Automated Imaging of Cultured Bacteria in Picoliter Droplets by Coherent Anti‐Stokes Raman Scattering and Two‐Photon Excited Fluorescence Microscopy

ABSTRACT We present a novel and fully automated approach that combines an image‐based fuzzy logic controller (FLC) to precisely position pre‐selected microfluidic picoliter droplets containing cultured bacteria with nonlinear microscopy for detailed analysis of droplet content. Using coherent anti‐Stokes Raman scattering (CARS) at the aliphatic CH‐stretching vibration of methylene groups at 2841 cm −1 and two‐photon excited fluorescence (TPEF) microscopy, time series and z‐stack images of up to 50 droplets were recorded. The autosampler adjusts the pressures at the inlet and outlet of the microfluidic chip in bright field mode to position a droplet in the center of the region of interest. Then, the pressures are kept constant, and CARS and TPEF images are acquired in laser scanning mode for up to 60 s. Once the laser scanning image acquisition is complete, the next droplet is automatically processed. The software‐based integration of the droplet autosampler and CARS/TPEF laser scanning microscopy is a powerful tool for fully automated investigation of microbial dynamics at the single‐droplet level and provides insights into bacterial morphology and motility, offering diffraction‐limited spatial resolution and a medium throughput of thousands of droplets per day.