Auto-ICell: An Accessible and Cost-Effective Integrative Droplet Microfluidic System for Real-Time Single-Cell Morphological and Apoptotic Analysis

The Auto-ICell system, a novel, and cost-effective integrated droplet microfluidic system, is introduced for real-time analysis of single-cell morphology and apoptosis. This system integrates a 3D-printed microfluidic chip with image analysis algorithms, enabling the generation of uniform droplet reactors and immediate image analysis. The system employs a color-based image analysis algorithm in the bright field for droplet content analysis. Meanwhile, in the fluorescence field, cell apoptosis is quantitatively measured through a combination of deep-learning-enabled multiple fluorescent channel analysis and a live/dead cell stain kit. Breast cancer cells are encapsulated within uniform droplets, with diameters ranging from 70 {\mu}m to 240 {\mu}m, generated at a high throughput of 1,500 droplets per minute. Real-time image analysis results are displayed within 2 seconds on a custom graphical user interface (GUI). The system provides an automatic calculation of the distribution and ratio of encapsulated dyes in the bright field, and in the fluorescent field, cell blebbing and cell circularity are observed and quantified respectively. The Auto-ICell system is non-invasive and provides online detection, offering a robust, time-efficient, user-friendly, and cost-effective solution for single-cell analysis. It significantly enhances the detection throughput of droplet single-cell analysis by reducing setup costs and improving operational performance. This study highlights the potential of the Auto-ICell system in advancing biological research and personalized disease treatment, with promising applications in cell culture, biochemical microreactors, drug carriers, cell-based assays, synthetic biology, and point-of-care diagnostics.

Deep Learning Approach for Large-Scale, Real-Time Quantification of Green Fluorescent Protein-Labeled Biological Samples in Microreactors

Absolute quantification of biological samples entails determining expression levels in precise numerical copies, offering enhanced accuracy and superior performance for rare templates. However, existing methodologies suffer from significant limitations: flow cytometers are both costly and intricate, while fluorescence imaging relying on software tools or manual counting is time-consuming and prone to inaccuracies. In this study, we have devised a comprehensive deep-learning-enabled pipeline that enables the automated segmentation and classification of GFP (green fluorescent protein)-labeled microreactors, facilitating real-time absolute quantification. Our findings demonstrate the efficacy of this technique in accurately predicting the sizes and occupancy status of microreactors using standard laboratory fluorescence microscopes, thereby providing precise measurements of template concentrations. Notably, our approach exhibits an analysis speed of quantifying over 2,000 microreactors (across 10 images) within remarkably 2.5 seconds, and a dynamic range spanning from 56.52 to 1569.43 copies per micron-liter. Furthermore, our Deep-dGFP algorithm showcases remarkable generalization capabilities, as it can be directly applied to various GFP-labeling scenarios, including droplet-based, microwell-based, and agarose-based biological applications. To the best of our knowledge, this represents the first successful implementation of an all-in-one image analysis algorithm in droplet digital PCR (polymerase chain reaction), microwell digital PCR, droplet single-cell sequencing, agarose digital PCR, and bacterial quantification, without necessitating any transfer learning steps, modifications, or retraining procedures. We firmly believe that our Deep-dGFP technique will be readily embraced by biomedical laboratories and holds potential for further development in related clinical applications.