Intracellular gallium nitride microrod laser

The study addresses limitations of conventional fluorescent dyes for cell labeling—namely broad emission spectra, photobleaching, and low efficiency—which hinder multiplexed tracking of large cell populations. Intracellular lasers offer narrow linewidths and high signal-to-noise ratios, but prior inorganic implementations (e.g., CdS, InGaAsP, AlGaInP) raise toxicity concerns. The research question is whether biocompatible GaN microrods can function as intracellular lasers with low thresholds and stable emission suitable for labeling and tracking living cells. The work aims to fabricate GaN microrods, internalize them into HeLa cells, assess biocompatibility, and characterize their lasing behavior under intracellular conditions.

Prior work shows intracellular microlasers can act as barcode-like probes with much narrower linewidths than fluorescent dyes, enhancing tracking fidelity. Inorganic resonators such as CdS nanowires, AlGaP multiquantum well nanodisks, and InGaAsP nanodisks have demonstrated strong lasing but involve toxic or uncertainly biocompatible materials. GaN is highlighted as a nontoxic, biocompatible semiconductor with high refractive index and optical gain, making it a promising candidate for intracellular lasers. Earlier GaN micro/nanostructures on rigid substrates have achieved low lasing thresholds; this study extends such capabilities to intracellular environments using microrods that can be released from graphene substrates and internalized by cells.

• Growth of GaN microrods: GaN microrods were grown on CVD-synthesized graphene transferred onto SiO₂/Si substrates using MOVPE without metal catalysts. A two-step temperature process (750–850 °C for 3 min; 950–1050 °C for 30 min) was used, followed by heating at 1100 °C for 10 min in hydrogen. Precursors: TMGa, DTBSi, and NH₃ with flow ranges of 15–30, 1–3, and 100–500 sccm; nitrogen carrier gas; reactor pressure 300 Torr with H₂/N₂ gas mixture. Resulting microrod areal density across graphene was ~1×10⁷ cm⁻². The weak van der Waals interaction between GaN and graphene enabled facile detachment.
• Cell culture: HeLa cells were cultured in modified Eagle’s medium with 10% fetal bovine serum, 1% GlutaMAX, and 1% penicillin/streptomycin at 37 °C, 5% CO₂.
• Microrod sterilization and internalization: As-grown microrods on graphene/SiO₂/Si were sterilized in 95% ethanol for 1 h. Microrods were detached by sonication for 1 min in 1 ml culture medium. The suspension concentration was adjusted to 10⁷ ml⁻¹, and 200 µl was added to a culture dish. Internalization into cells occurred naturally via endocytosis within hours.
• Fluorescence imaging and CLSM: Cytoplasm and nuclei were labeled with calcein AM and DAPI, respectively. Imaging used an inverted microscope (Olympus IX73) with EMCCD (Andor) and XYZ automated stage; epifluorescence excitation with white LED (Lumencor); 40×/0.6 NA objective; appropriate dichroics/filters (Chroma 49002 EGFP for calcein AM; 49000 DAPI for DAPI). 3D confocal laser scanning microscopy was used to confirm intracellular localization (Z-projections and cross-sections).
• Lasing measurements (micro-PL): Room-temperature µ-PL spectroscopy using the 355 nm third harmonic of a Nd:YAG laser (10 Hz, 10 ns pulses). The beam was focused to an ~8 µm spot with a UV objective (NA 0.50). A motorized stage positioned samples; emitted light analyzed with a monochromator and CCD.
• Viability assays: Phase-contrast time-lapse OM to observe migration and division over two weeks. Live/dead staining used calcein AM (live) and ethidium homodimer-1 (dead). Post-lasing exposure viability was also assessed after focused beam exposure (10 mJ/cm², 2 s).

• Efficient internalization: Time-lapse imaging showed endocytosis of GaN microrods within minutes once contacted by filopodia/lamellipodia; >70% of 53 cells contained microrods.
• Confirmed intracellular localization: CLSM Z-projection and cross-sectional images showed microrods within the cytoplasm.
• Biocompatibility and normal function: Cells containing microrods migrated and underwent normal mitosis over 2 weeks; microrods remained in one daughter cell post-division. Calcein AM staining positive for all cells; ethidium homodimer negative, indicating viability. Cells remained alive after focused laser exposure at 10 mJ/cm² for 2 s.
• Intracellular lasing: Under 355 nm pulsed excitation, below threshold a broad near-band-edge emission (~370 nm) was observed; above threshold, sharp peaks appeared indicative of lasing.
• Lasing threshold: Ith ≈ 270 kW/cm², comparable to high-quality GaN micro/nanostructures on single-crystal substrates.
• Resonator characteristics: Mode spacing Δλ ≈ 1.11 nm consistent with Fabry–Perot oscillation along the rod axis; inferred cavity length L ≈ 12.3 µm, matching SEM. Average Q-factor ≈ 620 (from ~0.6 nm linewidth). WGM lasing excluded (expected WGM spacing ~10 nm for 1.5 µm diameter rods).
• Power dependence: Integrated PL slope below threshold ≈ 3.7; above threshold ≈ 10.4.
• Dimensions: SEM showed typical diameter 1.5 ± 1 µm and length 10 ± 3 µm; mode-spacing analysis gave average length 11.5 µm with SD 2.4 µm.
• Environmental robustness and individuality: Lasing spectra varied among microrods (center wavelength, spacing, and modal intensities), enabling unique spectral barcodes for tracking. Refractive index variations in the cellular environment affected different transverse modes similarly, preserving spectral profiles for tagging.
• Optical indices: High refractive index of GaN (n ≈ 2.6) and cellular medium (n ≈ 1.36) contributed to low intracellular threshold.

The study demonstrates that GaN microrods can be internalized by mammalian cells and operate as stable, low-threshold Fabry–Perot microlasers in the intracellular environment without compromising cell viability or normal cellular activities. The high optical quality of MOVPE-grown GaN on graphene and its high refractive index enable efficient lasing despite the low-index cellular milieu. The distinct and stable lasing spectra of individual microrods provide a practical basis for barcode-like cell labeling and tracking. The observed threshold and Q-factors align with or approach those reported for GaN structures on crystalline substrates, validating the graphene-assisted growth and release strategy. Furthermore, the spectral resilience to refractive index fluctuations suggests robustness for dynamic intracellular environments, enhancing applicability for long-term tracking. Overall, the findings address the need for narrow-linewidth, high-SNR intracellular probes while leveraging a biocompatible material system.

GaN microrod lasers grown on graphene by MOVPE can be readily detached, dispersed, and internalized into HeLa cells, where they lase with low threshold (~270 kW/cm²) and exhibit distinct, robust spectra suitable for cell labeling and tracking. Cells maintain viability, migration, and division over weeks, indicating good biocompatibility. While UV excitation is effective, it is not ideal for long-term or in vivo applications; future work will develop InGaN nanorod intracellular lasers to enable visible-range emission by tuning In composition. GaN-based intracellular microlasers thus present a promising platform for multiplexed cellular tagging and tracking.

The demonstrated lasers operate at UV excitation (355 nm), which may be unsuitable for long-term experiments and in vivo imaging due to potential phototoxicity. The study focuses on HeLa cells; broader biocompatibility across cell types and in vivo contexts was not assessed. Spectral analyses indicate multi-transverse-mode behavior, which can complicate spectra in some cases.