Genetically Encoded SRS Probes

• Genetically encoded SRS probes are molecular tags introduced via DNA that generate Raman signals in living cells for targeted imaging.
• These probes leverage infrared fluorescent proteins with biliverdin-binding to achieve electronic pre-resonant enhancement and high sensitivity.
• They enable super-multiplexed Raman imaging with performance comparable to synthetic dyes, facilitating precise subcellular mapping.

Genetically encoded stimulated Raman scattering (SRS) probes are molecular tags introduced into living cells via DNA-encoded protein sequences, designed to generate SRS signals analogous to fluorescent proteins in optical microscopy. Infrared fluorescent proteins (IRFPs), enabled by biliverdin-binding chromophores, exploit electronic pre-resonant (epr) enhancement to achieve SRS cross-sections and multiplexing capabilities on par with synthetic dyes, thereby allowing specific genetic targeting and visualization of subcellular regions. This approach addresses a foundational limitation in SRS microscopy: the lack of robust, genetically encodable tags suitable for Raman-based imaging.

1. Physical Principles of SRS and Electronic Pre-Resonant Enhancement

SRS microscopy leverages inelastic light scattering to probe vibrational modes within molecules, offering unique contrast based on molecular bond vibrations. The stimulated Raman-loss (SRL) intensity under typical, non-saturated conditions follows

$$S_{\rm SRS} \propto I_{\rm pump} \, I_{\rm Stokes} \, \sigma_R \, N \, L$$

where $I_{\rm pump}$ and $I_{\rm Stokes}$ denote excitation pulse intensities, $\sigma_R$ the spontaneous Raman cross-section, $N$ the scatterer density, and $L$ the focal volume interaction length.

Electronic pre-resonant enhancement occurs when the pump laser frequency $\omega_p$ is detuned by $\Delta = \omega_e - \omega_p$ beneath an electronic transition $\omega_e$, boosting the effective Raman cross-section by a factor scaling with $\Delta^{-2}$:

$$\sigma_R^{\rm epr} \sim \sigma_R^{(0)} \cdot \Delta^{-2}$$

According to Albrecht’s A-term theory,

$$F_A(\omega_p, \omega_v) = \frac{(\omega_p - \omega_v)^2 + \omega_p^2}{(\omega_e - \omega_p)^2}$$

and the enhancement of cross-section is proportional to $F_A^2$. Experimental parameters (pump at 820 nm, probe mode $\omega_v \sim 1650$ cm$^{-1}$) yield $F_A \approx 13.6$ for mRhubarb720, $5.2$ for emIRFP670, and $2.5$ for mCherry, correlating directly with SRL amplitude.

2. Photophysical and Vibrational Properties of IRFP SRS Probes

IRFPs utilize biliverdin IXα (BV) as their chromophore, covalently attached via a cysteine residue within the GAF domain. Structural spectral features (Table S1) include:

• mRhubarb720: $\lambda_{\max} = 703$ nm, $\epsilon = 72.5$ mM$^{-1}$cm$^{-1}$
• emIRFP670: $\lambda_{\max} = 644$ nm, $\epsilon = 87.4$ mM$^{-1}$cm$^{-1}$
• mIRFP670nano3: $\lambda_{\max} = 645$ nm, $\epsilon = 129$ mM$^{-1}$cm$^{-1}$

Raman-active C=C and C=N stretches of BV manifest as strong peaks at $\sim 1620$ cm$^{-1}$ (mRhubarb720, solution 1 mM in Tris pH 8), with full width at half maximum $\approx 20$ cm$^{-1}$. Under identical concentration (1 mM), pump (6 mW) and Stokes (15 mW), SRS readout of mRhubarb720 yields $\Delta I / I = 2.6 \times 10^{-5}$, with shot-noise–limited detection threshold at $\Delta I / I = 1.9 \times 10^{-7}$—equivalent to a detection limit of $7.2 \mu$M. Extrapolated to 24 mW/120 mW laser powers, this detection limit decreases to $452$ nM, in line with high-performance synthetic dyes (e.g., ATTO740, $250$ nM).

3. Genetic Engineering and Targeting in Mammalian Cells

Gene encoding for the IRFP probe, specifically mRhubarb720, is fused in-frame to the C-terminus of human histone H2B via a linker, sub-cloned into standard mammalian vectors driven by CMV or EF-1$\alpha$ promoters. Transfection into HeLa cells is achieved through Fugene6. Nuclear targeting fidelity is validated by exclusive nuclear fluorescence (widefield epi, PMT detection) and DIC overlays with SRL at $1620$ cm$^{-1}$ (“heart-shaped” nucleus morphology).

4. SRS Microscopy: Instrumentation and Imaging Protocols

Typical SRS imaging uses an 820 nm pump and a tunable Stokes arm (e.g., 956 nm for 1740 cm$^{-1}$, 948 nm for 1640 cm$^{-1}$), with spectral focusing: $\sim100$ fs pulses chirped to $\sim3$ ps by optical glass rods. System repetition rate is 80 MHz, with Stokes amplitude modulated at 2.5 MHz via acousto-optic modulator.

Detection schemes include:

• Transmission SRL: Si photodiode + lock-in amplifier (2.5 MHz)
• Forward fluorescence: PMT (600–750 nm bandpass)
• Epi-fluorescence: PMT (confocal pinhole)

Live-cell compatibility: sample powers of 7 mW (pump) plus 12 mW (Stokes); pixel dwell time $0.01$ ms; $463\times463$ pixel fields across $50\times50 \mu$m in $2$ s/frame; 60$\times$, 1.27 NA water immersion objective. Spectral region centered on $1640$ cm$^{-1}$ provides resonance matching while maintaining sufficient red-detuning from BV electronic absorption to suppress one-photon excitation (attenuated by thermal factor $\sim 10^4$).

5. Comparative Performance: IRFPs Versus Synthetic Probes

Quantitative analysis (Fig 2b–c) under equal excitation/concentration reveals mRhubarb720 SRL amplitude is $\sim7\times$ that of emIRFP670 and mIRFP670nano3, and $\sim10\times$ mCherry, mirroring $F_A^2$ predictions (ratios: 1:1/7:1/29).

Against synthetic dyes:

• mRhubarb720, shot-noise limited detection, 452 nM (ATTO740: 250 nM)
• Super-multiplexed epr-CRS (ATTO dyes): 30–50 molecules in focus, 24 resolvable colors
• mRhubarb720 enables genetic encoding of tags with comparable SRS metrics

6. Imaging Results and Super-Multiplexing Potential

HeLa nuclei expressing H2B–mRhubarb720 generate clearly demarcated SRL images at $1620$ cm$^{-1}$ (0.08 mV amplitude; $\sim10 \mu$M local concentration). Imaging executed at 2 s/frame ($463\times463$ pixels), alternating between $1620$ and $1660$ cm$^{-1}$ for background correction. While single-color IRFP imaging is demonstrated, the $\sim20$ cm$^{-1}$ vibrational linewidth of each IRFP probe suggests that “super-multiplexing”—substantially exceeding the 5-color limit of fluorescence methods—is feasible once additional IRFPs with unique vibrational modes can be deployed. This suggests a future capacity for highly parallel Raman-based organelle mapping.

7. Limitations, Photobleaching, and Prospective Developments

Photostability remains a bottleneck: one-photon absorption (BV thermal tail $\rightarrow$ S$_1$) and two-photon absorption (via pump + Stokes) under epr-SRS initiate irreversible photobleaching within $\sim10$ frames ($\sim1$ ms total exposure at 800 pulses/ms). The bleaching rate aligns with $\sim10\%$ excitation per pulse (cross-section $5\times10^{-20}$ cm$^2$ at 820 nm), implicating triplet-state involvement.

Improvement strategies include:

• Employing lower repetition-rate sources or rapid galvanometric scanners to minimize pulse delivery per pixel
• Engineering IRFPs for reduced two-photon cross-sections (increased molecular symmetry, rigidity)
• Incorporating vibrational tags in the cell-silent window (C$\equiv$C, nitriles) via non-canonical amino acids or modified chromophores

A plausible implication is that overcoming photobleaching and engineering a diverse palette of IRFP vibrational tags will enable scalable, genetically encoded, multiplexed Raman cell imaging. Efforts in probe design and instrumentation optimization are underway to address these major technical challenges.