Enriching microlaser-labelled cells by commercial flow cytometry-based cell sorting systems

Technical Summary: Enriching Microlaser-Labelled Cells by Commercial Flow Cytometry-Based Cell Sorting Systems

Problem Statement
Whispering-gallery mode (WGM) microlasers, typically polystyrene particles (LPs) doped with fluorescent dye, offer unique advantages for high-resolution cellular tagging, single-cell tracking, and biosensing due to their bright, spectrally narrow emission. However, a critical bottleneck exists in applying these tools to biomedical and tissue-engineering contexts: the inability to reproducibly control the number of LPs within a cell population. While thousands of labelled cells can be identified in culture, ensuring a defined purity and composition of the underlying cell population regarding LP labelling density is essential for advanced applications. Existing methods for sorting cells based on the spectral barcodes of internalized LPs rely on custom-built microfluidic setups and complex optical systems, which are difficult to build, maintain, and operate, limiting their accessibility to specialized core facilities.

Methodology
The authors developed a workflow utilizing widely available commercial flow cytometry and cell sorting systems to purify cell populations containing large LPs without custom microfluidics.

• Particle Selection and Characterization: Two types of commercial LPs (LP-A and LP-B) were compared. Their lasing performance, photoluminescence (PL) brightness, size distribution, and uniformity were characterized using optical pumping, flow cytometry (FSC-A, SSC-A, Red-PL), and brightfield microscopy.
• Surface Modification: To enhance LP-cell association, LP surfaces were modified with poly(ethyleneimine) (PEI) or lipofectamine to reverse their surface charge from negative to positive, promoting interaction with the negatively charged cell membrane.
• Sorting Strategy: HEK293T cells were incubated with LPs and sorted using a BD FACSAria Fusion. The gating strategy relied on forward- and side-scattering properties (FSC-A, SSC-A) to distinguish between bare LPs, LP-free cells (LP-), and LP-containing cells (LP+). Viability was ensured by excluding DAPI-positive events (dead cells). Singlet discrimination was performed to exclude aggregates.
• Validation and Analysis: Sorted populations were validated using imaging flow cytometry. The physical interaction between LPs and cells was analyzed via Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) to determine internalization versus surface attachment.
• Tissue Engineering Application: Sorted LP+ and LP- cells were mixed at defined ratios and seeded into ultra-low attachment plates to form multi-cellular spheroids. These spheroids were scanned using a hyperspectral confocal microscope to detect lasing signals and calculate LP diameters based on emission spectra.

Key Results

• LP Performance and Uniformity: LP-B exhibited superior lasing performance, showing robust lasing at low pump energies (4 nJ) compared to LP-A, which required higher energies and showed lower lasing efficiency. Flow cytometry revealed that LP-B had higher brightness and a narrower size distribution, whereas LP-A showed broader heterogeneity in size and gain medium content.
• Surface Modification and Uptake: Uncoated LPs showed variable attachment; LP-A attached to or was internalized by cells, while LP-B showed minimal attachment. Coating with PEI or lipofectamine significantly improved cell association. PEI-coated LPs showed the strongest interaction, with LPs localized exclusively on cells after washing.
• Sorting Efficiency and Cell Viability: The commercial sorter successfully separated LP+, LP-, and bare LP populations. However, manufacturer-dependent differences were observed: LP-A showed higher internalization efficiency (19.2% of events were LP+) compared to LP-B (5.1%). While cells associated with LP-A maintained proliferation rates similar to controls, cells associated with LP-B (both LP+ and LP- fractions) exhibited a ~20% reduction in confluency after 5 days, suggesting a potential inhibitory effect of LP-B on cell growth.
• Mechanism of Interaction: SEM and TEM imaging revealed distinct interaction mechanisms. LP-A were fully internalized, with the cell membrane completely engulfing the particle and placing it in direct contact with the cytosol. In contrast, LP-B were only partially engulfed, forming a membrane cup around the particle without direct contact with the cytosol.
• Spheroid Engineering: The sorting approach enabled the generation of spheroids with defined ratios of LP-containing and LP-free cells. Lasing signals were detectable up to 300 µm deep within the spheroids, and individual LP diameters could be calculated from the emission spectra, even in optically dense tissue.

Significance and Claims
The paper claims to establish a foundation for larger tissue engineering studies by demonstrating that commercially available flow cytometry systems can be effectively adapted for the purification and analysis of cells labelled with large microlasers. The authors assert that this scatter-based approach eliminates the need for complex, custom-built microfluidic setups, making high-resolution optical tagging and single-cell tracking more accessible.

Furthermore, the study highlights the importance of characterizing LP properties (uniformity, brightness) and surface chemistry, as these factors critically influence lasing performance, cellular uptake mechanisms, and cell viability. The ability to precisely control the density of LPs within spheroids opens new avenues for deep-tissue sensing and the systematic investigation of cell-particle interactions. Finally, the authors suggest that this framework provides a robust model for studying the physiological impact of environmental microplastics on human health, given the ability to track and monitor these particles at the single-cell level.