Journal Article

Three-dimensional digital PCR through light-sheet imaging of optically cleared emulsion

Wang, Jianbin, Zhang, Fangli, Sun, Yue, Du, Meijie, Fei, Peng, Chen, Zitian, Huang, Yanyi, Liao, Peiyu, Nie, Jun, Jiang, Mengcheng

2020

Overview

The realization of the vast potential of digital PCR (dPCR) to provide extremely accurate and sensitive measurements in the clinical setting has thus far been hindered by challenges such as assay robustness and high costs. Here we introduce a lossless and contamination-free dPCR technology, termed CLEAR-dPCR, which addresses these challenges by completing the dPCR sample preparation, PCR, and readout all in one tube. Optical clearing of the droplet dPCR emulsion was combined with emerging light-sheet fluorescence microscopy, to acquire a three-dimensional (3D) image of a half million droplets sealed in a tube in seconds. CLEAR-dPCR provides ultrahigh-throughput readout results in situ and fundamentally eliminates the possibility of either sample loss or contamination. This approach exhibits improved accuracy over existing dPCR platforms and enables a greatly increased dynamic range to be comparable to that of real-time quantitative PCR.

• Publisher: National Academy of Sciences
• Subject: Applied Physical Sciences / Biological Sciences / Contamination / Digital imaging / DNA - blood / DNA Copy Number Variations - genetics / Droplets / Emulsions / Emulsions - chemistry / Equipment Design / Female / Fluorescence / Fluorescence microscopy / Genetics / Humans / Image acquisition / Imaging, Three-Dimensional - methods / Laser microscopy / Light sheets / Microscopy, Fluorescence - methods / Physical Sciences / Polymerase Chain Reaction - methods / Pregnancy / Prenatal Diagnosis - methods / Sample preparation / Tuberous Sclerosis - genetics