**2.1 3D detection of droplets**

Aforementioned systems assist in the highly sensitive detection of droplets, quantifying the signal that reflects the distribution of the fluorescence intensity in droplets within a focal plane of the optical lens. In order to get the spatio-temporal distribution of the fluorescent signal 3D image acquisition is required [28]. Therefore, a novel merge of droplet microfluidics with the light-sheet fluorescent microscopy (LSFM) was proposed, with the aim to achieve high-throughput compartmentalization, manipulation and three-dimensional imaging of the sample. This was realized via integration of the orthogonal plane illumination into the optofluidic system. Each droplet that moves through the detection area of the device was scanned by a lasersheet, three-dimensionally reconstructed and analyzed. It consisted of an upright droplet microfluidic chip and a horizontal laser-sheet illumination path, where the laser-sheet penetrated the micro-channel transversely. As one droplet flowed down through the detection region, it was optically detected in sections by the thin lasersheet in a way of automatic fluid scan. The fluorescent signals from the sequentially illuminated planes of the droplet were then measured by the microscope's objective from the side facet of the chip and recorded using a high sensitivity CMOS camera. The optical microscope with a large field of view objective recorded the fluorescent signals with a high acquisition rate of 500 fps, and z step-size of 3.5 μm were reached.

To track fluorescence within every droplet, the solution of colloidal particles doped by a fluorescent dye was used during encapsulation. The 3D detection of such droplets, formed with the smaller and smaller sizes (from 1.5 mm to 0.5 mm), is demonstrated in. Each droplet consists of over 500 3D stacked plane images from different depths of the encapsulated particle clusters. Generally, this technology opens great potential for various lab-on-a-chip studies, such as embryo sorting and organoids growth monitoring, etc.

#### **Figure 2.**

*PCR microfluidics with microscopic analysis of fluorescence development. Adapted with permission from ref. [27].*
