Simultaneous bright-field and fluorescence lensless imaging with high excitation light extinction for microfluidics applications

Abstract

Lensless imaging fluorescence applications are limited by the unavoidable trade-off between the object-sensor distance and the excitation light attenuation at the detection plane. Indeed, a shorter object-sensor distance reduces the spreading of fluorescent signals but at the cost of losing excitation light attenuation provided by filter thickness. Accordingly, the combination of four synergistic pathways to attain further attenuation of excitation light is proposed: the optimization of total internal reflection by microfluidic chip geometry modification, cross-polarization extinction, interchangeable absorption longpass filters, and the CMOS sensor built-in bandpass Bayer filters. The advantages of such a combined setup are demonstrated with a microfluidic application, as we managed to keep the object-sensor distance as short as 600 µm and still distinguish the fluorescence of individual droplets, flowing as close as 650 µm to each other, while attenuating excitation light to an OD 5.9 level. This approach also allowed simultaneous fluorescence and bright-field observation of on-chip droplet fluorescence during flow at a rate of 30 fps. The enhanced capabilities in our scheme pave the way to further lensless fluorescence applications requiring parallelism, lower detection thresholds, and real-time acquisition, such as fluorescence biosensing.