Multiplexed fluorescence and scatter detection with single cell resolution using on-chip fiber optics for droplet microfluidic applications

The study addresses the challenge of multiparametric optical sensing in droplet-based microfluidics, where conventional fluorescence microscopy setups are bulky, expensive, and technically complex, limiting adoption in research and diagnostics. While flow cytometry provides high-throughput, multiparametric optical analysis of single cells, translating its principles to analyze single cells within water-in-oil droplets is difficult due to refractive index mismatches and droplet-to-cell size disparities. Prior droplet assays often rely on off-chip optics or double emulsions, each with significant limitations. The purpose of the work is to develop and validate OptiDrop, an on-chip optofluidic platform that integrates optical fibers for illumination and signal collection to simultaneously measure scatter and multiple fluorescence channels from droplets and their encapsulated contents with single-cell resolution. The platform aims to reduce cost and complexity, increase flexibility, and enable broader use of droplet microfluidics in research and point-of-care diagnostics. As a biological demonstration, the platform is used to measure differential surface expression of MHC I and II on mouse embryonic fibroblasts upon IFNγ stimulation using fluorescently labeled antibodies.

The paper reviews common approaches for droplet content detection, noting that fluorescence microscopy with high-speed imaging dominates but requires complex alignment of free-space optics (lenses, dichroics) and costly instrumentation with limited multiplexing flexibility. Attempts to miniaturize flow cytometry principles onto lab-on-chip devices for single-cell analysis exist, yet extending them to cells within droplets remains challenging because light traverses interfaces of differing refractive indices and droplet dimensions exceed cell size. Prior studies have shown detection of scatter, absorbance, and fluorescence from dyes and cell populations in droplets, but have lacked the sensitivity and multiplexing at single-cell resolution needed for high-throughput analysis. Using double emulsion droplets in standard flow cytometers is a powerful alternative but is difficult to handle and incompatible with many current droplet workflows. Integration of micro-optical components (lenses, waveguides, optical fibers) into microfluidic chips has been proposed, with optical fibers offering low cost, easy integration, broad wavelength compatibility, and low transmission loss. These insights motivate an on-chip, fiber-based approach for multiplexed, sensitive droplet readouts.

The OptiDrop platform integrates microfluidics, on-chip optical fibers, and photon detection electronics. A flow-focusing microfluidic junction generates stable, monodisperse water-in-oil droplets. At a downstream interrogation site, coplanar fiber guide grooves surround the central flow channel; fibers are placed with the incident laser fiber and collection fibers oriented at 45 degrees to the interrogation point. A 488 nm fiber-coupled laser provides excitation. Scattered and fluorescent light are collected by the angled fibers and routed to individual photomultiplier tubes (PMTs) equipped with appropriate bandpass (and neutral density for scatter) filters. PMTs output TTL pulses that are integrated by a pulse counter (FPGA-based) over time windows to yield intensity peak traces per channel corresponding to each droplet passage. The benchtop unit (approximately 20×12×4 inches) includes the chip, fiber-coupled optics, PMTs, an FPGA pulse counting module with live data display, and syringe pumps. The current bill of materials is approximately INR 10,00,000 (~USD 12,500), largely driven by a 100 mW laser, three PMTs, and pumps; lower-cost components can be substituted depending on application needs. Droplet formation and interrogation were characterized across oil and aqueous flow rates to study scatter signatures and timing metrics. The scatter channel uses a forward collection fiber at 45 degrees; the two-peak scatter signature (leading and lagging peaks) delineates droplet boundaries. Flow-rate dependence of intra-peak distance (dwell time) and inter-droplet spacing was quantified. To optimize optical coupling, the oil phase refractive index was tuned toward that of the aqueous phase by adding 3-bromobenzotrifluoride (0–30%) to Novec dSURF oil, controlling scatter intensity and transmission. Fluorescence performance was characterized using droplets containing Rhodamine 123 at varying concentrations, measuring peak height and width under fixed and varying flow conditions. The platform supports up to four fluorescence collection fibers/PMTs; signals are recorded simultaneously with scatter. For biological validation, cells (mouse embryonic fibroblasts) were encapsulated and stained with fluorescently labeled antibodies to measure MHC I and MHC II expression with and without IFNγ stimulation, enabling assessment of differential cell surface biomarker expression at single-cell resolution within droplets.

- The OptiDrop chip with on-chip optical fibers enables simultaneous detection of droplet scatter and multiple fluorescence signals with single-cell resolution using a single 488 nm laser and multiple PMTs. - Scatter detection at 45 degrees shows a robust, characteristic two-peak profile (leading and lagging peaks) marking droplet entry and exit from the excitation beam; these effectively define droplet boundaries for peak-gated fluorescence analysis. - Droplet dwell time (intra-peak distance) decreases with increasing oil flow rate and is independent of aqueous flow rate; inter-droplet spacing decreases (droplet frequency increases) with increasing aqueous flow rate, allowing controlled operation in the 20–100 Hz range (electronics can support ~10× higher frequencies, but fluidics currently limit throughput for larger droplets). - Refractive index tuning of the oil by adding 3-bromobenzotrifluoride (0–30%) reduces the refractive index contrast, decreasing scatter intensity and increasing light transmission into the droplet; this allows assay-dependent optimization of scatter vs transmission. - Fluorescence readouts from dye droplets show: (i) at fixed flow rates, peak height scales with Rhodamine 123 concentration while peak width remains constant; (ii) at fixed dye concentration, peak height is stable across oil flow rates while peak width increases with longer dwell times at slower oil flow. - The platform, validated with dyes and intensity standard beads, demonstrated biological utility by sensitively detecting differential surface expression of MHC I and MHC II on mouse embryonic fibroblasts in response to IFNγ stimulation using fluorescent antibody labeling.

By integrating illumination and collection fibers on-chip and positioning them at defined angles, the platform overcomes optical challenges posed by refractive index transitions in droplet systems and eliminates bulky free-space optics. The two-peak scatter signature provides precise droplet boundary detection, enabling accurate assignment of multiplexed fluorescence signals to individual droplets and their cellular contents. Flow-rate dependencies of dwell time and inter-droplet spacing allow users to tune assay timing and throughput. Refractive index matching of the oil enhances excitation and emission capture efficiency, improving sensitivity. Demonstration of differential MHC I/II expression in IFNγ-stimulated cells encapsulated in droplets shows that OptiDrop achieves the sensitivity and single-cell resolution required for biomarker analysis, bridging the capabilities of flow cytometry with droplet microfluidics. The system’s modularity (swap-in optical filters, adjustable number of PMTs) and reduced cost/footprint make it adaptable for varied assays and potential point-of-care applications.

The OptiDrop platform presents a compact, low-cost, and customizable on-chip optofluidic solution for multiplexed scatter and fluorescence detection in droplet microfluidics with single-cell resolution. It reliably identifies droplet boundaries via a distinctive scatter profile and quantifies fluorescence from dyes and cell-associated fluorophores, demonstrated by sensitive detection of IFNγ-induced changes in MHC I and II expression on cells within droplets. By avoiding free-space optics and leveraging fiber-based collection with PMTs, the system simplifies alignment, enhances flexibility, and reduces cost while maintaining high sensitivity. Future work could expand channel multiplexing, integrate on-chip sorting or additional droplet manipulation modules, further optimize refractive index matching and optics for different fluorophore sets, and scale throughput with improved fluidics or smaller droplet regimes to match the electronics’ capacity.

- Throughput is currently constrained by droplet fluidics for larger droplets; although the electronics could support approximately tenfold higher frequencies, the practical operation range is about 20–100 Hz in the present setup. - The reported implementation uses a single 488 nm laser and three PMTs in demonstrations; while customizable, the number of simultaneous optical parameters may be limited by available detection channels and optical filter configurations in a given setup. - Detailed biological protocol parameters (e.g., staining conditions, exact fluorescence quantitation) are not provided in the excerpt, which may limit immediate replication without the full methods section.