High-throughput and uniform large field-of-view multichannel fluorescence microscopy with super-thin dichroism for a dPCR gene chip.

With the rapid development of digital precision medicine, the digital polymerase chain reaction (dPCR) deoxyribonucleic acid (DNA) gene chip integrates more channels with smaller size and larger area, which leads to a higher technical requirement for commercial optical fluorescence microscopy. The multitime image splicing method is widely used for DNA detection. However, it consumes time and has visible seamless image results. This work has demonstrated the design and fabrication of a three channel reversed and reduced fluorescence microscopic imaging system with high-resolution and large field of view for one-time imaging. We introduced the super ultra-thin dichroic mirror into the space between the objective lens and the gene chip to achieve a uniform illumination and a strong signal for the large area gene chip. The fabricated new fluorescence microscopy can take a one-time imaging for the 28×18 dPCR gene chip with more than 20,000 multi micro-droplets within FAM, HEX, and ROX fluorescence channels. The optical system was designed with a numerical aperture (NA) of 0.106. Modulation transfer function (MTF) is higher than 0.675 at 70 lp/mm, and the function resolution capability is 10 µm with the whole magnification of -0.65. The fly's eye lens-based illumination system was tested with a uniform output of over 90% in the whole 34.7 chip area. The design was tested, and the experimental results showed that this new system provides a fast, efficient, and professional optical imaging method for detection of the new emerged digital PCR gene chip, which has larger area and more channels.