A new class of nano/microsized imaging probes has emerged that harness narrowband laser emission to enable massively multiplexed cell tagging. These laser particles (LPs) are particularly promising for single-cell analysis for several reasons. First, they provide a means for narrowband laser emission from inside cells, This enables cell tagging by spectral barcoding, which is far more reliable than barcoding based on intensity levels or imaging features that are of limited use in tissue due to wavelength-dependent absorption and scattering. Second, these spectral barcodes can be repeatedly read in a nondestructive manner. This allows cell tracking over time and space in biological systems, as well as the retention of individual cellular identity across different analytical platforms, which enables the association of spatial information and other image-derived phenotypes with molecular profiles.
Several potential designs have been reported, including fluorescent polystyrene particles20, 21, upconverting nanoparticle microlasers22, perovskite nanowires23, and plasmonic nanoparticles24. Our recent study employed silica-coated Ⅲ-Ⅴ semiconductor microdisk LPs with diameters of ~2 μm and total thicknesses of ~400 nm25. Our microdisk LPs have several key properties that make them more suitable for cellular barcoding than the previous designs, including relatively small sizes (~0.1% of the cell volume), stability in aqueous environments, biocompatibility, and tunable wavelengths over a wide range. Silica-coated microdisk LPs were readily internalized into a variety of cell types through macropinocytosis and had no appreciable effect on cell viability, cell cycle time, or motility when internalized inside cells25. Using five different semiconductor alloy compositions and stochastically varying the microdisk diameter through nanofabrication enabled single-mode emission over a wide range in the tissue-penetrating NIR-Ⅱ window, from 1170 to 1580 nm, with subnanometer linewidths (Fig. 2a). Given 1-nm bins within which LP emission is stable, this range provides more than 400 unique colors, with room for further expansion using different materials (e.g., InGaAsP and InAlGaAs for NIR-Ⅰ and NIR-Ⅱ, InGaN for visible). The use of the NIR range also preserves compatibility with conventional fluorescent probes in the visible wavelengths that can label different cellular features (e.g., nuclei, membrane). The availability of hundreds of spectrally distinct colors for multiplexed imaging enables longitudinal cell tracking even in dense, scattering tissues. In our study25, we used microdisk LPs to track thousands of individual cancer cells over several days in a 3D tumor spheroid invasion model. Our data enabled the classification of individual cells according to their motility and the identification of cells moving together in small packs within the spheroid by analyzing spatial correlations in velocities.
Despite progress in the development of multiplexed imaging probes, the number of resolvable colors, or available barcodes, is still far fewer than desired for most applications (~10, 000 cells per 1 mm3 of tissue volume). To tag cells uniquely, the number of barcodes should exceed the number of cells. Spectral barcoding with LPs is a practical and scalable approach to break through this limitation. LPs with different emissions can be combined to readily achieve millions of unique barcodes (Fig. 2b). One approach is the uptake of multiple LPs in each cell (Fig. 2c), as long as LPs can be colocalized to specific cells by segmentation of cell boundaries. However, this approach would not be suitable for dividing cells since barcode fidelity would be lost upon LP segregation. A more tractable approach is using multiple LPs stacked together in a single particle to generate an emission composed of multiple laser lines25 (Fig. 2d). To associate spatial information with single-cell analyses, the emission spectra of LP tagged cells can be read during imaging to catalog the position and identity of cells within tissue and following tissue dissociation at an appropriate step before subsequent analyses (Fig. 2e). For plate-based sequencing, LP emission can be measured during or after the sorting of single cells into individual microwells. LP cell tagging is also compatible with high-throughput methods, including microdroplet-based26 and split-pool barcoding27, which introduce cell-specific DNA barcodes for highly parallel analysis as long as an association between the optical LP barcode and the DNA barcode is made.
Further development of the LP technology could enable the phenotypic and molecular characterization of every cell in a tissue, limited only by light penetration during imaging. One critical area of research is cancer biology. A major question is how, when, and which cancer cells within a heterogeneous tumor become metastatic. Live imaging and multiplexed cell tracking with LPs in dense tumor tissue can delineate key steps in the metastatic cascade, including migration, intravasation, and extravasation, as well as interactions with the tumor microenvironment. Linking these cellular phenotypes with molecular profiles could address the largely unexplored roles of cellular cooperation and non-cell-autonomous behaviors at the single-cell level. Analysis of interacting ligands and receptors on individual cells may lead to the construction of a comprehensive, spatially resolved cell-cell interaction network to reveal functional mechanisms in metastatic tumor progression. Beyond research applications, similar experiments conducted on patient-derived tumor models could identify biomarkers for diagnosis or targets for therapy.
Another application of single-cell analyses, particularly scRNA-seq, is unbiased cell classification, which has been successful in identifying new cell types in a number of different tissues. One reason for their success is a data-driven approach that is unencumbered by previous biases towards cellular identity. Techniques that integrate multiple omics layers of data (e.g., transcriptomic and epigenomic) provide a more complete picture of a cell's molecular framework that is thus more powerful than using any single layer alone3. Cellular phenotypes captured by imaging can be considered additional layers of information that reflect a cell's native environment. Since cells do not naturally exist in isolation, this information is essential for understanding cellular state, function, and cell-cell interactions in their native tissue environment. A recent initiative by an international consortium to characterize every cell in the human body, the Human Cell Atlas, has highlighted the need for spatial information to generate comprehensive reference maps as a basis for diagnosing, monitoring, and treating disease28. Spatially resolved single-cell analysis will likely transform applications such as tissue engineering and precision medicine. Comprehensive datasets combining imaging and molecular profiles could also be used to train machine-learning algorithms for image-based cell classification and in silico diagnostics.
In conclusion, advances in cellular imaging and molecular analyses have pushed the envelope of what can be observed and measured, but their impact has been hampered by the inability to employ these technologies in tandem on the same cells. Laser particles are a novel class of multiplexed imaging probes with the potential to bridge these technologies by enabling massively multiplexed imaging, cell tagging, and comprehensive single-cell analysis.