Super-resolution through out-of-focus imaging in lens-based microscopy (Conference Presentation)

Abstract

The limited space-bandwidth-product of microscopy systems in general forces users to sacrifice either resolution or field-of-view (FOV). Here we introduce a wide-field and high-resolution imaging method that uses a stack of out-of-focus images of the specimen to increase the space-bandwidth-product of lens-based microscopes. Although modern microscope objective-lenses are designed for high-resolution imaging and can achieve a relatively large FOV, often the active area of the imager chip sets a limitation. To best utilize the full field-of-view of an objective-lens in our microscope, we first added a demagnification camera adaptor (e.g., 0.35×) to match the CCD sensor chip active area to the FOV of a 10X objective-lens (~5 mm2) that has an NA of 0.3. This demagnification, while increasing the FOV, downgrades the image resolution and results in pixelation. We illustrate that this spatial undersampling can be overcome through an iterative pixel super-resolution algorithm that uses a stack of out-of-focus images of the sample to restore a high-resolution image across a large FOV. We demonstrated the success of this approach using a resolution test-target and showed that our technique reduces the number of measurements required to achieve the same effective space-bandwidth-product using e.g., lateral scanning and digital stitching of different FOVs. Phase retrieval capability of this approach is also demonstrated by reconstructing unstained Papanicolaou (Pap) smear samples without the need for phase-contrast objective-lenses. This technique might be useful to maximize the throughput of lens-based optical imaging systems and inspire new microscopy designs that utilize auto-focusing steps to increase resolution.