Tunable red to green emissions in upconversion nanoparticles allow precise identification of single particles, enabling accurate, long-term multicolor tracking in complex live cell environments.
(Nanowerk Spotlight) Live cell imaging relies on the ability to follow individual molecules as they move through membranes, interact with receptors, or enter the cell. Single particle tracking, a method that enables this, is widely used to monitor the real time dynamics of proteins and receptors.
But researchers still face two persistent problems: First, the fluorescent dyes commonly used in these experiments degrade under light exposure, limiting the time window for observation. Second, identifying and tracking multiple types of molecules simultaneously, especially in crowded and complex environments, requires imaging systems that often involve specialized optics and time consuming post processing.
The challenge deepens when probes are distinguished by brightness. Fluorescence intensity can vary depending on a particle’s size, environment, or orientation, making it difficult to reliably assign identity. Ratiometric imaging offers a potential solution by using the ratio of two emission wavelengths from the same particle instead of relying on total brightness. This approach avoids spectral overlap and allows for faster, simpler imaging. However, most ratiometric probes are organic dyes with overlapping emission spectra and limited photostability, which restricts their usefulness in long term or multicolor imaging experiments.
The team designed a strategy to tune the red to green emission ratio in individual particles, creating stable spectral fingerprints that allow each type of particle to be uniquely identified. This makes it possible to track multiple particle populations in live cells over time without relying on complex optical systems or brightness based classification.
These nanoparticles are doped with ytterbium and erbium ions. When excited with near infrared light at 980 nanometers, ytterbium ions absorb the energy and transfer it to erbium ions, which then emit visible light. Green light results from a two photon process. Red light comes from a three photon pathway that requires more energy input. By controlling the number of excited ytterbium ions and how energy flows through the system, the researchers were able to shift the emission balance toward either red or green light.
This balance, known as the red to green or R to G ratio, provides a reliable way to distinguish particles. Because it reflects internal energy dynamics rather than external brightness, the ratio remains stable even when particles vary in size or are exposed to different conditions.
Mechanisms of tunable emissions of Yb, lanthanide-doped upconversion nanoparticles. According to the rate-equation model, at a constant particle volume, decreased spatial separation and higher density of excited Yb3+ ions around Er3+ ions lead to an increased third-photon energy transfer rate (kET3→6n′1) from Yb3+ to Er3+, favoring red emission; conversely, when this rate is lower, green emission becomes dominant. The parameter kUC2 corresponds to the transition rate from energy level 2 (2H11/2) to the ground state, typically treated as a constant. The solid gray arrow represents the transitions from energy level 3 (2H11/2) to 6 (4G7/2), driven by energy transfer from excited Yb3+ ions, while the dashed gray arrow indicates back energy transfer from Er3+ to Yb3+. The green and red arrows depict green and red emissions, respectively. The dashed purple arrow represents non-radiative relaxations between energy levels. (Image: reprinted with permission by Wiley-VCH Verlag)
The team developed a theoretical model showing that the R to G ratio depends on two key factors: The first is the efficiency of energy transfer from a third excited ytterbium ion to an already excited erbium ion. The second is the overall number of excited ytterbium ions in the system. When both are high, red emission becomes dominant. When they are low, green emission takes over.
To test this, the researchers created a series of nanoparticles with varying concentrations of ytterbium and erbium ions. They used a core shell shell structure to keep the overall particle size consistent, which allowed them to isolate the effects of doping levels.
Microscopy confirmed the particles were uniform, and photoluminescence measurements showed that increasing ytterbium concentration increased the R to G ratio, while increasing erbium concentration decreased it. This behavior matched the predictions from the model and revealed more than a tenfold range of tunability.
Single particle imaging confirmed that the R to G ratio was a more reliable identifier than brightness. The team used a dual channel microscope to observe individual particles and showed that, while total intensity varied, the R to G ratio remained consistent for each particle type. They used this ratio to distinguish five particle types with a misidentification rate below five percent, a level of accuracy not achievable with brightness based methods alone.
To support the experimental data, the researchers ran Monte Carlo simulations that modeled energy transfer events at the level of individual ions. These simulations showed that higher excitation power increases the number of excited ytterbium ions, which shifts the emission pattern toward red. At lower power, or with more erbium ions acting as energy sinks, green emission becomes dominant.
These findings confirmed that the R to G ratio reflects how energy is distributed among pathways rather than absolute output.
For use in biological systems, the nanoparticles were coated with polyethylene glycol to increase stability and compatibility with living cells. The R to G ratio remained stable across buffer solutions and illumination conditions, and the particles resisted photobleaching during extended imaging. This made them suitable for tracking in both model membrane systems and live cells.
In a supported lipid bilayer, the researchers mixed five types of particles and tracked their motion. Brightness alone could not separate the populations, but the R to G ratio clearly distinguished them. Each particle type formed a distinct cluster based on its ratio, and the identities remained stable during movement. This showed that ratiometric labeling works under conditions that simulate the cell membrane environment.
The method was then applied to live MG63 cells. Two particle types were used to label biotin receptors on the cell surface. Over ten minutes of imaging, the team followed the motion of these receptors with nanometer scale precision. The data revealed four types of behavior. Some receptors moved freely, others became confined in membrane regions, some resumed motion after confinement, and others displayed directed movement inside the cell. These patterns correspond to different stages of endocytosis and intracellular transport, and were resolved clearly using the R to G based tracking method.
This study demonstrates a practical way to encode particle identity using internal energy transfer rather than external labels. By tuning the red to green emission ratio, the researchers created a system that resists photobleaching, avoids spectral overlap, and allows simultaneous tracking of multiple targets with minimal hardware complexity. The ability to control and predict emission behavior through composition makes the approach highly adaptable for different biological systems.
Rather than relying on external filters, separate dyes, or complex signal processing, this method embeds identity in the particle’s own emission profile. It offers a scalable, stable, and interpretable solution for single particle imaging, with direct applications in live cell tracking, receptor dynamics, and other high precision bioimaging studies.
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