Metal‐Enhanced Fluorescence for the Biologist's Cellular Imaging Toolkit: Design Principles and Recent Applications

ABSTRACT

Fluorescence microscopy is essential in modern cell biology but remains constrained by photobleaching, autofluorescence, and the intrinsic quantum yields of emitters. Metal‐enhanced fluorescence (MEF) is a photophysical phenomenon in which interactions between luminescent species and metal nanostructures markedly increase emission, enabling a route to brighter, high‐contrast, noninvasive bioimaging by reshaping photophysical pathways without modifying fluorophore chemistry. This review translates MEF fundamentals into an experimental playbook for biologists, distinguishing MEF mechanisms and explaining how distance, spectral overlap, and nanoparticle morphology govern whether emission is boosted or quenched. We synthesize recent live‐cell applications—using gold and silver nanoparticles—to illustrate gains in signal‐to‐noise at lower excitation power, improved photostability, and opportunities where small‐molecule dyes often suffer low quantum yield, and provide practical guidelines for pairing dyes with metal nanostructures to lower the barrier to adopting MEF in cellular imaging.

Metal‐enhanced fluorescence (MEF) harnesses plasmonic interactions between fluorophores and metal nanostructures to increase excitation and/or radiative decay rates, yielding brighter, more photostable fluorescence signals for cellular imaging. This review highlights how nanoparticle size, shape, distance, and spectral overlap govern enhancement versus quenching, and provides practical design guidelines for bioimaging applications.

1. Introduction

Fluorescence microscopy is a powerful tool for the noninvasive visualization of biological samples [1, 2]. Its versatility and effectiveness have made it an invaluable technique for the detection and sensing of molecules, facilitating fundamental investigations across all forms of biology, including the study of signaling pathways and disease diagnosis. Live‐cell fluorescence microscopy can assist with determining and characterizing the spatiotemporal dynamics of molecules, supramolecular assemblies, organelles, and whole cells. For instance, fluorescence imaging permits the in situ monitoring of cell secretion events with sensitivity and resolution; this is crucial to understanding the fundamental cell biology that underlies cell–cell communication, migration, proliferation, and differentiation, since abnormal variations in cell secretion patterns can lead to cell dysfunction and diseases [3]. In classic fluorescence microscopy, a specimen is decorated with fluorophores that bind to specific proteins or other structures. Illuminated at a specific excitation wavelength (λ_Ex), fluorophores emit light (with peak emission wavelength, λ_Em) [4, 5, 6]. Collecting that emitted light produces high‐contrast images. Despite the high sensitivity of this technique, where single molecules can readily be detected [7], the opportunity to improve health outcomes by incrementally enhancing diagnostic capabilities and our fundamental understanding of disease mechanisms drives a constant pursuit of accessing new imaging modalities and reducing the limits of detection [8, 9]. Recent improvements in fluorescence microscopy have primarily depended on advances in hardware and optical setups (e.g., leading to higher camera sensitivity), as well as an explosion in computing power, data‐processing algorithms, and machine learning [10, 11]. Nonetheless, the detection of a fluorophore is still limited by its intrinsic spectroscopic properties, such as extinction coefficient, quantum yield, photostability, and photoblinking [4]. This is true for organic (molecular) fluorophores, quantum dots (QDs), upconversion nanoparticles, and fluorescent proteins. Autofluorescence of biologics can also interfere with fluorophore detection (most organic dyes have fluorescence lifetimes between 2 and 5 ns, which are close to cellular autofluorescence), while excessive excitation energy both photobleaches the reporting fluorescent molecule and induces reactive oxygen species (ROS) formation leading to cellular phototoxicity [12, 13, 14, 15]. These drawbacks—low quantum yields, photobleaching, autofluorescence—limit imaging sensitivity and are the driving forces behind efforts to amplify fluorescence and engineer novel imaging labels with improved optical properties.

In this context, metal‐enhanced fluorescence (MEF) can be conveniently used to favorably modify the spectral properties of fluorophores without the need to synthetically alter their chemical structures [4, 16, 17, 18, 19, 20, 21]. MEF originates from the ability of metal (e.g., silver‐AgNP, gold‐AuNP, etc.) nanoparticles to interact with light along the visible and near‐infrared regions of the electromagnetic spectrum [22, 23, 24, 25, 26], dramatically affecting the photophysical processes underlying the emission of fluorescent molecules within a near‐field range [27, 28]. MEF—and, in general, the understanding of the interaction between a fluorophore and a metal nanoparticle—has been the subject of intense research over the last 25 years, with major contributions from the Lakowicz and Geddes groups. Many excellent reviews cover MEF theory from a theoretical perspective [16, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39]. However, practical approaches and applications of MEF for biologists are still an emerging area of applied research. With this contribution, we aim to make this topic more accessible to biologists and facilitate a more direct and effective use of MEF in their research. Following an overview of the basic theory of MEF, we summarize key contributions to the field that shape the current landscape of metal‐enhanced live cell imaging. This review focuses only on MEF‐assisted cellular imaging and its design principles, and does not cover plasmonically enhanced assay biosensing (i.e., using gold or silver nanoparticles to amplify the detection signals in biosensing assays), or SERS (Surface‐Enhanced Raman Scattering, an analytical technique that amplifies the Raman signals of molecules adsorbed onto metal surfaces or nanoparticles to detect and identify even trace amounts of substances), for which we recommend some relevant reviews [40, 41, 42, 43, 44, 45, 46, 47].

2. Principles of Metal‐Enhanced Fluorescence

After a fluorophore is excited, the excess energy can be released through either radiative (i.e., by emitting a photon, observed as fluorescence propagating into the far field) or nonradiative decay. In the latter case, the excited fluorophore loses energy without emitting light. This can occur through various mechanisms, such as internal conversion (where the energy of an excited state is converted to vibrational energy within the molecule and may be released as heat) or collisions between fluorophores and quenchers. The rates at which radiative and nonradiative decay occur are indicated by Γ and k_nr , respectively. The balance between radiative and nonradiative transitions determines the overall fluorescence efficiency of the fluorophore, with efficient fluorophores having a higher probability of radiative transitions and less energy loss through nonradiative processes. Γ and k_nr define both the quantum yield of fluorescence (Φ_F), which denotes the emission's efficiency by quantifying the ratio of emitted photons to absorbed photons, and the fluorescence lifetime (τ), which represents the average [48] time during which a fluorophore exists in its excited state prior to returning to the ground state, according to Equations 1 and 2:

$${\mathrm{\Phi }}_F=\frac{\mathrm{\Gamma }}{\mathrm{\Gamma }+k_{nr}}$$ (1)

$$\tau =\frac{1}{\mathrm{\Gamma }+k_{nr}}$$ (2)

where Γ represents the radiative decay rate for the dye, and k_nr is the combined rate constant for all nonradiative decay pathways. Importantly, Γ depends on the fluorophore's extinction coefficient and does not change substantially in different environments or most experiments. This is because the more a molecule ‘likes’ absorbing light (i.e., high extinction coefficient), the faster it will radiatively decay. The concepts of quantum yield and lifetime are most effectively illustrated using a simplified Jablonski diagram (Figure 1a) that focuses exclusively on the processes involved in returning to the ground state, on the fluorophore's emissive rate (Γ) and its rate of nonradiative decay (k_nr ).

FIGURE 1.

(a) Simplified Jablonski diagram to illustrate the concepts of radiative and nonradiative decay. Metal nanoparticles may enhance fluorescence intensity by increasing the rate of fluorophore excitation or by increasing the effective quantum yield through the formation of plasmophores. E is the rate of excitation without metal and E_m is the additional excitation in the presence of the metal. Γ is the radiative decay rate in the absence of the metal and Γ_m is the radiative decay rate due to the metal. Figure 1a provides a general illustration of the two MEF mechanisms for cases in which Kasha’s rule (see reference no. 55 for more details) is maintained and spectral distortions are negligible. (b) Representation of the localized surface plasmon resonance (SPR) explained as the collective oscillation of conduction band electrons on the surfaces of metal nanostructures in response to an incident electromagnetic field.

While it might seem contradictory that the radiative decay rate remains stable in different environments (e.g., a different solvent), yet the quantum yield can vary, the explanation lies in understanding the broader context of a fluorophore's behaviors in different surroundings: since the radiative decay rate is nearly constant (in the absence of metals), the quantum yield can only be increased by decreasing the value of k_nr , achievable, for example, by changing the polarity or viscosity of the medium. To conclude this brief overview of classical fluorescence, we emphasize that changes in Γ and k_nr would result in changes in quantum yield and lifetime in the same direction, as illustrated by Equations 1 and 2.

When metal nanoparticles are located in proximity to fluorophores, their surface plasmons can modify emission beyond what classical fluorescence predicts. MEF primarily arises from near‐field interactions between molecules and metal nanoparticles, enhancing brightness by either increasing the rate at which fluorophores are excited (the excitation rate) or by raising the rate at which they emit light (the radiative decay rate), or in some instances, both effects may be observed. Plasmon excitation, often used synonymously with surface plasmon resonance or SPR, can be explained as the collective oscillation of conduction band electrons on the surfaces of metal nanostructures in response to incident photons of specific wavelengths (Figure 1b). Picture nanoscale metal particles: when light of the right color strikes them, they become excited, and their electrons oscillate in unison—a collective motion known as plasmon excitation, like a synchronized dance triggered when metal nanoparticles interact with light. The spectral signature of plasmons can be controlled by adjusting the size, shape and, if applicable, the arrangement of the nanostructures on a surface (a central advantage of plasmonics, as this tunability can be achieved synthetically to match a given fluorophore and biological application, rather than by modifying fixed excitation or detection hardware) [49, 50, 51, 52, 53]. This effect results in an enhancement of the electromagnetic field in the vicinity (typically within the near‐field region, on the order of tens of nanometers) of the nanoparticle surface. Consequently, the likelihood of exciting fluorophores nearby is enhanced. In other words, the local electric field concentrated around the illuminated metal nanoparticle results in an increased excitation (E + E_m, where E is the rate of excitation without metal and E_m is the additional excitation in the presence of metal) of fluorophores near the metals (Figure 1a). Thus, fluorophores in the proximity of metal nanoparticles experience increased excitation rates. With more molecules raised to higher energy states and, therefore, with more molecules returning to their ground states by emitting photons, a higher fluorescence intensity is obtained. This component of MEF will result in increased brightness without changing the quantum yield or lifetime, allowing for lower incident intensities (thus minimizing phototoxicity, reducing photobleaching, and preserving cell viability) and lower background noise.

In parallel, metal surfaces enhance the fluorophores’ radiative decay rate (Γ + Γ_m, where Γ_m is the additional rate due to the metal). This occurs through the formation of a radiative complex between the nanoparticle and fluorophore, which we refer to as a ‘plasmophore’. This term is meant to encompass the combined nature of the emitting species—part metal, part fluorophore [54]. The observed emission retains essentially the same spectrum as the fluorophore; [55] however, the lifetime is decreased (vide infra) and falls typically within the range of plasmon decay rates (near 50 fs). In our view, since this emission has properties of both the fluorophore and the metal, considering the fluorophore‐metal complex as the emitting entity is the most appropriate approach. As illustrated in Figure 1a, this plasmophore typically exhibits a notably higher radiative decay rate (Γ + Γ_m) compared to the corresponding fluorophore alone (Γ). Alternatively, one can also view this enhancement from the perspective of the fluorophore itself, describing it as molecular emission being boosted (increased emission) by the electromagnetic field generated through the SPR. It follows that Φ_F and τ need to be changed to:

$${\mathrm{\Phi }}_F=\frac{\mathrm{\Gamma }+{\mathrm{\Gamma }}_m}{\mathrm{\Gamma }+{\mathrm{\Gamma }}_m+k_{nr}}$$ (3)

$$\tau =\frac{1}{\mathrm{\Gamma }+{\mathrm{\Gamma }}_m+k_{nr}}$$ (4)

causing Φ_F and τ to move in opposite directions in response to the addition of Γ_m.

Much of the existing literature on MEF acknowledges the influence of factors such as nanoparticle size, shape, interparticle spacing and fluorophore‐nanoparticle distance. With respect to the latter, it is generally agreed that for a fluorophore to experience a maximum increase in the radiative decay rate (highest Γ_m), it should be ideally placed between 10 and 25 nm from the metal surface [56]. However, it is important to note that the exact distance for optimal MEF can vary based on factors such as the specific fluorophore, the type of metal, and the experimental conditions, and that there is no strict numerical ‘maximum distance’ that universally applies. For instance, fluorophores up to about 200 nm away from AgNP may experience MEF. As the distance between the fluorophore and the metal nanoparticle increases, the interaction between them weakens, leading to a decrease in MEF, until effects (enhanced electric field and Γ_m) become negligible and the fluorophore behaves as if it were in free space (classical fluorescence). While there might not be an exact numerical limit, distances between 10 and 25 nm typically achieve the highest MEF.

The size and shape of the metal nanoparticles play significant roles in determining luminescence enhancement. In fact, for small particles, the absorption component dominates, while for larger ones, the scattering cross‐section is more prevalent. Moreover, nanoscale polyhedra often exhibit greater scattering than larger spherical nanoparticles, or they may scatter across a range of wavelengths that are more convenient for achieving MEF with a specific dye. For plasmophoric MEF, a strong scattering component of the metal's extinction spectrum at the fluorophore's emission wavelengths is crucial.

Lastly, we note that it is critical to understand the importance of the synergistic interaction between nanoparticles and dyes. For example, the common assumption that larger nanoparticles are better for MEF can be misleading because it overlooks the importance of the spectral properties of the metal and the fluorophore. A simple guiding principle is that when the fluorophore's absorption spectrum aligns with the SPR of metal nanoparticles (i.e., the absorption component of the nanoparticle extinction spectrum), the fluorophore's excitation rate increases. This is primarily due to FRET (Förster resonance energy transfer) at short (within ∼10 nm) distances between nanoparticles and dyes [57]. If, instead, it is the fluorophore's emission spectrum that aligns with the plasmon band, at distances above ∼10 nm FRET ceases to be effective, but the emission experiences a boost due to the Purcell effect, which increases the radiative decay rate by plasmophoric coupling. On the other hand, if the emission spectrum of the dye aligns with the metal nanoparticles’ SPR and the distance between them is small (on average, less than 10 nm), the emission is quenched (the emission of the fluorophore is simply absorbed by the nanoparticles, unless the latter are primarily scatterers). Further, at such close distances, additional nonradiative decay channels become significant, and fluorescence quenching also occurs due to energy transfer from the excited fluorophore to the metals. The outcome of this particular scenario is that quenching dominates, reducing fluorescence intensity.

Let's examine some recent examples to clarify these principles more practically. For each of the examples selected, after a brief synopsis of the paper's objectives and main findings, we focused on the experimental design and, particularly, the choice of metal nanoparticle/fluorophore system used (e.g., Why was a specific metal nanostructure chosen to work with a specific fluorescent probe? Why silver versus gold? Was the metal nanoparticle shape important?) in the hope of affording more clarity and to synthesize readily‐available knowledge to facilitate future experimental design and enable broader applications of MEF in biological research.

3. Recent Examples of MEF in Bioimaging

Leading‐edge research building MEF into the design of materials for biomedical applications has produced numerous examples in which one or both of the increased excitation and plasmophoric MEF mechanisms have been observed, using different combinations of small‐molecule dyes, fluorescent proteins, and other biomolecules and nanostructures of various types, sizes, and morphologies.

Recent work by Huang et al. well exemplifies the opportunity to better understand and optimize the use of MEF in practical biological and biomedical contexts [58]. Here, a polymer‐coated mixture (Figure 2a) of gold nanorods (AuNR) and short‐wave infrared (SWIR) small molecule dyes achieved MEF (up to 45‐fold enhancement factor) for in vivo bioimaging of ovarian cancer down to sub‐millimetre in size and demonstrated potential for future applications in real‐time fluorescence‐imaging guided surgery.

FIGURE 2.

(a) Scheme and TEM image of the AuNR–dye nanocomposite and (b) relative position of the SWIR dye absorption (dotted gray line), emission (dotted black line), and AuNR (red line) extinction spectrum as normalized by the peak absorption or emission. Adapted from [ref. 58] with permission.

The benefits of employing longer‐wavelength excitation in the near‐infrared (NIR) window for bioimaging are well known (e.g., deeper tissue penetration and lower autofluorescence, properties that can counteract the degradation of imaging quality due to tissue scattering and absorption) [59]. Nonetheless, the application of organic small molecules (e.g., indocyanine green/Cardiogreen, methylene blue, IRDye800CW, CH1055) [60] or inorganic NIR‐absorbing/emitting probes [59] (e.g., lanthanide‐doped nanoparticles, quantum dots, nanodiamonds, carbon dots) that absorb and emit in the NIR is limited by their low quantum yields and poor photostability—both of which can be mediated by employing biocompatible gold (and theoretically other metal) nanostructures to achieve MEF. In this example, the selection of a commercially available small molecule organic SWIR (short‐wave infrared, 900–1700 nm) dye, IR‐E1050 (Φ_F = 0.2%), with peak absorption and emission at 700 nm and 1000 nm, respectively, created a significant overlap between the dye emission band and the longitudinal plasmon [61] in the extinction spectrum of 80 nm AuNR (Figure 2b).

This configuration (i.e., overlap between the fluorophore's emission spectrum with the surface plasmon band) enabled the plasmophoric MEF mechanism but does not guarantee a net enhancement will be observed in similar systems employing different nanostructure morphologies, as the overlap of the dye emission spectrum with the absorption component of the nanoparticle extinction spectrum can result in quenching. Herein, the dye/polyelectrolyte layer is only 2–3 nm, as shown in the transmission electron microscopy image in Figure 2. In this case, however, it is the high aspect ratio (i.e., the length‐to‐width ratio) of AuNR, as compared to spherical nanoparticles, that drastically increases the scattering component of the extinction spectrum that overlaps with dye emission, leading to fluorescence enhancement via plasmophoric coupling. The balance between scattering and absorption in AuNR depends on several factors, including their aspect ratio and size. Absorption predominates at smaller sizes or lower aspect ratios, while scattering becomes more significant at larger sizes or higher aspect ratios (this case). This condition should be an essential consideration for researchers who intend to employ spherical or quasispherical nanostructures—where the scattering component of the extinction spectrum tends to be both less prominent and overwhelmed by nanoparticle absorption, typically making these morphologies more suitable for pursuing MEF via excitation rate enhancement (which instead requires overlap between nanoparticle extinction and dye absorption)—and highlights the importance of selecting viable combinations of dyes and nanostructures with complementary spectroscopic properties to optimize MEF.

Key Takeaway

Combining dyes and nanostructures with complementary spectroscopic properties is crucial to optimizing MEF. Nanostructures that scatter light efficiently (e.g., larger, high aspect‐ratio or polyhedral particles) in the wavelength range of dye emission are more likely to induce plasmophoric MEF. Spherical nanostructures are more suitably paired with fluorophores whose absorption overlaps with SPR, which will enhance molecular excitation.

In another interesting example [62], a commercial small molecule and low quantum yield NIR dye (IRDye800CW) was combined with marginally smaller AuNR displaying a longitudinal plasmon band centred at ∼800 nm (in the previous example, the SPR of AuNR was 980 nm) to enable enhanced confocal fluorescence imaging and more sensitive in vitro detection of adenosine triphosphate (ATP) in HeLa cell lysates, rat brain microdialysis samples and living HeLa cells. A key feature in this system is that both the absorption [λ_Abs (max) ∼790 nm] and emission [λ_Em (max) ∼810 nm] spectra of the dye are entirely encompassed by the broader AuNR extinction spectrum, theoretically allowing for both MEF mechanisms (increased excitation rate and increased quantum yield of the fluorophore). Nevertheless, evidence of an AuNR‐mediated 30‐fold increase in the radiative decay rate and corresponding enhancement of the fluorescence quantum yield from 0.08 to 0.61 in the presence of ATP suggests that plasmophoric coupling was the dominant MEF mechanism at work. Herein, the significant scattering capability of rods, coupled with the inherently low quantum yield of IRDye800CW, provides considerable room for enhancement (toward achieving the maximum quantum yield of 1).

This work also thoroughly examined the effect of the AuNR‐dye separation distance on MEF efficiency, and various lengths of polyethylene glycol (PEG) linker were incorporated into the DNA/aptamer sequence used to conjugate the metal nanostructures and fluorophores. By tuning the AuNR‐dye distance, Yu et al. were able to optimize the observed fluorescence enhancement factor. This delivered highly improved sensitivity and lower LODs for ATP detection than previously reported fluorescence methods.

The dominance of the plasmophoric MEF mechanism in this system conveys an additional advantage for bioimaging applications in that the increased radiative decay rate (notably, the nonradiative decay rate was almost unchanged) and correspondingly lower fluorescence lifetime (see Equations 3 and 4) greatly improved the dye's photostability, which generally allows for longer irradiation times/lower cumulative exposure to the excitation light, and a reduced likelihoods of both dye degradation (i.e., decreased probability of undergoing photochemical reactions that could degrade the fluorophore) and generation of ROS that can be hazardous to biological samples.

Both previous examples highlight that the use of low quantum yield fluorophores offering other desirable spectroscopic properties (e.g., NIR or SWIR absorption and emission) can be facilitated by selecting noble metal nanostructures with extinction spectra positioned to drive plasmophoric coupling and realize increased quantum yields.

Key Takeaway

Significant spectral overlap between fluorophore absorption and nanoparticle SPR is not required to observe MEF by the plasmophoric mechanism, which can be used to increase fluorescence quantum yield and fluorophore photostability.

As noted above, MEF efficiency is also influenced by the spatial separation between the nanostructure and the fluorophore, permitting optimization of the enhancement factor by i) Manipulating the relative concentrations of the two components, which is a simple experimental approach (for instance, controlling the dye‐to‐AuNR number ratio [dye surface density] as done by Huang et al. [58]) and/or by 2) Fixing the nanostructure‐fluorophore distance by incorporating a synthetic tether, a linker or core‐shell element into the system, which adds an optional layer of complexity to the design but also provides greater control and can sometimes offer dual functionality (e.g., the length of the dynamic self‐hybridizing DNA loop structure in the previous example by Yu et al. [62] also determines the AuNR‐dye separation). Endeavoring to create a noninvasive nanoprobe that leverages MEF to enable highly sensitive, real‐time fluorescence imaging of exogenous and endogenous H₂S in living cells, Luo and coworkers controlled the nanostructure‐fluorophore distance by manipulating the thickness of the silica layer in a core‐shell AuNR@silica nanocomposite (Figure 3) conjugated to the porphyrin dye meso‐tetrakis(4‐carboxylphenyl)porphyrin (TCPP, whose Φ_F is typically around 0.10 in aqueous solutions) [63]. Here, the silica matrix was used to coat Au nanorods with an aspect ratio of 2.3 (average length and diameter of 44 and 19 nm, respectively) and enabled facile surface modification of the metal nanoparticles while improving their biocompatibility. TCPP fluorescence was initially quenched by copper ion (Cu²⁺) coordination, but was reactivated in the presence of H₂S, where the stronger affinity of Cu²⁺ for S²⁻ dissociated the TCPP‐Cu complex, restoring the fluorophore's emission. The latter is simultaneously enhanced by AuNR@silica (Figure 3a).

FIGURE 3.

(a) Schematic illustration of the metal‐enhanced fluorescence nanoprobes Au@silica‐TCPP‐Cu²⁺ for detection of H₂S. (b) UV–vis spectra of AuNRs (a) and TCPP (b) and fluorescence spectrum of TCPP ( c ). (c) UV‐vis spectra of Au@silica (a), silica‐TCPP (b) and Au@silica‐TCPP (c). Adapted from [ref. 63] with permission.

The wide spectral separation between dye absorption [λ_Abs (max) ∼420 nm] and both the transverse and longitudinal plasmon bands of the AuNR (centered at approximately 520 and 655 nm respectively, Figure 3b) intrinsically minimized the contribution of the increased excitation MEF mechanism (which requires that the absorption spectrum of the fluorophore overlaps with the SPR) toward the overall fluorescence enhancement observed. Conversely, this system was engineered such that plasmophoric MEF (i.e., increased emission), enabled by the overlap between dye emission [λ_Em (max) ∼650 nm] and AuNR scattering, was the dominant mechanism of fluorescence enhancement. Accordingly, Luo and coworkers observed changes in the fluorescence lifetime (Equation 4) as a function of nanostructure‐fluorophore separation, controlled by varying the thickness of the silica shell to 34, 22, and 15 nm. While all three AuNR@silica achieved MEF, reducing the silica shell thickness from 34 nm to 22 nm decreased the lifetime and maximized fluorescence enhancement at approximately 5‐fold relative to silica‐TCPP in the absence of AuNR, implying an increase in the radiative decay rate. Interestingly, an even smaller nanostructure‐fluorophore distance of ∼15 nm further reduced the fluorescence lifetime, but the overall enhancement deteriorated, implying an increase in k_nr in Equations 3 and 4, which is consistent with a new nonradiative decay pathway (energy transfer from the excited state dye to AuNR) becoming available at smaller nanostructure‐fluorophore distances [64].

As noted in the Introduction, MEF also depends on the nanoparticle morphology. Gao et al. observed MEF using gold nanostructures of different sizes and shapes coupled to the fluorophore Cy5.5, which has an emission maximum around 700 nm, for in situ imaging of intracellular microRNAs (miRNAs) [65]. Due to its low concentration at the single‐cell level, the real‐time monitoring of miRNAs in live cells is typically limited by photobleaching or weak fluorescence signals. In this example, silica‐coated gold nanostars (AuNST@SiO₂) with ∼150 nm AuNST diameter and adjustable shell thickness were decorated with covalently‐linked, Cy5.5‐labelled DNA. In this system, the fluorescence of Cy5.5‐labelled DNA was initially quenched by partial hybridization with a second DNA strand tagged with a BHQ‐3 (Black Hole Quencher‐3) FRET quencher. The probe was then attached to AuNST@SiO₂. If present, the target analyte miRNA‐21 can fully hybridize with the Cy5.5‐DNA strand, displacing the quenching DNA‐BHQ‐3 and turning on fluorescence, which was subsequently enhanced by MEF. The distance between Cy5.5 and AuNSTs can be regulated by changing the silica shell thickness. Optimization of the latter at 22 nm (similar to Yu et al. [62]) achieved a 21‐fold fluorescence enhancement and afforded 0.21 pM LOD for intracellular imaging of miRNA‐21 in both normal (L02) and cancer (MCF‐7 and HeLa) cell lines, enabling discrimination of aberrant miRNA‐21 expression levels and recognition of tumor cells within co‐cultured mixtures, which could have important implications for clinical diagnosis. Before delving into the mechanism of MEF in this specific system, it is important to briefly discuss the Authors’ choice of using Au ‘nanostars’, which are a type of gold nanoparticle characterized by their star‐like shape with multiple pointed tips. The sharp tips and branched morphology of gold nanostars lead to strong plasmonic effects and create regions where the electromagnetic field can be intensely concentrated. These areas are known as “hot spots” and are typical of nonspherical nanoparticles, such as nanorods, nanoplates (nanotriangles), nanocubes, and nanostars. Simply put, plasmonic ‘hot spots’ are regions at the tips, vertices, sharp corners, edges, etc., where the local electromagnetic field is significantly enhanced due to the unique geometries of these particles compared to flat surfaces or spherical nanoparticles. With their multiple sharp tips and branched structures, gold nanostars produce highly enhanced fields at their pointed regions. In this case, and like Yu et al., the Authors designed a system where both the absorption [λ_Abs (max) ∼687 nm] and emission [λ_Em (max) ∼701 nm] of the dye are encompassed by the broad nanoparticle extinction spectrum (centered at 704 nm), theoretically allowing for both MEF mechanisms to occur simultaneously. On the one hand, finite‐difference time‐domain simulations (FDTD, see [66] for a lay explanation) revealed plasmonic ‘hot spots’ of the intensely concentrated electronic field around the AuNST sharp vertices that contribute to enhanced excitation of Cy5.5. On the other hand, the Authors’ semi‐quantitative analysis suggesting a reduced Cy5.5 fluorescence lifetime in the presence of AuNST@SiO₂ points to some contribution from plasmophoric MEF, which is consistent with the large size and nonspherical morphology of AuNST that offers a high degree of light scattering (recall from the introduction that for plasmophoric MEF a strong scattering component of the metal nanoparticle's extinction spectrum at the fluorophore's emission wavelengths is essential). Previously, the same corresponding authors reported a system in which uncoated gold nanobipyramids (AuNBPs) ∼50 nm in length were tethered to a Cy5.5‐labelled DNA hairpin for the intracellular analysis of telomerase activity [67]. Telomerase activity is highly expressed in over 90% of human cancer cells, while it is absent or significantly reduced in normal cells. This differential expression makes telomerase a valuable cancer marker. Here, optimizing the length of the nanoparticle‐dye linker to 49 oligonucleotide bases achieved a 10.4‐fold fluorescence enhancement in the presence of the target telomerase. This arrangement enabled the in situ detection of telomerase activity down to 23 HeLa cells (with a dynamic range of 40‐1200 HeLa cells) and discrimination between normal and cancer cells in co‐cultured mixtures. In this example, the AuNBP extinction spectrum (∼700 nm) was much narrower than that of AuNST@SiO₂ but still enveloped both the absorption and emission profiles of Cy5.5, making it difficult to distinguish the relative contributions of the two MEF mechanisms without the aid of single‐molecule techniques [21] or a detailed analysis of changes to the fluorescence lifetime and quantum yield. Regardless of the relative contributions of the two MEF mechanisms toward overall fluorescence enhancement, these examples demonstrate that nanostructure‐fluorophore separation is a key parameter for optimizing MEF of small or macrocyclic fluorophores; deciding whether or how best to employ it for individual biological applications of MEF should be an important case‐by‐case consideration for experimental design.

Key Takeaway

Controlling the ratio of fluorophores to nanostructures and/or their spatial separation in the experimental design (e.g., with a tether or linker, or a core/shell arrangement) can enable optimization of the MEF effect for different classes of common dyes.

Synthetic dyes and molecular fluorophores are not the only species whose emission can be enhanced by noble metal nanostructures. Recent work by Can et al. has shown that photoluminescent nitrogen‐doped carbon quantum dots (CQDs) embedded in nanocomposite materials comprising quasispherical gold nanoparticles (AuNP; 23 ± 7 nm) can also exhibit the beneficial properties of MEF up to 1000‐fold overall luminescence enhancement [68]. In recent years, carbon quantum dots (carbon‐based nanoparticles, CQDs) have emerged as low‐cost, sustainable nanoprobes for fluorescence imaging and microscopy [69, 70, 71]. Depending on preparation, their surfaces bear amino (NH₂), carboxy (COOH), or hydroxy (OH) groups, and they offer tunable emission and excitation, good biocompatibility, low toxicity and inherent water solubility—unlike many synthetic dyes. Consequently, CQDs are increasingly replacing traditional organic dyes and semiconductor quantum dots in various biomedical applications [72, 73, 74, 75]. In this example, N‐doped CQDs‐AuNPs hybrids were prepared with a simple one‐pot solid synthesis and used for imaging human dermal fibroblasts (HDF) and A459 lung epithelial cells. The emission spectrum [λ_Em (max) ∼457 nm] of CQDs has significant overlap with the AuNP SPR [λ_Abs (max) ∼524 nm] while CQD absorption is primarily in the UV region [λ_Abs (max) ∼357 nm], suggesting minimal contribution from the increased excitation MEF mechanism for excitation wavelengths below 450 nm. While no attempt to control the gold‐CQD distance was reported in this case (yielding a less precise mixture of distances), the simple one‐pot sample preparation in water and probe design, along with the high staining efficiency and cytocompatibility exhibited by the CQD‐AuNP hybrid in both healthy and cancer cell lines, are all promising for the continued development of bioimaging applications.

Key Takeaway

In addition to synthetic dyes, the principles of MEF can also be applied to the design of nanocomposite systems that enhance the utility of carbon‐based luminescent materials for bioimaging applications.

In addition to gold nanostructures, plasmonic silver nanostructures (AgNP) can also be used to achieve MEF and may offer advantages over gold nanostructures depending on the nature of the experimental investigation. For example, our group has reported several examples of MEF by AgNP [21, 76, 77, 78], including a seminal example using spherical 3 nm AgNP to study lysosome dynamics via live‐cell fluorescence microscopy. We demonstrated that AgNP fed to RAW macrophages are small enough to accumulate within lysosomes without impacting their functionality and are effective at enhancing fluorescence confocal imaging of cells labelled with several green‐emitting dyes such as lysosome‐targeted Alexa Fluor 488‐conjugated dextran, boron dipyrromethene (BODIPY)‐cholesterol, and DQ‐BSA (Figure 4) [79]. Further, we showed that AgNP inside lysosomes enhanced the fluorescence of GFP fused to the cytosolic tail of LAMP1, a lysosomal membrane protein, confirming the possibility of achieving MEF in live cells across the lysosomal membrane. This approach enabled us to reduce the excitation energy while maintaining a good signal‐to‐noise ratio, allowing for dynamic studies of lysosomes without affecting their motility. Notably, AgNP did not alter lysosomal properties, such as pH, degradative capacity, and membrane integrity, suggesting that lysosomal loading of AgNP is a valuable approach for studying their dynamics while minimizing photobleaching and phototoxicity. Additionally, leveraging MEF to enable the use of green‐emitting fluorophores at low laser power opens the door to colocalization experiments with red‐emitting dyes, a topic of ongoing research in our lab.

FIGURE 4.

(top panel) AgNP‐enhanced fluorescence of BODIPY‐cholesterol, DQ‐BSA and Alexa Fluor 488‐conjugates in RAW macrophages. (A, C, and E) RAW cells were fed vehicle, 50‐µl AgNP, or 250‐µl AgNP, followed by 10‐µg/ml BODIPY‐cholesterol for 40 min and chased for 10 min (A), or 10‐µg/ml DQ‐BSA for 1 h (C), or 100‐µg/ml Alexa Fluor 488‐conjugated dextran (E). Cells were then imaged by spinning disk confocal microscopy. Images are shown in grayscale (top) or false color (bottom), where white‐yellow is the highest intensity and black‐blue is the lowest intensity. Scale bar = 10 µm. (B, D, and F) Quantification of fluorescence intensity of BODIPY‐cholesterol per cell (B), or DQ‐BSA per cell (D), or Alexa Fluor 488‐dextran per cell (F), normalized to cells without AgNP. Shown is the mean ± SEM from N = 4 experiments, where 15–30 cells were quantified per condition per experiment. One‐way ANOVA and Tukey's post hoc test were used to compare means, * p < 0.05, ** p < 0.01. (bottom panel) Normalized steady‐state absorption (20°C, CH₃CN) and emission spectra of BODIPY493/503 (20°C, CH₃CN, λ_Ex = 460 nm), and extinction spectrum of AgNP (20°C, water). Adapted from [ref. 79].

In terms of spectral properties, small, spherical AgNP ‘seeds’ scatter less light than larger nonspherical nanostructures, and their extinction spectrum [λ_Abs (max) ∼390 nm, see Figure 4] is well‐known to be dominated by nanoparticle absorption [80], making them ideal candidates for amplifying fluorescence via the increased excitation MEF mechanism. We paired these AgNP with multiple green‐emitting dyes whose absorption (centered at 500 nm, Figure 4) overlaps with nanoparticle extinction. While dye emission (∼515/520 nm) exhibits less overlap with AgNP extinction, the plasmophoric MEF may have contributed to the 2‐ to 6‐fold fluorescence enhancement observed with different green‐emitting dyes. Having established this proof of concept, it may even be possible to further enhance the fluorescence of green‐emitting dyes in live cells using spherical AgNP and/or AuNP that exhibit moderately red‐shifted extinction spectra while remaining small enough to avoid impact on intracellular and/or intra‐lysosomal processes. Overall, our work showcased the versatility of AgNP in enhancing multiple green‐emitting fluorophores without causing cytotoxicity or altering lysosomal properties, creating a versatile tool to monitor lysosome motility and study the endo‐lysosomal pathway at reduced laser power (5‐fold lower laser power and 10‐fold shorter exposure time than standard imaging conditions) to avoid excessive light energy that could damage fluorescent probes and be toxic to living samples.

Key Takeaway

Like plasmonic gold nanostructures, silver nanostructures are another practical tool for utilizing MEF in biological systems that can enable the use of green‐emitting fluorophores at low excitation power, co‐labelling with red‐emitting fluorophores and may be advantageous in cases where smaller plasmonic nanostructures are needed to utilize MEF without impacting intracellular processes.

Antonescu and coworkers have now extended the utility of this approach to study lysosomal nutrient sensing and response mechanisms toward a deeper understanding of cellular adaptation to metabolic stress. In brief, spherical 3 nm AgNP loaded into the lysosomes of ARPE‐19 epithelial cells transfected with doxycycline‐inducible DEPDC5‐eGFP expression functioned as a nonperturbing tool to monitor and enhance eGFP fluorescence by live‐cell spinning disc confocal microscopy, at a low doxycycline concentration. Endogenous levels of expression of DEPDC5 is quite low, estimated to be <4000 copies per cell, such that study of DEPDC5‐eGFP at levels that approximate endogenous expression makes detection of the eGFP signal without MEF very challenging. This approach identified a substantial overlap of DEPDC5‐eGFP with lysosomes that was not diminished by amino acid starvation or subsequent nutrient replenishment, suggesting that regulation of DEPDC5 conformation by amino acid signaling plays a critical role in regulating the functions of glycogen synthase kinase 3 (GSK3ß) that can impact cell physiology and survival [81].

Table 1 summarizes and compares the discussed applications and includes additional representative examples, organized to facilitate cross‐study comparisons.

TABLE 1.

Representative applications of metal‐enhanced fluorescence (MEF) in cell imaging.

Biological model or imaging context	Fluorophore (type, λEm)	Metal nanostructure	Fluorophore‐metal separation	Reported enhancement factor	Ref.
Tumor imaging SKOV‐3, OVCAR‐8	SWIR dye IR‐E1050 λEm ≈ 1000 nm	Au nanorods	Polymer layer	Up to 45×	[58]
ATP sensing HeLa	IRDye800CW λEm ≈ 810 nm	Au nanorods	DNA aptamers	Up to 9×	[62]
H2S sensing A549, H1299	TCPP porphyrin λEm ≈ 650 nm	Au nanorods	Silica shell	Up to 5×	[63]
miRNA sensing HeLa, MCF‐7, L‐02	Cy5.5 λEm ≈ 700 nm	Au nanostars	Silica shell	Up to 21×	[65]
Telomerase sensing HeLa, L‐02	Cy5.5 λEm ≈ 700 nm	Au nanorods Au nanobipyramids	DNA strands	Up to 10.4×	[67]
HDF, A549	N‐doped Carbon Dots λEm ≈ 450 nm	Au, quasispherical	Not controlled (carbon matrix)	Up to ∼1000×	[68]
RAW 264.7	Alexa Fluor 488 BODIPY DQ‐BSA GFP λEm ≈ 515 nm	Ag spheres	Not controlled	Up to 8× (dye‐dependent)	[79]
miRNA sensing HeLa, MCF‐7, CCC‐HPF‐1	Cy5 λEm ≈ 670 nm	Au nanorods	DNA strands	Up to 8.3×	[82]
miRNA sensing HepG2, H9C2	Cy5 λEm ≈ 670 nm	Au nanorods (quencher) Au nanoclusters (enhancer)	DNA strands	OFF—ON	[83]
Single‐molecule fluorescence measurements of ribosomal initiation complex	Cy3 λEm ≈ 570 nm Cy5 λEm ≈ 670 nm	Ag spheres	PEG, Biotin–streptavidin–mRNA Not controlled	Up to 4.2× (Cy3) Up to 5.5× (Cy5)	[84]

4. Key Takeaways for Applying MEF in Bioimaging

To translate the concepts analyzed in this short review into further practical use, we highlight a set of guiding considerations for applying plasmonic metal nanostructures in bioimaging. These points combine theoretical principles with practical design lessons from the cited recent studies and are intended as a quick reference. Together, they offer a concise framework for selecting effective dye–nanoparticle pairings and tailoring experiments to specific imaging goals.

Consideration	Guideline / Insight
1. NP size, shape and optical properties	Optimize dimensions (readily tunable synthetically) to match the plasmon resonance and scattering profile to the fluorophore's emission/excitation. Select material (e.g., Au, Ag) and structure (nanoshells, rods, nanoplates, with high aspect‐ratio, polyhedral, etc.) to minimize absorption losses. When absorption dominates, the excitation energy is more likely dissipated non‐radiatively, leading to quenching.
2. Fluorophore–NP distance	Maintain a critical separation (typically 10–25 nm); too close leads to quenching, too far reduces enhancement.
3. Dye's quantum yield	Fluorophores with low‐to‐moderate ΦF show the greatest relative MEF gains, while high ΦF (> 0.8) benefit less, with smaller enhancement (less room for improvement), but still improved photostability and reduced photobleaching.
4. Biological compatibility	Surface functionalization (dextran, peptides, silica shells, etc.) should support stability, targeting, and non‐toxicity in live‐cell systems.
5. Application‐driven design	Consider whether the goal is sensitivity (single‐molecule detection), long‐term tracking (photostability) or multiplexing; select nanoparticle–dye pairs accordingly.

MEF is most powerful when implemented as a design‐driven strategy, in which nanoparticle size, shape, and material are tuned to match the fluorophore's optical properties and favor scattering over absorption, and the dye is positioned at an optimal separation from the metal surface. Under these conditions, MEF is particularly effective for fluorophores with low‐to‐moderate quantum yields, where increased radiative decay rates and improved photostability provide clear advantages. By contrast, MEF is not competitive when absorption losses dominate (and quenching occurs), when excessive separation suppresses enhancement, or when dyes have very high intrinsic quantum yields, which show only limited additional brightness gains.

5. Conclusions

Optimizing MEF hinges on complementary dye–nanostructure pairing. We emphasize that what ultimately determines enhancement or quenching depends on the specific pairing between the nanoparticle and the fluorophore, not the choice of materials alone. It is the relationship—the spatial and spectral match—between the two that governs the outcome. Nanostructures that scatter light efficiently within the dye's emission band—e.g., high‐aspect‐ratio or polyhedral particles—promote plasmophoric MEF by boosting radiative rates. Conversely, spherical nanostructures pair better with dyes whose absorption overlaps the SPR, enhancing excitation. Significant spectral overlap between fluorophore absorption and SPR isn't required for plasmophoric MEF, which can increase quantum yield and photostability. Nanoparticle size and shape—easily tuned synthetically—are key design levers. Optimization isn't “gold versus silver” or “this dye versus that,” but deliberate nanoparticle‐fluorophore pairing—matched in size, shape, composition, and spectral overlap—that ultimately determines whether fluorescence is boosted or suppressed.

The intrinsic quantum yield of a fluorophore also plays a role in determining how much benefit it can actually derive from MEF. Dyes with low‐to‐moderate Φ_F values (typically below 0.3) often display the most dramatic relative enhancements. This is because plasmonic coupling effectively pushes their quantum efficiency closer to unity. By contrast, dyes with already high Φ_F values (above ∼0.8), such as Alexa Fluor series dyes, emit efficiently on their own. In these cases, MEF cannot drastically increase quantum yield further, so the relative enhancement in brightness is smaller (often in the range of 1.5‐3 fold compared to the 10‐50 fold achievable with lower Φ_F dyes). Nevertheless, even for these highly efficient fluorophores, coupling to metal nanostructures can still provide meaningful benefits, including increased brightness under low excitation conditions and significantly improved photostability/reduced photobleaching.

Controlling fluorophore: nanostructure ratio and spacing (e.g., via linkers/tethers or core–shell arrangements) enables MEF optimization across dye classes and does not require elaborate or inaccessible methods. Simple strategies can be highly effective! Coating metal nanoparticles with a thin silica layer and tuning the thickness of this silica shell—something that can be achieved with well‐established wet‐chemistry methods—offers straightforward, reproducible control of dye‐metal spacing—thick enough to avoid short‐range quenching yet close enough for plasmonic enhancement. Defined‐length DNA or molecular tethers can likewise set dyes at precise, nanometer‐scale distances from the nanoparticle surface and fix the number of dyes per particle. The ratio of multiple fluorophores per nanoparticle (the “loading”) is easily tuned by varying dye concentration during conjugation/embedding (silica shells are particularly useful here, as dyes can be incorporated during synthesis, with the amount of fluorophore added dictating the final loading), or by attaching at defined numbers and positions via DNA or polymers. These core–shell and linker strategies are scalable, biocompatible, and rely on standard wet‐chemistry and bioconjugation methods, rather than complex nanofabrication.

Beyond synthetic dyes and biomolecular fluorophores, MEF principles also apply to nanocomposites with emerging carbon‐based luminescent materials, such as carbon quantum dots—tunable in emission, low‐cost, intrinsically hydrophilic, and biocompatible. Their modest quantum yields can limit sensitivity in biological applications, but coupling them to plasmonic nanostructures makes it possible to boost their brightness and help overcome these limitations. Practical options include embedding carbon dots within silica shells grown around Ag or Au cores (e.g., CQDs entrapped within Ag@SiO₂ nanoshells [85] have shown strong fluorescence enhancement from optimized spacing) or light doping carbon nanomaterials with trace metals (as in one of the examples cited here). These hybrid nanocomposite systems not only broaden the palette of emitters that benefit from MEF but provide a bridge between classical dye imaging and greener carbon‐based probes.

This short review highlights both the practical power of plasmonic metal nanostructures for live‐cell bioimaging and the need for clear, accessible design principles for experimental adoption. Our aim has been to distill some takeaways to help researchers choose effective dye–nanoparticle combinations. Looking forward, MEF raises important considerations for multiplexed fluorescence imaging in biological systems. The increased brightness and improved photostability afforded by MEF can be advantageous for multi‐channel experiments, particularly when imaging low‐quantum‐yield fluorophores or operating at reduced excitation intensities, alleviating photobleaching and phototoxicity during extended or multicolor acquisition. In practice, however, these benefits are not uniformly realized across all dyes in a multiplexed setting. MEF enhancement is highly sensitive to fluorophore‐nanostructure distance and to spectral overlap with the plasmon resonance, as discussed throughout this review. As a consequence, different fluorophores within a co‐stained sample may experience different degrees of enhancement, potentially leading to channel imbalance or distorted relative signal intensities. Moreover, fluorophores that already exhibit high quantum yields or are spectrally mismatched to the plasmonic response may show minimal benefit or even quenching under otherwise identical conditions. These considerations help explain why most MEF studies to date have focused on single‐fluorophore systems. This reflects fundamental photophysical constraints rather than a lack of interest. While proof‐of‐concept demonstrations of selective enhancement of one fluorophore over another—such as dual‐color systems in which a single channel is intentionally enhanced, or multicolor configurations that are spatially separated—exist, broadly applicable and quantitative multi‐channel MEF imaging has not yet been realized. Together, these limitations define a realistic frontier for the field and emphasize the importance of deliberate photophysical and experimental design, which motivates the framework and guidance presented in this review. MEF is a readily available, implementable strategy that can deliver substantial improvements in brightness, photostability, sensitivity—pushing the boundaries of bioimaging—and, with clear design and application‐driven guidelines, is ready for immediate adoption in routine workflows. It is our hope that this work will help bridge the gap in translation between fundamental plasmonics and real‐world bioimaging—a challenge that remains central to our ongoing investigations and collaborations.

Author Contributions

S.I. conceived the study. G.K.H. performed the investigation. G.K.H. and S.I. curated the data and conducted formal analysis. S.I. validated the results and provided resources. G.K.H. and S.I. wrote the original draft. S.I. reviewed and edited the manuscript, prepared the visualizations, supervised the project, administered the project, and secured funding.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data sharing is not applicable to this article, as no datasets were generated or analyzed during the current study.