Extended Depth of Field Optics for Precise Image Analysis in Microfluidic Flow Cytometry

Abstract

We have examined a method to address the defocusing problem on target samples in a microfluidic pathway by an optical approach and report our experiment. An imaging optics has been constructed for extension of the depth of focused field. This system consists of four parts: (1) a low numerical aperture (NA; i.e., large depth of field) objective lens; (2) a zoom lens; (3) a light-emitting diode (LED) illumination source; and (4) a charge-coupled device (CCD) camera. As a low NA objective lens contributes to the extension of the depth of field and a zoom lens contributes to the optimization of pixel resolution on an image sensor of a camera, the same resolution as that of a 40× objective lens was acquired by the combination of a 10× objective lens and a 4× zoom lens as the spatial resolution of the latter combination was within the size of pixels of the CCD camera. As a result, improved depth of field was obtained at any magnification from 10× to 40×, and it was indicated that an extended depth of field optics for image-based microfluidic pathways such as in flow cytometry can be constructed using a low NA objective lens and a zoom lens.