Flat-Field Illumination Microscopy for Large Field-of-View Quantitative Imaging

Abstract

Fluorescence imaging is a powerful tool for understanding of molecular mechanisms of cellular processes and functions. It is desirable to have a robust imaging method that enables us to obtain quantitative information. However, in laser-based fluorescence imaging, the nonuniform illumination distribution by the Gaussian-shaped beam results in severe problems for quantitative analysis of images. Additionally, it limits the field-of-view and leads to rapid photobleaching at the center of the beam. The solution for the uneven distribution is to have an even illumination beam. The method proposed is the reshaping of the Gaussian illumination profile into a Flattop illumination profile by using refractive optics. Our results showed that the flat-field illumination yielded far more leveled intensities and much wider field-of-view compared to Gaussian profile. We demonstrated epi- and TIRF illumination with multiple wavelengths. Our method is likely to combine with high-throughput imaging as well.