and 410.3ms for the\nsample with two PVA layers (n=32), indicating nearly a 10-fold increase in photobleaching\nlifetime. This enhancement demonstrates that PVA layers markedly improve\nphotostability, thereby facilitating effective single-molecule imaging as in previous single-\nmolecule studies [Gensch, T. et al, J. Phys. Chem. A 109, 6652-6658 (2005); Liu, Q. et\nal, J. Phys. Chem. A 124, 2497-2504 (2020)]. All single-molecule data shown in Figs. 4D-\nI of the manuscript were acquired using the PVA-sandwich configuration. Without PVA,\nmost signals bleached within 100ms, making single-molecule detection difficult. We also\nanalyzed the signal amplitude of each time trace (Fig. R1D) and found that the distribution\nfollows a log-normal profile, consistent with previously published reports on single-\nmolecule fluorescence intensities [Wang, X. et al, Int. J. Mol. Sci. 20, 1410 (2019); Mutch\net al, Biophys. J. 92, 2926 (2007)]. All experiments were conducted with a 1ms-exposure\ntime, matching the imaging condition used in the manuscript. Only traces lasting longer\nthan 30ms were included in the analysis. We added the results and discussions in the\nmanuscript as follows:\n“Under these conditions, we performed single-molecule imaging of RANMP-T (1 nM) in\na PMMA matrix and successfully obtained single-molecule images and single-step\nphotobleaching traces (Figs. 4D-E and Supplementary Fig. 12E). Orange and blue\narrows in Fig. 4D correspond to photobleaching of M1 and M2 molecules, where abrupt\nphotobleaching events in the time trace confirm their single-molecule nature. We then\ncompared traces from a sample with no PVA layer (Supplementary Figs. 12E-G) to those\nfrom a PVA-PMMA-PVA configuration (Fig. 4C). Both samples exhibited clear single-step\nbleaching events and show diverse range of bleaching times (Fig. 4E and Supplementary\nFig. 12E). For quantitative analysis, we constructed histograms of photobleaching\ndurations and amplitude (Figs. 4F-G and Supplementary 12F-G). The distributions of\nphotobleaching time and signal amplitude were well-fitted to single exponential32 and log-\nnormal functions33, respectively, consistent with single-molecule behavior. The extracted\ntime constants () were 42.2 ms for the sample without PVA (n=63) and 410.3 ms for the\nPVA-PMMA-PVA configuration (n=32), indicating a nearly 10-fold increase in\nphotobleaching lifetime—consistent to previous single-molecule studies that used PVA\nto suppress photobleaching.34”\nFurthermore, we provided additional supporting data in Fig. S12C and S12D (as shown\nbelow). Fig. S12C shows a representative multi-step photobleaching trace obtained at a\nconcentration of 10 nM—ten times higher than that used for the single-step\nphotobleaching results in Fig. 4. The discrete steps in the trace are consistent with the\npresence of multiple molecules within a focal spot, each bleaching independently. This\n2\nhighlights the sensitivity of our method to resolve individual bleaching events. Fig. S12D\npresents a half-moon photobleaching pattern in an image constructed by raster scanning.\nThis pattern, reported in the first SREF study [Xiong, H. et al, Nat. Photonics 13, 412-417\n(2019)], is considered a hallmark of single-molecule distribution. The combination of half-\nmoon pattern and single-step bleaching has been proposed as strong evidence for single-\nmolecule detection. We included the related discussion in the manuscript as follows:\n“Single-molecule behaviors were also indicated in multi-step photobleaching event\n(Supplementary Fig. 12C) obtained at a concentration of 10 nM, 10 times more\nconcentrated than our single step photobleaching results in Fig. 4. The multistep trace is\nconsistent with multiple molecules within a focal spot where discrete emitters bleach in\nsteps—demonstrating the sensitivity of our method to resolve individual bleaching\nevents. Also, supplementary Fig. 12D presents a half-moon photobleaching pattern in an\nimage constructed by raster scanning, which serves as a signature of single-molecule\ndetection.”\nWe sincerely thank the reviewer for raising this point, which motivated us to acquire\nadditional data that substantially reinforce our conclusions. The new results are now\nincluded in Fig. R1, as well as in Figs. 4E-F and Figs. S12C-G.\nPVA layer (left panels) and PVA-PMMA-PVA sample (right panels). (A) Sample\ngeometries of RANMP-T samples for ER-SRS single-molecule detection. (B)\nRepresentative traces with single-step photobleaching. (C) Histograms of photobleaching\ntimes based on 63 molecules from no PVA sample (left) and 32 molecules from double\nPVA layer sample (right). The decay constant () was determined from a single-\nexponential fitting to the histogram (red line). (D) Histograms of single-molecule ER-SRS\namplitudes. Each amplitude value per trace was calculated by “(Average intensity before\n4\nphotobleaching) – (Average intensity after photobleaching)”. The red line represents a\nlog-normal fit to the amplitude distribution. Exposure time: 1ms. Only traces lasting\nlonger than 30ms were included in the histograms.\nFigure S12. (C) Image of molecules and their multi-step photobleaching trace (red\narrow) obtained at a concentration of 10 nM. Powers were set as P =500 W, P =6\npump Stokes\nmW. (D) Half-moon photobleaching pattern indicative of single-molecule distribution.\nFurthermore, the application scope seems quite limited here due to high background, and\nthe reliance on specific molecular structures, which are prone to aggregation. For\nbroader utility, it is essential to clarify what novel and unique measurements this\ntechnique enables that are not achievable with existing methods. Are the\nmeasurements compatible with biological cell imaging as the authors outline in the\noutlook part? Maybe some preliminary data would be more convincing.\nWe thank the reviewer for raising this important point regarding the broad applicability of\nour method. As the reviewer noted, our long-term aim is to enable multiplexed biological\nimaging. As we detail in the point #1 below, the signal-to-background level of ER-SRS\n(Fig.2F and Fig.S9H) is comparable to that of SREF and BonFIRE, both of which have\nbeen successfully applied to biological systems [Shi et al, Nat Comm 10:4764 (2019);\nWang et al, Nat Photon 17:846-855 (2023)].\nThe Pdot nanoparticles in Fig. 3 are compatible with immunostaining protocols, as\ndemonstrated in our previous study [Jeong et al, ACS Nano 17: 4800-4812 (2023)].\nFurthermore, the incorporation of organelle-targeting moieties—such as cationic groups\nfor mitochondria or basic groups for lysosomes— into the RANMP framework holds\nstrong potential to transform these hydrophobic probes into lipophilic, organelle-specific\nmarkers [Yang, Bai & Hu, Nat Comm 15: 2578 (2024)]. Although we were unable to\ninclude preliminary biological data within the limited timeframe for revision, we are\nactively pursuing these directions in ongoing works.\nRegarding the concern about molecular aggregation, we note that the RANMP molecules\nemployed in our study were deliberately designed with bulky alkyl substituents to\nsterically hinder close molecular packing (Fig. S6B; see our response to the reviewer\ncomment #8 below for details). Thus, we believe that aggregation does not pose a\nlimitation in our current system.\nbulky alkyl chain (green; R and R in Fig. 2A) in the side view. DFT calculation (B3LYP/6-\n1 2\n31G(d,p)) was used to obtain the optimized molecular structure in S .\nReviewer #2’s independent and positive assessment of RANMP:\n“While RANMP might need some effort to make (which is comparable to sophisticated\nfluorescence sensors), I expect its utility in broad sensing and imaging applications.”\nWe share this perspective and believe that applying well-established strategies from the\nfluorescence imaging field to RANMP will open new opportunities for multiplexed imaging\nbeyond the reach of current fluorescence-based methods. We added the perspective at\nDiscussion section in the manuscript as follows:\n“Many strategies used in fluorescence sensors can be applied to RANMP. For instance,\nthe incorporation of organelle-targeting moieties—such as cationic groups for\nmitochondria or basic groups for lysosomes— into the RANMP framework holds strong\npotential to convert these hydrophobic probes into lipophilic, organelle-specific\nmarkers42. Additionally, introducing hydrophilic functional groups, such as phosphates,\ncould broaden the applicability of RANMP probes by improving their solubility. While the\nplanar core of RANMP facilitates π–π stacking and potential aggregation, the bulky\nalkyl chains (R and R in Fig. 2A) can be strategically modified to further suppress\n1 2\nundesired stacking. (Supplementary Fig. 6B).”\n“The development of multiplexed RANMP probes with single-molecule sensitivity may\nunlock new possibilities for spectral identification of molecular targets in spatial\nomics46,47 and super-resolution microscopy48,49. The PSMA-encapsulated Pdot in this\nstudy also offers a convenient, general route for boosting the S/B of ER-SRS probes,\nfor biocompatibility and live-cell applications and for efficient targeting in biological\nsamples by using the carboxylic groups on the particle surface30.”\nBelow are my major comments:\n1. One of the key advantages of the EPR-SRS microscopy is the achieved high S/B\nwhile significantly enhancing Raman imaging sensitivity. This capability has the\npotential to enable a wide range of applications, including highly multiplexed imaging in\ntissues and live biological cells. In the introduction, the authors strongly criticized about\nthe potential background issues for SREF and BonFIRE, both of which are\n6\nfluorescence-detected vibrational excitation schemes. For instance, in the abstract, they\nindicated that:“However, these methods involve complex sample preparation or\nproduce high backgrounds, limiting their practicality. However, some of these claims are\nnot entirely accurate. For example, the authors discussed the limit of NIR dyes for\nBonFIRE. However, possibility of visible (VIS)-absorbing dyes has also been\ndemonstrated. Nonetheless, given the emphasis on background suppression, one\nwould expect ER-SRS to achieve significantly improved S/B ratios. The authors\nmentioned in multiple locations that they achieved optimal S/B, yet no specific values\nare provided. For the best-performing molecule, RANMP-T, the estimated S/B\nseems to be only around 0.2-0.3. Some other molecules are even worse. This\nbackground would require even more complicated instrumentation to remove the\nbackground, which in turn significantly limits the method’s practicality for general\nimaging applications.\nWe sincerely thank the reviewer for the insightful comment regarding the Introduction\nsection of our manuscript. In response, we have revised the sentence in the abstract\n“However, these methods involve complex sample preparation or produce high\nbackgrounds, limiting their practicality” to “However, these methods often require\nelaborate sample preparation or sometimes generate background signals when\nunintended processes lead to fluorescence emission.”, to tone down the statement.\nFurthermore, we appreciate the reviewer’s suggestion on quantifying the S/B ratio of ER-\nSRS signals. We fully acknowledge that ER-SRS also suffers from background stemming\nfrom high electronic resonance and therefore motivate us to optimize S/B ratio under\nvarious wavelengths with our independently tunable SRS instrument.\nWhile it is true that ER-SRS in the film state (Fig. 2E; see below) generally exhibits S/B\nratio ranging from 0.1 to 1.2, we would like to highlight that RANMP-T in the PVA-PMMA-\nPVA film of 10 nM (red line in Fig. S12B; see below) achieved an S/B ratio of\napproximately 0.6, serving as a key motivation for pursuing single-molecule detection\nusing ER-SRS. Also, the new solution data set, which was motivated by the reviewer\ncomment #8, exhibited S/B of 0.8 for RANMP-T and 0.2 for RANMP (Fig. S9F). In Pdot\nsamples where aggregation-induced quenching could further suppress the background,\nS/B ratios were 1.2 and 0.9 for RANMP-4Cl and RANMP-T, respectively (Fig. 3C and Fig.\nS11C).\nFor comparison, S/B ratio of SREF ranges from 0.3 to 0.5 depending on the isotope, and\nthat of BonFIRE ranges from 0.08 to 0.6 depending on the solvent. ER-SRS\ndemonstrates a comparable S/B ratio (0.1–1.2), indicating that its performance is not\nparticularly inferior to these existing methods and sometimes better (e.g.1.2 in RANMP-\n4Cl film, 0.8 in RANMP-T solution, 1.2 and 0.9 in Pdots with RANMP-4Cl and RANMP-\nT, respectively).\nWe added the related details in the manuscript as follows:\n“At the optimized conditions, the S/B values ranged from 0.1 to 1.2 (Fig. 2E), comparable\nto or exceeding those reported for SREF and BonFIRE. Also, ER-SRS signals showed\n200-400x amplification compared to EPR-SRS of Rho800 [(pump, Stokes) = (838.4 nm,\n7\n1,031.2 nm)] (Fig. 2F). ER-SRS spectra of RANMP and RANMP-T solutions in ortho-\ndichlorobenzene (o-DCB) under optimized conditions yielded S/B ratios of 0.1 and 0.8,\nrespectively. These spectra showed approximately 577-fold and 1,152-fold signal\namplification compared to EPR-SRS of Rho800 for RANMP and RANMP-T solutions,\nrespectively (Supplementary Figs. 9E–H).”\n“Under the optimized conditions, the S/B ratios were 1.2 and 0.9 for RANMP-4Cl and\nRANMP-T Pdots, respectively.”\nFigure 2. (E) ER-SRS spectra of the four RANMPs at the optimal combination of pump\nand Stokes wavelengths. Lines indicate spline interpolation. (F) Summary of ER-SRS\nspectra at the optimal condition in E in terms of the absorption max ( ), wavelength\nmax\ndifference of the pump wavelength from the absorption max ( -  ), signal\nmax pump\nenhancement and S/B ratio in comparison to the EPR-SRS signal of Rho800 with fixed\nStokes wavelength at 1,031.2 nm as in previous studies.\nFigure S9. ER-SRS spectra of RANMP series in o-DCB solution: (E) RANMP, (F)\nRANMP-T. The SR signal of solution was measured under pump laser power at 200 µW,\n( + 21 nm), the solution of RANMP-T exhibited approximately a 1,152-fold signal\nmax\nenhancement compared to EPR-SRS of Rho800. Also, RANMP solution showed a 577-\nfold signal enhancement compared to Rho800 EPR-SRS under its optimal condition at\n( + 25 nm). (G) UV-vis absorption spectra of RANMP solution and RANMP-T solution\nmax\nin o-DCB. (H) Summary of ER-SRS spectra of RANMP and RANMP-T in film and solution\nstates.\nFigure S12. (B) Optimization of wavelength combinations for ER-SRS of RANMP-T\nembedded in PVA-PMMA-PVA film (10 nM) with powers set as P =500 W, P =6\npump Stokes\nmW. The maximum signal-to-background ratio was observed at  -40.9 nm, where\nmax\n =749 nm and  =897.9 nm.\npump Stokes\nThe background originates mainly from the pump beam because we measured\nstimulated Raman gain (SRG) of the Stokes beam while modulating the pump (Fig. 2C;\nsee below). We chose SRG because the Stokes wavelength range was better matched\nto the sensitivity of the diode detector. We attribute the primary source of background to\nstimulated emission where the pump promotes molecules into the excited state and the\nStokes beam induces stimulated emission from those excited molecules. Since the\nstimulated emission signal is included in the modulation-transferred Stokes signal, it\ninherently leads to significant background. To further address the issue of background\narising from the pump, we are now actively considering transitioning to a stimulated\nRaman loss (SRL) detection scheme in future studies.\nby the Gaussian function when the Stokes pulse precedes the pump. Red line is a fit to\nthe trace by the exponential function when pump pulse precedes Stokes.\n2. Related to this comment, equally importantly, what is the signal to noise (S/B) ratio?\nHere the detector shot noise appears to be less a concern, but background fluctuation\nseems to be the dominant limiting factor. All spectra shown only have single point\nmeasurements. No statistical repeats were shown to indicate measurement variance.\nThe severe background fluctuations observed in the data appear to significantly constrain\nS/N and, consequently, the actual detectability of the signal. The authors successfully\nfit the measured curves based on the prior knowledge about the spectral shape. However,\na key question remains: what would be the typical fitting confidence if no prior information\nwere available? Specifically, in a scenario where the resonance frequency, linewidth, and\nother parameters are not predetermined—closer to real conditions in unknown\nenvironments—how robust would the signal extraction be? Addressing this issue would\nbe crucial for demonstrating the method’s practical applicability. In addition, it is also\nchallenging to evaluate how much measurement fluctuation contributed to the analysis in\npure SRG for computing the actual signal sizes.\nWe thank the reviewer for the comment. Many spectra in this study were obtained by\naveraging multiple measured spectra (five times in most cases). To make this clear, we\nnow present the mean ± standard deviation in the spectral data in Fig.1D, Fig.2E, Fig.3B,\nFig.4B, Fig. S9E and Fig. S9F (Fig. R2; see below) to indicate measurement variance.\nFig. S9E and Fig. S9F (Fig. R2; shown below) were newly acquired during revision and\nthe corresponding ER-SRS spectra were obtained in solution using ortho-\ndichlorobenzene (o-DCB) as the solvent at a concentration of 1 mM. We added these\ndetails at Methods section, specifically under “ER-SRS Microscopy”.\n1\nWe appreciate the reviewer for raising the concern about background fluctuations. In\nour ER-SRS measurements, we employed stimulated Raman gain detection, which is\ninherently susceptible to background signals arising from the pump, as discussed in our\nresponse to the reviewer comment #1. Therefore, the observed background fluctuations\n1\n1\nare likely due to the intensity fluctuation of the pump OPO. Moving forward, we are\noptimistic that our planned shift to SRL detection will help reduce these background\nfluctuations. We added related details in the Discussion section as follows:\n“The S/B ratios of solution, film and single-molecule samples could be further enhanced\nby adopting stimulated Raman loss (SRL) detection, which effectively suppresses the\npump-induced background that dominates in the current SRG scheme (red line, Fig. 2C).”\nIn addition, the reviewer raised concern regarding fitting the spectra with a prior\nknowledge. In fact, we did not perform any spectral fitting, except for Fig. 4A, where a\nVoigt profile was used to more clearly represent the peak shape at the low concentration\nof 1 nM [Shi, L. et al. Nat Commun 10, 764 (2019)]. For all the other data, we used\nsimple spline interpolation without any additional constraints or prior assumptions. No\nfurther data processing was applied. We apologize for not explicitly mentioning the details\nof how we generated spectra in the manuscript. Since it was based on a very simple\nmethod that is commonly used in SRS studies [Xu, Y. et al, Applied Spectroscopy 75(1),\n34-45 (2021); Shi, L. et al, Nat Biotechnol 40, 364-373 (2022); Shah, F.A. et al, Sci Rep\n13, 12301 (2023)], we initially believed it was not necessary to specify. However, we\nagree that this information should be included for clarity and reproducibility. We now\nexplicitly state the use of spline interpolation and use of no further analysis in the captions\nof Fig. 1D, 2E and 3B, as well as in the Methods section, specifically under “ER-SRS\nMicroscopy”, as follows:\n“We did not use any spectral fitting to our spectra except for Fig. 4A, but used spline\ninterpolation without any additional data processing. Fig. 4A was the only spectrum that\nwe fitted with a Voigt function50.”\nWe thank the reviewer for giving us an opportunity of highlighting the simplicity of our ER-\nSRS spectra.\n3. How is the pure SRG signal calculated? In Fig. 3B, the authors illustrated how the peak\nheight was determined. However, looking at Fig. S8A, I would not be able to obtain the\nsame conclusion shown in (B). To me, the SRG is close between 1 mW and 2 mW, and\nthen somehow there is a abrupt jump in the background (can the authors explain why?)\nfor 5 mW data. The signals remain about the same across 5 mW to 15 mW data.\nWe apologize for not explicitly describing the method used to calculate the pure SRG\nsignal in the manuscript. As stated in Fig. 3B (shown below), the pure SRG was\ncalculated as “peak height – background”. Our definition for pure SRG is in line with “pure\nSREF” in Xiong et al Nat Photon 2019 and “pure (BonFIRE) signal” in Wang et al Nat\nPhoton 2023. We will now explicitly state the definition of pure SRG as “peak height –\nbackground” in the revised manuscript, especially in the caption of Fig. 3B.\nIn Fig. S8A (shown below), at 1 mW Stokes power (blue line), the peak at 2214 cm⁻¹ is\noverwhelmed by the background, making it undetectable. Therefore, this data point was\nexcluded from the quantification in Fig. S8B and is not shown in the plot, which displays\ndata starting from 2 mW. As Stokes power increases, both the signal and background\ngenerally increase. The abrupt increase is observed when the power rises from 2 mW to\n1\n2\n5 mW results from a 2.5-fold increase in Stokes power, indicating a linear dependence of\nthe signal on Stokes power.\nThe reviewer noted that the signal appears to remain about the same across 5 mW to 15\nmW. However, we would like to clarify that the signal increases up to around 8 mW, after\nwhich it becomes saturated. Thus, the apparent plateau occurs in the range of 8 mW to\n15 mW, which we attribute to saturation effects at higher powers that suppress further\nsignal enhancement. In the updated version of the Fig. S8B (shown below), we updated\nthe x-axis to reflect the actual measured values (2, 5, 8, 10, and 15 mW) instead of the\nprevious fixed intervals (0, 5, 10, and 15 mW) to improve clarity.\nFigure 3. (B) ER-SRS spectra of RANMP-T under various wavelength combinations of\npump and Stokes for optimizing the S/B ratio. Powers are set as 1 and 8 mW for pump\nand Stokes beam, respectively. Pure SRG is calculated as “peak height – background”.\nLines indicate spline interpolation.\nFigure S8. Stokes power dependence of ER-SRS spectra of RANMP-T film (5.8 mg/mL,\n1\n(calculated as “peak height – background”) exhibits linear dependence up to 8 mW, with\nsaturation occurring beyond 10 mW.\n4. Related, the background change seems very random. For example, in Fig. 3B, +37 nm\nhas ok background, but a decent peak; then +57 nm has a very small background, also\nvery smaller peak. This is understandable. But at + 77 nm, both the peak and background\nrestored. Could the authors provide more insights about the variations here?\nWe thank the reviewer for the valuable comment. The sharp increase in signal\nobserved at +77 nm ( =784.4 nm) is attributed to the proximity of pump wavelength\npump\nto the second absorption maximum of the RANMP-T Pdot, which occurs at 778.2 nm in\nits UV-visible absorption spectrum (Fig. R3). Therefore, pump near second absorption\nmaximum enhances electronic resonance, thereby leading to a stronger signal at this\nwavelength. We have clarified this point in the revised manuscript to avoid confusion,\nsaying “The sharp signal enhancement observed at  + 77 nm ( =784.4 nm) is\nmax pump\nattributed to the proximity of the pump wavelength to the second absorption maximum\nof the RANMP-T Pdot (778.2 nm), enhancing electronic resonance effect.”.\nFigure R3. Sharp signal enhancement observed at +77 nm ( =784.4 nm), attributed\npump\nto the proximity to the second absorption maximum of RANMP-T Pdot (778.2 nm) in the\nUV-visible absorption spectrum. Enhanced electronic resonance near this absorption\npeak leads to stronger signal intensity.\n5. Excitation under the resonance condition would induce more severe photobleaching.\nThe authors showed longer survival time for RANMP-T compared to Rho800 films, most\nobviously in two PVA layers. If this is the requirement, it would make the general\napplication very challenging. What would the curve look like if there is no PVA layers at\nall, as in a more general case? Additionally, why is photobleaching fitted as a double-\nexponential decay? The authors emphasized that the key of achieving ER-SRS is to use\nlow quantum yield molecules with high intersystem crossing rate (ISC). In this case,\nRANMP series are expected to have 100-1000 fold higher ISC rate compared to Rh800.\nHowever, it is known that triplet state would lead to much quicker photobleaching,\nwhich suggest RANMP should fundamentally photo-bleach much faster than Rh800 under\nambient conditions.\n1\nthe use of PVA; however, the signal of Rho800 in this condition was too weak to be\nreliably measured, rendering a direct comparison meaningless (Fig. R4A). In the same\ncondition with no PVA layer, RANMP-T signal was reliably measured (Fig. R4B).\nHowever, due to the failure of detecting signal from Rho800, the corresponding data were\nnot included in supplementary figure.\nFig. S13 presents the photobleaching curve obtained from the film prepared from RANMP\ndissolved in chloroform that evaporates after spin coating. Inhomogeneous local\nenvironments of molecules in films lead to a multi-exponential decay of photophysical\nprocesses because molecules in different local environments decay differently.\nTherefore, the decay behavior cannot be adequately described by single-exponential\nfunction and instead follows a double-exponential decay. This mismatch between the\nsingle-exponential fitting and the actual data can be clearly observed in Fig. R4C below,\nwhere the fitting result does not adequately capture the decay behavior.\nIn contrast, the distribution of photobleaching times of single RANMP-T molecules in a\nPMMA matrix follow a single-exponential decay (Fig. R1C on page 3 of this letter; Figs.\n4F and S12F), reflecting the reduced variability in molecular interactions and\nphotophysical properties afforded by the homogeneous environment. During revision, we\nobtained this new data set of single-molecule ER-SRS traces with no PVA layer. Thus,\nin the revised manuscript, we replaced our previous single-molecule data on a sample\nwith a single PVA layer in the initial submission to the new no-PVA data (Figs. S12E-G).\nA B\nC\n)\n.\nu\n.\na\n(\ny\nt\nis\nn\ne\nt\nn\ni\nd\ne\nz\nila\nm\nr\no\nN\nFigure R4. Photobleaching curves of Rho800 and RANMP-T. (A) Raw signal of Rho800\nmeasured without a PVA layer at (pump, Stokes) = (749 nm, 899.2 nm). The signal\nchange was too weak to be reliably measured, preventing meaningful analysis. No\n1\n5\nfurther data processing was applied. (B) Raw signal of RANMP-T under the same no-\nPVA condition measured at (pump, Stokes) = (749 nm, 897.9 nm). The signal exhibited\nstable and detectable changes. No further data processing was applied. (C)\nPhotobleaching curve of RANMP-T without a PVA layer (blue) fitted with a single-\nexponential decay function (red).\nAs the reviewer correctly pointed out, photobleaching is closely associated with the\ntriplet state. According to our theoretical calculations, k /k ratios for RANMP series\nISC r\nare found to be much larger than that of Rho800, indicating that intersystem crossing\n(ISC) is much faster than radiative decay from S1 state and the fluorescence quantum\nyields of RANMP series are negligibly small. In addition, phosphorescence of RANMP\nseries was not significantly measurable, which leads to conclude that triplet states of\nRANMP series have short triplet state lifetimes, likely due to nonradiative processes\nincluding thermal relaxation (i.e., intersystem crossing from T to S state).\nThe\n1 0\nextremely short triplet lifetime also makes RANMP-T highly susceptible to thermal\nquenching, which in turn can slow down photobleaching. This point is also discussed in\nthe Discussion section in the manuscript:\n“Our attempts to measure the triplet-state lifetime of RANMP-T film were unsuccessful,\nas no phosphorescence signal was detectable (data not shown), suggesting an\nextremely short triplet-state lifetime. This implies that the triplet state of RANMP, likely\nin the NIR region, is highly susceptible to thermal quenching. Efficient triplet-state\nquenching may therefore contribute to the slower photobleaching observed in the ER-\nSRS signal of RANMP. To further suppress triplet-mediated radical formation leading to\nphotobleaching, strategies used in fluorescence imaging—such as adding reducing\nagents, antioxidants and triplet state quenchers—could be adapted for ER-SRS.”\n6. Additionally, how about photo-toxicity? Triplet state would evoke significant\ngeneration of singlet oxygen as the author also mentioned in the application of PDT,\nand further limits the general applicability especially in biological cells as an imaging\nagent.\nWe thank the reviewer for raising an important point regarding potential phototoxicity\ncaused by triplet-state-induced singlet oxygen generation. We fully acknowledge that\nsuch effects can limit the applicability of imaging agents in biological systems.\nHowever, we would like to mention that the triplet-state lifetime was too short to\nproduce detectable phosphorescence signal (see our response in the reviewer\ncomment #5 above). Thus, the short triplet-state lifetime is likely to result in insignificant\ngeneration of singlet oxygen.\nNonetheless, singlet oxygen generation is actively utilized in biological imaging—such\nas miniSOG and KillerRed—also rely on singlet oxygen generation from the triplet state.\nFor example, miniSOG (mini Singlet Oxygen Generator) is a small (106-residue)\nfluorescent flavoprotein that efficiently produces singlet oxygen upon blue light\nillumination. Despite this, it is widely used as a genetically encoded tag for correlated\nlight and electron microscopy and has been successfully applied in various biological\ncontexts including cells, tissues, and organisms such as mice. Similarly, KillerRed is\n1\n6\nanother singlet oxygen-generating protein that is commonly used in cell biology. It can\nbe directly expressed in cells and easily targeted to specific subcellular compartments,\ntaking advantage of light-induced reactive oxygen species for imaging or photodynamic\napplications. These examples demonstrate that, although singlet oxygen generation\ncan potentially cause phototoxicity, such effects can be managed or even utilized\nbeneficially in biological applications.\n7. In this regard, are RANMP molecules compatible with water and hence cellular\nenvironment measurement? The structures of the RANMP should make them highly\nprone for aggregation, which should be a severe issue in aqueous environments.\nWe thank the reviewer for the thoughtful comment. RANMP molecules are hydrophobic\n(e.g. soluble in chloroform, but not soluble in DMSO), rendering them inherently\nincompatible with aqueous environments. However, this hydrophobic nature has high\npotential for effective targeting of lipophilic environments, such as cellular membranes,\nby attaching organelle-targeting moieties [Yang, Bai & Hu, Nat Comm 15: 2578 (2024)].\nAlso, our Pdot nanoparticles encapsulate RANMPs with PSMA matrix making the\nparticles biocompatible.\nNonetheless, we fully acknowledge the importance of improving water compatibility for\nbroader biological applications. To this end, we are currently exploring molecular design\nstrategies that incorporate hydrophilic components, with the goal of enhancing aqueous\nsolubility while preserving the desirable properties of RANMP.\n8. Related, in Fig. S4, the authors showed an about 50-100 nm bathochromic shift in film\ncompared to the solution, which I assume is in DMSO. The significant redshift,\naccompanied by an increased shoulder peak, strongly suggests J-aggregate formation,\nwhich is more pronounced in Pdot. The authors reported enhancement fold based on film\nmeasurement in Fig. 2F, which raises the issue of quantification accuracy. Can the\nauthors also show comparison from solutions? Additionally, instead of normalized abs,\nmaybe absolute-intensity abs spectra would be more informative for quantitative\nunderstandings.\nWe thank the reviewer for the valuable comments. Fig. S4 presents the UV-visible\nabsorption spectrum of a RANMP solution in chloroform, as RANMPs are insoluble in\nDMSO. To improve clarity, we have added the solvent information to the caption of Fig.\nS4 in the revised manuscript.\nRegarding aggregation, the planar structure of RANMP facilitates efficient  stacking,\nwhich can lead to aggregation. This planar nature of the molecule ensures aggregation\nboth in thin films and in polymer dots (Pdots). In films, spin coating promotes well-aligned\nmolecular stacking, resulting in ordered aggregation. In contrast, Pdots are formed by\nconfining the molecules within a polymer matrix, leading to aggregation with random\norientation. This disordered aggregation in Pdots induces aggregation-induced\nquenching, which suppresses fluorescence and in turn facilitates amplification of the ER-\nSRS signal, enabling single-particle imaging. While some crystalline domains may exist\n1\n7\nand contribute to a redshift in the spectrum, the overall morphology of the Pdots is largely\namorphous.\nIt should be noted that our quantum chemical calculations, based on density functional\ntheory (DFT) methods (B3LYP/6-31G(d,p)), were performed on simplified structures in\nwhich the bulky alkyl chains were shortened to methyl groups, retaining only the planar\nconjugated core. As a result, the calculations may overestimate molecular planarity and\npacking propensity. In reality, as shown in Fig. S6B, the full RANMP molecules contain\nbulky alkyl chains that can sterically hinder  stacking and likely suppress the\nformation of ideal J-aggregates. We added a new figure on the full molecular structure\nin Fig. S6B and included the details at Methods section, specifically under “Quantum\nchemical calculations”:\n“DFT calculations were performed on simplified structures in which the bulky alkyl\nchains (R and R in Fig. 2A) were shortened to methyl groups, retaining only the planar\n1 2\nconjugated core. As a result, the calculations may overestimate molecular planarity and\npacking propensity. The full RANMP molecules contain bulky alkyl chains that can\nsterically hinder  stacking (Supplementary Fig. 6B).”\nWe included a related discussion at Discussion section in the manuscript as follows:\n“Many strategies in fluorescence sensors can be applied to RANMP. For instance, the\nincorporation of organelle-targeting moieties—such as cationic groups for mitochondria\nor basic groups for lysosomes— into the RANMP framework holds strong potential to\ntransform these hydrophobic probes into lipophilic, organelle-specific markers42.\nFollowing strategies for making organic fluorophores more hydrophilic such as attaching\nphosphate groups may expand the applicability of RANMP probes. The planar core of\nRANMP has potential to induce aggregation via stacking, but the bulky alkyl chain\n(R and R in Fig. 2A) can be tuned to further prevent molecular stacking.”\n1 2\nFigure S6. (B) Molecular structure of full RANMP-4Cl including planar core (orange) and\nbulky alkyl chain (green; R and R in Fig. 2A) in the side view. DFT calculations were\n1 2\nperformed on simplified structures in which the bulky alkyl chains were shortened to\nmethyl groups, retaining only the planar conjugated core.\nWe also attempted to measure the ER-SRS signal in solution. However, chloroform's low\nboiling point (61.2°C) led to rapid evaporation at room temperature, which posed a major\nchallenge for data acquisition. Specifically, the solvent evaporated within approximately\n10 seconds after sample preparation.\n1\ndichlorobenzene (o-DCB), which has a higher boiling point than chloroform. First, we\noptimized the ER-SRS signal by measuring across a range of excitation wavelengths\ncentered around the maximum absorption wavelength of 715 nm. Through this\noptimization process, we found that the optimal condition occurred at  + 21 nm. Under\nmax\nthis optimal condition, the ER-SRS signal of RANMP-T exhibited an approximately 1,152-\nfold enhancement compared to the EPR-SRS signal of Rho800. This enhancement was\ncalculated based on the sample concentration and laser power settings. The new solution\ndata is added to Fig. S9E-F as below.\nAs recommended, we have included the absolute-intensity absorption spectra in the (Fig.\nS4A, shown below) in addition to the previously normalized spectra. As reviewer\nsuggested, this provides a more quantitative representation of the absorption features in\nthe solution state. We hope these additions addresses reviewer’s concern and enhances\nthe clarity of the spectral interpretation.\nFigure S9. ER-SRS spectra of RANMP series in o-DCB solution: (E) RANMP, (F) RANMP-\nT. The SR signal of solution was measured under pump laser power at 200 µW, Stokes power\nat 8 mW and sample concentration at 1 mM. Under the optimal condition ( + 21 nm), the\nmax\nsolution of RANMP-T exhibited approximately a 1,152-fold signal enhancement compared to\nEPR-SRS of Rho800. Also, RANMP solution showed a 577-fold signal enhancement\ncompared to Rho800 EPR-SRS under its optimal condition at ( + 25nm). (G) UV-vis\nmax\nabsorption spectra of RANMP solution and RANMP-T solution in o-DCB. (H) Summary of\nER-SRS spectra of RANMP and RANMP-T in film and solution states.\n1\nNormalized UV-vis absorption spectra of RANMP series in chloroform solution (top left),\nfilm (top right), absolute intensity in chloroform solution (bottom left) and Pdots (bottom\nright). (B) Raman peaks of CN modes in film samples. Raman shift frequencies are\n2,218.8 cm-1, 2,220.4 cm-1, 2,220.0 cm-1 and 2,214.2 cm-1 for RANMP, RANMP-4F,\nRANMP-4Cl and RANMP-T, respectively.\n9. In this regard, was the wavelength used for solution measurements significantly\ndifferent from that in film? How does this change the detectability?\nWe are thankful for the reviewer’s suggestion on solution measurements that indeed\nimproved the signal enhancement as well as S/B ratio (Fig. S9H). We observed\napproximately 500-fold and 1000-fold signal enhancement in RANMP and RANMP-T\n2\n0\nsolutions in o-DCB, respectively, when compared to EPR-SRS of Rho800 (Fig. S9).\nThese enhancements are more than twice as high as those obtained in the film\ncondition, indicating that further amplification is achievable in the solution phase. The\nenhancement was calculated by considering the laser powers (200 µW pump and 8\nmW Stokes) and the sample concentration (1 mM in solution). While S/B of RANMP in\nsolution and film state were similar, S/B of RANMP-T solution (0.8) was substantially\nimproved than that of film (0.3).\nThe optimal pump wavelength for RANMP-T solution appeared at λ + 21 nm,\nmax\nwhereas in film, it appeared at λ - 43 nm. This difference is likely due to its proximity\nmax\nto the second absorption maximum (~720 nm) of film, which is absent in the solution\nspectrum.\nFor RANMP, the optimal pump wavelength in solution and film was found at λ +15\nmax\nnm and λ - 37.7 nm, respectively. Due to the wavelength tuning range limitation of\nmax\nour laser system (660–990 nm), we were unable to fully explore the lower wavelength\nregime for the RANMP solution (maximum absorption at 680 nm). Thus, it is possible\nthat we may have missed the true optimal excitation condition in solution.\nFigure S9. ER-SRS spectra of RANMP series in o-DCB solution: (E) RANMP, (F) RANMP-\nT. The SR signal of solution was measured under pump laser power at 200 µW, Stokes power\nat 8 mW and sample concentration at 1 mM. Under the optimal condition ( + 21 nm), the\nmax\nsolution of RANMP-T exhibited approximately a 1,152-fold signal enhancement compared to\nEPR-SRS of Rho800. Also, RANMP solution showed a 577-fold signal enhancement\ncompared to Rho800 EPR-SRS under its optimal condition at ( + 25nm). (G) UV-vis\nmax\nabsorption spectra of RANMP solution and RANMP-T solution in o-DCB. (H) Summary of\nER-SRS spectra of RANMP and RANMP-T in film and solution states.\n10. The authors corresponded their 1 nM solution detection to ~ 3 molecules? Based on\ntheir setup parameters (e.g. objective NA=1.2), single molecule equivalent should be well\nabove 1 nM.\n2\nsamples whose thickness exceeds the focal depth, the single-molecule equivalent\nconcentration is typically well above 1 nM. However, in our study, we used a spin-\ncoated PMMA film (20 nm thickness) prepared from a 1 nM solution, whose thickness is\nsignificantly less than the focal depth. Based on the estimated laser focal area and the\nfilm thickness, we now calculate that approximately six molecules are expected to be\npresent within the effective focal volume under our experimental conditions.\nIn the original manuscript, we had stated that ~3 molecules were present in the focal\nvolume. The initial estimation was based on the Sparrow criterion, using a prefactor of\n0.47. In response to the reviewer’s insightful comment, we have revised the calculation\nusing the Rayleigh criterion, a more widely accepted standard, with a prefactor of 0.61.\nFollowing is the equation that we used for the calculation.\nThe calculation is supported by the images of PMMA films at concentrations of 5 nM\n(Fig. R5A) and 10 nM (Fig. R5B), indicating high molecular density. Based on these\nobservations, we found it difficult to clearly identify isolated single molecules above 1\nnM in our 20 nm-thick film samples.\nFig R5. Images of PMMA films containing different concentrations of RANMP-T. (A)\nMolecules observed at a concentration of 5 nM. (B) Molecules observed at a\nconcentration of 10 nM. Measurements were performed at (pump, Stokes) = (749 nm,\n897.9 nm). Scale bar: 1 μm.\n11. Importantly, in “single-molecule” data, the signals vanished with temporal delay of\njust 1 ps (Fig. 4E). However, the pulse width here is 2 ps, I would expect delaying the\npulse overlap by 1 ps, would only decrease the signal by maybe 30%, rather than causing\nit to disappear entirely. Similarly, signals are nearly absent at 2236 cm-1 (Fig. 4E),\nwhereas Fig. 4A suggests that approximately 30% of the signal should still be present.\nThis again raises questions about how true SRG signal is determined. The limited\n2\n2\ndynamic range also raises a critical issue about confirming the single molecule identity in\nFig. 4, which was purely based on signal-step photobleaching on ER-SRS signal. While\nit is a standard procedure in fluorescence, the signal dynamic range there is significantly\nhigher. In addition, due to the aggregation-prone property of the RANMP, as evidence by\nabsorption spectra and also the structural basis, determining the detected spots are\nactual singal molecules, but not molecular aggregates, is a key control experiment. More\nunequivocal evidence would be needed to substantiate this importance claim in this\npaper.\nWe thank the reviewer for the insightful comment. Given that the FWHM of our laser\npulse is 1.66 ps, a simple calculation based on this value indicates that at a 1 ps time\ndelay, approximately 63.4% of the signal is lost, leaving 36.6% remaining. When\nanalyzing the measured time delay dependence spectrum shown in Fig. 2C (figure is\nshown below), the signal at 1 ps (blue line) is reduced to 20.23% of the maximum.\nThis significant decrease indicates that the molecular signal drops below the visibility\nthreshold in the image, rendering it undetectable.\nFigure 2. (C) Time trace of ER-SRS signal of RANMP-T. Blue curve is Gaussian fitting to\nthe trace when the Stokes pulse precedes the pump. Red line is exponential fitting to the\ntrace when pump pulse precedes Stokes.\nTo further support the claim that the observed signal originates from a single molecule,\nwe collected several tens of ER-SRS time traces and analyzed the distributions of\nphotobleaching time and signal amplitudes, which were well-fitted to single-exponential\ndecay model and log-normal model (Fig. 4D-I and Fig. S12E-G), consistent to single-\nmolecule behavior. Also, we have included the characteristic “half-moon”\nphotobleaching behavior in Fig. S12D. This spatially photobleached pattern is observed\nin single-molecule imaging as in SREF paper [Xiong, H. et al, Nature photonics 13, 412-\n417 (2019)]. In addition, to illustrate the photobleaching behavior observed when\nmultiple molecules are within the focal volume, we have included an example of multi-\nstep photobleaching and added in Fig. S12C. More details are included in pages 2-4 of\nthis letter.\n2\n3\n12. Related, in Fig. 3D, in off-resonance channel at 2226 cm-1, both sets of dots\ndisappear. However, based on Fig. 3E, wouldn’t that at least 50% of signal remain for\nRanmp-4Cl?\nWe thank the reviewer for the comment. In the spontaneous Raman spectrum,\napproximately 60% of the signal remains at 2226 cm⁻¹. However, in the ER-SRS\nspectrum, only about 30% of the signal is present at the same Raman shift (Fig. R6).\nTherefore, it is reasonable that the signal appears to vanish in the ER-SRS image due to\nthis significant reduction in intensity. We have replaced spontaneous Raman spectra in\nFig. 3E with ER-SRS spectra of Pdots for direct comparison of spectral and image data.\nAlso, the highly enhanced signal in ER-SRS spectra make the bandwidth assessment\nmore reliable than the spontaneous Raman spectra obtained under nonresonant\ncondition. We thank the reviewer for guiding us to present better spectra for the single-\nparticle images.\nFigure R6. (A) Spontaneous Raman of Pdots. (B) ER-SRS spectra of Pdots.\n13. The temporal profile in Fig. 2C looks very odd too. For true ER-SRS signals, the\ntemporal profile should be more symmetrical. This raises issues about the signal origins.\nIs this the characterization of signal or background?\nAs reviewer mentioned, the temporal profile of true ER-SRS signal should be\nsymmetrical. However, the temporal profile in Fig. 2C plotted with the unprocessed signal\nfrom lock-in amplifier that amplifies the pump-modulated intensity of the Stokes beam,\nthereby includes “background” contributions originating from stimulated emission, which\noccur only when the pump beam is preceding the Stokes beam. This explanation is\nprovided in the manuscript as follows:\n“When the Stokes pulse precedes the pump (blue line, Fig. 2C), the intensity follows a\nGaussian profile centered at time zero, consistent with SRS convolved with the\npicosecond pulse duration. In contrast, when the pump precedes the Stokes (red line,\nFig. 2C), the signal decays exponentially with a lifetime of 4.26 ps, which is on the order\nof that of stimulated emission. This asymmetrical time delay dependence indicates a\npump-dominated background.”\n2\n4\n14. It is also confusing that the authors first indicated a 4.26 ps lifetime of the signal,\nbut then indicated the “confirming that the signal reduction was due to the 218 short\nvibrational lifetime of nitrile groups (<1 ps)”. How is the time- delay measurement here\nprobe any vibrational lifetime of nitrile?\nWe apologize for the confusion. The 4.26 ps lifetime is related to background, not signal.\nThe 4.26 ps was measured by fitting the part of the time-delay curve when the pump\nprecedes the Stokes (i.e. red curve in Fig. 2C) with a single exponential. We provide a\nmore detailed description in the main text, specifying it as the lifetime of the background.\nWe assumed the lifetime of ER-SRS can be deduced from the time-delay measurement\nwhen the Stokes precedes the pump (i.e. blue curve in Fig. 2C). When the Stokes\nprecedes the pump only by 1 ps, the signal reduced to 20%, indicating lifetime shorter\nthan 1ps, consistent to the vibrational lifetime.\n15. For the origin of optimal S/B in ER-SRS, the authors claim that the key was the low\nquantum yield, so low electronic background. However, for ER-SRS investigations on\nRho800, pump wavelengths were kept redder compared to the absorption max of the\ndye. In RANMP, almost all optimal S/B were achieved by using Pump excitation bluer\nthan the absorption maximum. SRS is immune to fluorescence – why is low quantum\nyield the key? Alternatively, is the shifted pump excitation the reason for improved S/B?\nHave the authors explored Rho800 excitation at the blue absorption end?\nWe thank the reviewer for the helpful comment. The wavelength tuning range of our laser\nsystem (660–990 nm) inherently limited the available excitation wavelengths during the\noptimization process. Experiments using a pump wavelength of 700 nm were not\nperformed in Rho800, as the output power in this region is relatively low, making it difficult\nto obtain reliable measurements.\nAdditionally, under our current detection scheme based on Stokes gain, ER-SRS is\nsusceptible to pump-induced fluorescence that can be captured through the filter\ntransmitting the Stokes wavelength. Therefore, a low quantum yield (QY) is essential to\nminimize such background signals and ensure reliable ER-SRS measurements under\nour current experimental condition. This is illustrated in Fig. 1E, where Rho800 under\nrigorous resonance conditions generates high background, substantially lowering the\nsignal-to-background ratio (S/B) of ER-SRS (gray shaded area in Fig. 1E) compared to\nthose of EPR-SRS conditions.\n16. In film measurement, the concentrations also seemed extremely high. 5.8 mg/ml or\n10mg/ml would be estimated to about 5-10 mM in concentration? Would this number\nbecome even higher when films dry out? Since ER-SRS offers high sensitivity, why are\nsuch high conc. still needed for measurement? It again raises significant concerns about\nsevere aggregate formation.\nWe thank the reviewer for the helpful comment. The primary reason for forming a\nrelatively thick film was to ensure the accuracy and reliability of the UV-Vis absorption\nspectrum measurements, which were directly applied to the ER-SRS experiments. Since\nthin films produce weak absorption signals that make it difficult to obtain well-resolved\n2\npump wavelengths for ER-SRS.\n10 mg/mL is estimated to be approximately 5 mM in concentration. To make a uniform\nsurface, we used a spin coater instead of drop casting. The spin-coating method involves\nspinning the glass, which removes the excess solution by the centrifugal force and\nproduces films with uniform thickness. The film thickness was typically about 80-100 nm.\n17. There is no discussion on acquisition time related to the detection sensitivity,\nespecially for single molecule detection, and also the concentration curve in Fig. 4B. The\nonly report about time was “5 μm x 5 μm FOV in 62 s”. However, this depends on the\npixel numbers. If 10-by-10 pixels, the pixel dwell time is about 620 ms? This is extremely\nlong compared to typical acquisition time used in EPR-SRS, SREF and BonFIRE.\nWe apologize for the confusion. We have mentioned the exposure time (1ms) in\nthe figure caption, but we should have included the information in the manuscript as well.\nWe have added the explanation to the Methods section of the manuscript as follows:\n“For single-molecule imaging, we used a 1ms exposure time per pixel to balance signal\nstrength and imaging speed. This allows for rapid acquisition while minimizing sample\ndrift and photodamage, making it suitable for sensitive or time-limited samples.”\nCurrently, image acquisition is performed using a sample stage scanning method at a\nspeed of 250 mm/s. By switching to Galvano laser scanning, which enables scanning\nspeeds from 1500-3000 mm/s (based on IPG Photonics specifications1), the imaging\nspeed could be increased by up to tenfold. For example, acquiring a 5 µm × 5 µm\nimage, which currently takes 62 seconds, could be completed in approximately 6\nseconds using Galvano scanning. Our 1 ms exposure time is comparable to the 2 ms\nexposure times used in SREF and BonFIRE. Therefore, further improvement in the\nscanning method is expected to reduce the total acquisition time to a level comparable\nwith those of the previous techniques.\nReviewer #2 (Remarks to the Author):\nHow to achieve the ultimate single-molecule sensitivity of Raman spectroscopy in the\noptical far-field (without near-field surface enhancement) has been a long sought-after\ngoal in physical chemistry. Recent efforts of stimulated Raman excited fluorescence\n(SREF) have harnessed Raman-encoded fluorescence for this goal but are limited by\nthe shortcoming of fluorescence requirement.\nThis new work shows the fundamental (more basic) stimulated Raman scattering\nspectroscopy, without the help of fluorescence detection, itself is able to achieve this\nsensitivity for specially designed molecular systems. Raman-amplified nonfluorescent\n2\n6\nmolecular probe (RANMP) is a clever chemistry design. The quantum calculation\nagrees with experimental measurement, supporting the mechanism. The spectroscopy\nand imaging experiments are well presented. While RANMP might need some effort to\nmake (which is comparable to sophisticated fluorescence sensors), I expect its utility in\nbroad sensing and imaging applications. This study sends an important and strong\nmessage to the community. It shall inspire many new works in this field.\nWe sincerely thank the reviewer for the thoughtful and encouraging comments. We are\nparticularly pleased that the reviewer recognizes the novelty of achieving single-\nmolecule sensitivity using ER-SRS without relying on fluorescence, as well as the\nmolecular design of RANMP. We also appreciate the recognition of the agreement\nbetween theoretical calculations and experimental results, which we believe\nstrengthens the mechanistic understanding of our approach. While the synthesis of\nRANMP involves a certain level of complexity, we believe this is well justified by the\nunique photophysical properties and the single-molecule sensitivity.\nFinally, we are truly encouraged by the reviewer’s remarks on the broader utility and\npotential impact of our work. We hope that this study will indeed inspire further\ndevelopments in single-molecule spectroscopy and beyond.\nReviewer #3 (Remarks to the Author):\nOh and coworkers report on enhanced sensitivity in electronically resonant SRS\nimaging using a new set of designer Raman probes and an experimental system based\non two synchronized OPOs. The main finding is the achievement (for the first time) of\nsingle molecule sensitivity in SRS microscopy without resorting to SERS.\nOverall this manuscript is excellent. The study is detailed, provides all the necessary\nbackground and convincing findings. It can be accepted to Nature Communications\nprovided that the authors address the following (rather minor) comments:\n1. With an atomic number of 16, Sulfur is not really a heavy atom.\nWe thank the reviewer for their valuable comments and positive assessment. The\nreviewer is correct that sulfur, with an atomic number of 16, is not typically considered a\nheavy atom. We called Sulfur as a heavy atom in the context of conjugated organic\nmolecules absorbing optical light. It is common to refer to atoms heavier than carbon,\nnitrogen, and oxygen—such as sulfur—as \"heavy atoms.\" This terminology is often\nused to highlight the contrast in atomic properties within organic compounds. We used\nthe term in this conventional context, and we appreciate the opportunity to clarify this\npoint.\n2. Integration times (per pixel) for the measurements are stated in several places, but it\nis important to provide in the discussion details about the practicality of this method in\nterms of imaging (what can be achieved with pixel dwell times of up to ~1ms) and single\nmolecule spectroscopy (same, but on a time scale of 100s of ms).\n2\nmethod in terms of imaging and spectroscopy. In our ER-SRS imaging experiments, we\nemployed an exposure time of 1ms per pixel, which offers a practical balance between\nsignal strength and imaging speed. Although SRS signals are typically weak at the\nsingle-molecule level, the use of electronic resonance enhancement in our method\nallows us to optimize signal-to-background ratio even with short integration times.\nA 1ms exposure enables rapid image acquisition, making it feasible to generate ER-\nSRS images with high sensitivity within a reasonable total scan time, which is\nparticularly advantageous for minimizing sample drift and photodamage during\nacquisition. Additionally, the short exposure time is beneficial for observing dynamic\nprocesses or unstable samples, where long acquisition times could degrade spatial\naccuracy. Thus, the use of a 1ms exposure time demonstrates the potential of our\nmethod to be applied for sensitive or time-limited samples. For comparison, both SREF\nand BonFIRE operate at an exposure time of 2ms.\nCurrently, image acquisition is performed using a sample stage scanning method at a\nspeed of 250 mm/s. By switching to Galvano laser scanning, which enables scanning\nspeeds from 1500-3000 mm/s based on IPG Photonics specifications [Wang, W. et al,\nOptik 287, 171055 (2023)], the imaging speed could be increased by up to tenfold. For\nexample, acquiring a 5 µm × 5 µm image, which currently takes 62 seconds, could be\ncompleted in approximately 6 seconds using Galvano scanning. This improvement\nwould significantly reduce photobleaching.\nWe have added the explanation to the Methods section of the manuscript as follows:\n“For single-molecule imaging, we used a 1ms exposure time per pixel to balance signal\nstrength and imaging speed. This allows for rapid acquisition while minimizing sample\ndrift and photodamage, making it suitable for sensitive or time-limited samples.”\n3. Photobleaching is compelling evidence that the experiment detected single\nmolecules but not an ultimate proof. The authors should take note of that.\nWe appreciate the reviewer’s important comment and fully agree that, although\nphotobleaching behavior is compelling, it is not definitive proof of single-molecule\ndetection. Reviewer 1 raised a similar concern, and our detailed response to that point\nis provided on pages 2–4 of this letter.\nTo strengthen our validation of single-molecule sensitivity, we analyzed 30–60 ER-SRS\ntime traces of RANMP-T molecules (see Fig. R1, page 2). The observed\nphotobleaching lifetimes and signal amplitudes followed single-exponential and log-\nnormal distributions, respectively—signatures consistent with single-molecule origins.\nThese results are in good agreement with previous studies [Wennmalm et al, J. Phys.\nChem. B 103, 2516-2519 (1999); Mutch et al, Biophys. J. 92, 2926 (2007)]. We\nexamined two sample configurations (with and without PVA layers for photobleaching\nsuppression), both of which exhibited consistent single-molecule behaviors across\ndifferent environments. The detailed data are presented on pages 2-4 and in Fig. R1.\n2\nconcentration (10 nM), indicative of multiple molecules within a focal spot. Furthermore,\nwe identified a characteristic “half-moon” pattern in a raster-scanned image—a known\nhallmark of single-molecule localization. These results are shown in Fig. S12 and\ndiscussed in detail on page 2.\nWe sincerely appreciate the reviewer’s thoughtful feedback, which inspired us to\nstrengthen the manuscript with additional data. The updated results are now included in\nFigs. 4E–F and S12C–G.\n2\n9"}, {"round": 2, "reviewer_comments": {"reviewer_2": "I don't have further questions. The quality of the revised manuscript has exceeded my expectation.", "reviewer_3": "From this referee's perspective, the response by the authors suffices for publication of the manuscript in Nature Communications."}, "author_response": ""}]

Decision letter