Advancing Plant Cell Imaging: The Transformative Potential of Optimised HaloTag Technology

In recent years, single-molecule live-cell imaging technology has emerged as a crucial tool in life sciences due to its high resolution and throughput. Current labelling methods face limitations: organic dyes lack spectral specificity, quantum dots exhibit biological toxicity, and fluorescent proteins suffer from low brightness and rapid photobleaching. Monitoring the dynamic changes and functional activities of receptors and key enzymes on cell membranes remains technically challenging (Kumar et al. 2024; Wolfram et al. 2024). A novel hybrid labeling technology, combining ligand-conjugated dyes with genetic tags, overcomes these challenges by offering high brightness, stability, specificity, and sparse labeling adjustability. Among existing approaches, HaloTag Technology is one of the widely used methods for single-molecule live-cell imaging. As shown in Figure 1, this system utilises a genetically engineered protein tag derived from a modified dehalogenase enzyme. The system relies on stable, irreversible covalent bonding of the tag protein with synthetic ligands (Los et al. 2008). When fused with target proteins, it enables specific labeling with fluorophore-conjugated probes, offering a superior signal-to-noise ratio, modular flexibility for multicolour imaging, and exceptional stability for long-term live-cell imaging and dynamic tracking (Grimm et al. 2020).

HaloTag technology has been widely applied for protein localisation, dynamic tracking, and interaction analysis in mammalian cells (Kim et al. 2024; Tyagi et al. 2024). However, plant cell imaging faces challenges such as probe penetration barriers, autofluorescence interference, and insufficient expression of labelled proteins. These limitations make HaloTag technology a promising solution for overcoming these bottlenecks. Although previous studies have utilised this technology for precise protein localisation analysis, real-time molecular tracking, and protein-protein interaction detection, its potential in transient transformation and stable transgenic plants remains unclear (Minner-Meinen et al. 2021).

Recently, Qian et al. (2025) published a study in Plant Cell & Environment reporting that they successfully optimized HaloTag technology for plant research, demonstrating its advantages in super-resolution imaging, multicolor imaging, and spatiotemporal labeling differentiation (Figure 1). Their successful application of single-particle tracking highlights HaloTag's potential as a versatile tool for investigating spatiotemporal dynamics and various biological processes in live cells. Qian et al. (2025) explored the optimisation of HaloTag technology in transgenic plants, demonstrating that the HaloTag-fused protein maintains functional integrity in these plants and that the HaloTag can be efficiently labeled with dyes. Moreover, they optimized this system, significantly shortening the total experimental duration. In addition to its successful applications in model plants (N. benthamiana and Arabidopsis), the optimized system is predicted to be widely applicable in other plant species with minor modifications.

Since the signal loss due to the photobleaching of fluorescent proteins has significantly limited live-cell imaging in plant cells, Qian et al. (2025) demonstrated that HaloTag-fused proteins enable Structured Illumination Microscopy (SIM) imaging with superior resolution and higher brightness, providing clearer organelle visualisation and enhanced multicolor imaging capabilities. Additionally, HaloTag exhibits strong and stable fluorescence intensity, superior resistance to photobleaching, and enables prolonged continuous fluorescence imaging. More importantly, the monomeric properties of the HaloTag minimise its interference with the function of target proteins. Therefore, HaloTag expands the spatiotemporal dimensions of live-cell imaging and demonstrates great application prospects.

HaloTag technology also serves as a powerful tool for protein dynamics analysis, offering distinct advantages for imaging and tracking applications. Its exceptional brightness enables enhanced temporal resolution through reduced exposure times, which is particularly valuable for capturing rapid dynamics of plasma membrane (PM) proteins (Qian et al. 2025). The successful combination of HaloTag technology with single-particle tracking reveals the specific dynamics of membrane proteins and changes in the rearrangement of endoplasmic reticulum (ER)–plasma membrane (PM) connectivity, providing a powerful tool for analysing protein dynamics (Qian et al. 2025).

With the rise of plant synthetic biology and precision agriculture, HaloTag holds great promise in real-time monitoring of stress responses, gene editing dynamics, and disease-resistant protein networks. Future development directions include: (1) designing plant-specific photo-controllable probes for spatiotemporally precise labeling; (2) integrating multi-technology (e.g., photoactivatable fluorescent proteins [FPs], quantum dots, Sun Tag) to enhance live-cell imaging; and (3) developing reversible covalent ligands to enable repeated labeling and extend observation windows. By addressing these challenges, HaloTag could become a next-generation ‘molecular microscope’ to decode plant life processes, bridging fundamental research and agricultural innovation.

In conclusion, Qian et al. (2025) optimized HaloTag applications in plant systems for the first time, establishing its versatility as a superior live-cell imaging tool with unique advantages for plant science. This study provides novel insights into HaloTag technology and opens new perspectives for investigating unexplored biological questions. HaloTag is reshaping the paradigm of plant cell imaging with its precision, flexibility, and minimal interference. Despite existing technical challenges such as permeability and signal interference, interdisciplinary innovations are expected to establish this technology as an indispensable tool in plant science within the next decade.

The authors declare no conflicts of interest.