Enhanced Yellow Fluorescent Protein (EYFP)

• EYFP is an engineered autofluorescent protein with optimized emission properties and a lower saturation intensity, making it ideal for quantitative imaging.
• Its superior photophysical characteristics enable precise single-molecule detection and live cell tracking with reduced photodamage and high signal-to-noise ratios.
• EYFP’s versatile applications span advanced microscopy, nanoprobe labeling, and quantum sensing via optically addressable spin qubits, driving innovation in bioimaging.

Enhanced Yellow Fluorescent Protein (EYFP) is an engineered autofluorescent protein widely employed in cellular imaging, single-molecule detection, and emerging quantum sensing applications. Developed as a spectral variant of Green Fluorescent Protein (GFP), EYFP offers optimized emission properties, lower saturation intensity, and increased sensitivity in biological environments, supporting advances in cellular biophysics, quantum technologies, and multiphoton microscopy.

1. Photophysical Properties and Detection Metrics

EYFP exhibits favorable photophysical characteristics for quantitative imaging and single-molecule studies. In vitro, embedded EYFP molecules achieve maximum photon emission rates of $k \simeq 10^5$ photons/ms, indistinguishable from eGFP in terms of photobleaching and blinking times ($\tau_{bl} \simeq 3$ ms). Crucially, EYFP’s saturation intensity (%%%%2%%%% kW/cm²) is nearly half that of eGFP ($I_S = 13 \pm 5$ kW/cm²), reducing required excitation power and thus minimizing photodamage and background fluorescence originating from cellular autofluorescence (0704.3853).

The detected photon yield from an individual EYFP molecule, considering photobleaching and blinking, is described by:

$$N_{\text{det}}(t, I) = \eta_{\text{det}} \cdot k \frac{I/I_S}{1 + I/I_S} \left[1 - \exp\left(-\frac{t}{\tau_{bl}(1 + I/I_S)}\right)\right],$$

where $\eta_{\text{det}}$ is the overall detection efficiency, $k$ the emission rate, $I$ excitation intensity, $I_S$ saturation intensity, and $\tau_{bl}$ the characteristic time for photobleaching/blinking. In regimes where photobleaching is negligible ($t \ll \tau_{bl}(1 + I/I_S)$), the expression reduces to:

$$N_{\text{det}}(t, I) = \eta_{\text{det}} \cdot k t \frac{I/I_S}{1 + I/I_S}.$$

These equations formalize the direct link between photon budget and localization precision, demonstrating the centrality of photophysical kinetics and instrumentation.

Detection ratio ($R$), incorporating absorption cross section and detection efficiency, further underscores EYFP’s dominance. For one-photon excitation: $R \approx 1.8$ for eCFP, $8.7$ for eGFP, and $405$ for EYFP—over forty times greater than eGFP, validating EYFP’s application in environments with competing autofluorescence (0704.3853).

2. Single-Molecule Imaging in Live Cells

EYFP’s properties translate effectively to live cell contexts. Fusion constructs (e.g., with cardiac L-type Ca²⁺ channel or metabotropic glutamate receptors in COS-7 and HEK293 cells) remain detectable with signal-to-noise ratios $>$10. The robustness of EYFP’s fluorescence and photobleaching profile in vitro is mirrored in live cell imaging, facilitating quantitative stoichiometric analyses and dynamic tracking (0704.3853).

Wide-field fluorescence microscopy employing high numerical aperture objectives and sensitive CCDs allows for single-molecule resolution without requiring total internal reflection (TIR), thus avoiding excitation intensity gradients related to membrane topography.

Recent improvements using citrine—a more photostable EYFP variant—extend single-molecule trajectory lengths from 3–5 (EYFP) to up to 10 (citrine), facilitating longer observation windows within live cells (0704.3853).

3. Specific Probes and Photostable Labeling

Gold nanoparticle-based optical probes functionalized with GFP-nanobodies enable highly specific, non-fluorescent single-molecule tracking of EYFP fusion proteins in live cells (Leduc et al., 2013). The nanobody (camelid antibody fragment, $\sim$2 × 4 nm) binds EYFP’s β-barrel with $K_d \sim 10^{-23}$ nM, ensuring irreversibility under physiological conditions.

Photothermal detection—quantified by $S \propto \sigma_{abs} I_{pump}$—circumvents limitations of conventional fluorophores, offering perfect photostability and enabling tracking in crowded, confined domains such as adhesion sites or cytoskeletal assemblies. The local temperature elevation induced by the nanoprobe—$\Delta n = \frac{dn}{dT}\Delta T$—produces a refractive index change leveraged for detection, entirely bypassing issues of photobleaching (Leduc et al., 2013).

The specificity and minimal size of the gold nanoprobes, combined with the available GFP/EYFP-tagged protein library, open new avenues for long-term single-molecule imaging, quantitative dynamic analyses, and correlative electron-optical microscopy.

4. Excitation Pathways and Nonlinear Microscopy

EYFP supports advanced excitation schemes. Sequential two-photon fluorescence microscopy employing forbidden state transitions utilizes real (dark, typically triplet) intermediate states within EYFP to enable nonlinear red excitation (665 nm), culminating in green fluorescence (Wong-Campos et al., 18 Mar 2025). The two-step mechanism populates the intermediate state (T₁), then excites to a higher state (Tₙ), from which rapid intersystem crossing leads to radiative decay from S₁ → S₀.

The steady-state fluorescence emission power dependence deviates from the pure quadratic expected in virtual-state two-photon absorption, instead following:

$\text{Exponent} = 2 - (1 + k_2/k_{\text{ph}})$

where $k_2$ is the secondary excitation rate and $k_{\text{ph}}$ the intermediate state decay rate. Observed exponents of $1.61 \pm 0.06$ (EYFP, 665 nm) confirm dominant sequential excitation via real states (Wong-Campos et al., 18 Mar 2025).

This “2p-prime” method enables optical sectioning, deep tissue imaging with reduced scattering and phototoxicity, and compatibility with picosecond or continuous-wave excitation, lowering system complexity relative to ultrafast femtosecond laser requirements.

5. Quantum Sensing and Spin Qubit Applications

EYFP functions as an optically addressable spin qubit sensor, exploiting its metastable triplet state accessed via intersystem crossing and read out by Optically Activated Delayed Fluorescence (OADF) (Feder et al., 25 Nov 2024). The procedure utilizes a 912 nm near-infrared pulse to transfer triplet population to a higher triplet state (T₂), triggering rapid reverse intersystem crossing to singlet S₁ and subsequent delayed fluorescence from S₁ → S₀. This readout protocol yields spin contrast up to 44% for certain transitions.

Coherent control under microwave irradiation enables spin-lattice relaxation time $T_1 = (141 \pm 5)$ μs and coherence time $T_2 = (16 \pm 2)$ μs under Carr-Purcell-Meiboom-Gill decoupling at 80 K. Room-temperature optically detected magnetic resonance (ODMR) is attainable in aqueous solution, yielding spin contrast up to 3% and static (DC) field sensitivity $\leq 93$ pT·mol⁻¹/²·Hz⁻¹/²; AC field sensitivity reaches $183$ fT·mol⁻¹/²·Hz⁻¹/². EYFP can be genetically encoded and maintains contrast and coherence in mammalian (HEK 293T) intracellular environments (Feder et al., 25 Nov 2024).

Spin dynamics are described by a spin-1 Hamiltonian:

$$H = \hbar D S_{z}^2 + K E (S_{x}^2 - S_{y}^2) - \gamma_e \mathbf{S} \cdot \mathbf{B}$$

with $D \approx 2.356$ GHz, $E \approx 0.458$ GHz, and variables as defined (Feder et al., 25 Nov 2024). Powder-averaged spectra reveal orientation broadening, guiding magnetic field sensing protocols.

6. Excitonic Coupling, Photon Antibunching, and Quantum Design

Strong excitonic coupling and photon antibunching co-occur in yellow fluorescent protein dimers (Venus variant), challenging conventional expectations (Abrahams, 19 Aug 2025). Delocalized excitonic states, formed through strong chromophore coupling $J \simeq -34$ meV (as measured by circular dichroism and Davydov splitting), would typically favour superradiance and photon bunching. However, rapid dephasing (sub-picosecond) forces single-photon antibunching, as observed in antibunching–fluorescence correlation spectroscopy.

Population dynamics adhere to a Lindblad master equation:

$$\frac{d\hat{\rho}}{dt} = -\frac{i}{\hbar} [\hat{H}, \hat{\rho}] + \sum_\ell \mathcal{L}(\hat{\rho}, \gamma_\ell),$$

with dissipators representing pure dephasing and thermal transitions. The pure dephasing rate for Drude–Lorentz spectral density is

$$\gamma_\phi = \frac{2\lambda k_B T}{\hbar^2 \gamma_c},$$

where $\lambda$ is reorganization energy, $T$ temperature, $\gamma_c$ bath cutoff frequency.

A plausible implication is that related proteins such as EYFP may be tuned via chromophore geometry and interface design to exploit this “bioexciton motif” (Editor’s term), integrating strong excitonic coupling, rapid decoherence, and controlled emission statistics. Although coherence lifetimes at room temperature preclude quantum gate operations, cryogenic cooling may extend lifetimes sufficiently for molecular qubits and ultrafast optical gate explorations (Abrahams, 19 Aug 2025).

7. Methodological Advances and Future Directions

EYFP research has fostered methodological innovation in quantitative imaging, spin control, nanoprobe design, and nonlinear optical microscopy. Calibration techniques using detailed photon budget equations, advanced imaging configurations that eliminate excitation gradients, and robust probe designs highlight general strategies for precise single-molecule quantification and dynamic tracking (0704.3853, Leduc et al., 2013).

Two-step excitation utilizing real intermediary states minimizes instrumentation requirements for multiphoton microscopy, expanding applicability in thick and living tissues (Wong-Campos et al., 18 Mar 2025). The demonstration of fluorescent-protein spin qubits suggests future biocompatible quantum sensors, especially as protein engineering and environmental control improve coherence properties (Feder et al., 25 Nov 2024, Abrahams, 19 Aug 2025).

Further optimization of EYFP and cryptic motifs in related proteins may enable tailored combinations of brightness, photostability, emission statistics, and quantum coherence for advanced applications in bioimaging, nanoscale magnetometry, and hybrid optical-quantum systems.