An apparatus for qualitative assessment of the shading ratio of oblique illumination and real-time high-contrast imaging.

Abstract

Oblique illumination imaging can significantly improve the contrast of transparent thin samples. However, in traditional oblique illumination methods, either the condenser is offset or a block is added to the condenser, which makes it complicated and challenged to build a stable oblique illumination imaging. Herein, we present a method to measure the optimal shading ratio of oblique illumination in an inverted microscope, and develop an apparatus for stable high-speed high-contrast imaging with uniform brightness. At optimal shading ratio, the oblique illumination imaging has better imaging quality than differential interference contrast, which characteristic is independent on sample. In oblique illumination with low magnification objective, the images have uneven brightness. According to target brightness, we have developed a brightness unevenness correction algorithm to form uniform background brightness for oblique illumination. Integrating the algorithm with imaging acquisition, corrected oblique illumination microscopy is appropriate to observe living cells with high contrast.