---
title: Genetically Encoded SRS Probes
url: https://www.emergentmind.com/topics/genetically-encoded-srs-probes
type: topic
---

# Genetically Encoded SRS Probes

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.

Source: https://www.emergentmind.com/topics/genetically-encoded-srs-probes