**2. Optical methods**

The analysis of optical signals, *i.e.* photoluminescence and optical density (OD), is currently the conventional state-of-the-art method to detect and monitor the droplets in a microfluidic format. A schematic representation of a microfluidic platform equipped with an optical detection system and its operation is given in the following **Figure 1**. The complete system is capable to perform multiple tasks, which are divided into the separate modules, *e.g.* formation of the droplets, their transportation, incubation, merging, splitting, etc. This enables to mix the reagents in droplets and to tailor their composition on demand. Exemplary, the droplets are generated using *e.g.* flow focusing geometry, via controlled emulsification of the aqueous phase using oil containing surfactants for stabilization. The droplets are typically generated at few μm-scaled flow-focusing junction (*ca.* 10–30 μm) and are transported in a few mm long channel before droplets reach further manipulation structures [20]. Manipulation of the droplets content, size and interfacial properties are usually used for example to initiate multitude of chemical

*Real-Time Tracking of Individual Droplets in Multiphase Microfluidics DOI: http://dx.doi.org/10.5772/intechopen.106796*

**Figure 1.**

*Complete system for (a) droplet processing, (b) generation, (c) storage, (d) sorting, and (e) image analysis. Adapted with permission from refs. [18, 19].*

reactions [20] of interest. This is performed via fusion of the droplets that is controllably afforded using electro-coalescence [21]. For the multiparametric on-chip analysis, droplets may be split into the fractions at the multibranch junctions. The light microscope images show the respective processes of the droplets generation, merging, splitting, etc.

Efficient detection of thousands of droplets one-by-one and providing of the sufficiently high signal to noise ratio (SNR) is of extreme importance in droplets based systems. **Figure 1a** shows the schematic representation of the typical light path configuration across the microfluidic channel, applicable to detect droplets in microfluidic systems. For these purposes, the optical microscopy with the integrated illumination, *e.g.* a mercury arc or halogen lamp, integrated semiconductor lasers [22, 23], laser emitting diodes (LEDs) [24], are used as the light sources. A semiconductor laser is considered to be one of the most frequently used tool, being able to produce monochromatic, directed light beam, staying relatively compact. To enable the fluorescent analysis, setups are supplemented by the combinations of the filters and lenses for focusing and filtering excitation and emission beams. Apart from the light source, typical detection event involving fluorescence, *e.g.* to count droplets, employs pair of the dichroic mirrors (DM) to assure the precise excitation and emission of the light. Reagents in droplets are excited on the fly, during exposition to the light beam for *ca.* several milliseconds [25]. The intensity of the emission is proportional to the intensity of the incident light. Emitted light is directed by the respective optic elements, such as fibers and mirrors, towards detectors, which are represented by photomultiplier tubes (PMT) or avalanche diodes. The PMTs and diodes convert the light into an electrical signal for the detection of fluorescent events one-by-one. This configuration typically results in a high-throughput benchtop instrument. Although lasers provide great SNR for droplets detection, they miss the cost efficiency, low energy consumption and miniaturized integration, which is a specific peculiarity, for example of LEDs. It should be however noticed that for the sake of setup simplification (*e.g.* skipping

the excitation filters), only narrow-band LEDs are suitable. Overall, thanks to the aforementioned advantages LEDs are currently the most used technology for dynamic detection of the droplets in micro- and millifluific setups. Fluorescent microscopy with the integrated Charge Coupled Device (CCD) sensors and CMOS sensors are still actively employed to offer spatial monitoring of the droplets-fluorescence over time [18] (**Figure 1e**). This has high relevance for the realization of the droplet-based digital Polymerase Chain Reactions (PCR) and next generation sequencing for monitoring of *e.g.* circulating tumor DNA [26]. In this realization microscopy scanning of the multiple droplets that are stored in the incubation chamber is performed in time domain. Droplets with the positive amplification of the fluorescent signal are visually detected, as demonstrated in **Figure 2** [27].
