Exploration of chromatic aberration for multiplanar imaging: Proof of concept with implications for fast, efficient autofocus

Abstract

Image-based autofocus determines focus directly from the specimen (as opposed to reflective surface positioning with an offset), but sequential acquisition of a stack of images to measure resolution/sharpness and find best focus is slower than reflective positioning. Simultaneous imaging of multiple focal planes, which is also useful for 3D imaging of live cells, is faster but requires complicated optics. With color CCD cameras and white light sources commonly available, we asked if axial chromatic aberration can be utilized to acquire multiple focal planes simultaneously, and if it can be controlled through a range sufficient for practical use. For proof of concept, we theoretically and experimentally explored the focal differences between three narrow wavelength bands on a 3-chip color CCD camera with and without glass inserts of various thicknesses and dispersions. Ray tracing yielded changes in foci of 0.65-0.9 lm upon insertion of 12.5-mm thick glass samples for green (G, 522 nm) vs. blue (B, 462 nm) and green vs. red (G-R, 604 nm). On a microscope: (1) With no glass inserts, the differences in foci were 2.15 lm (G-B) and 0.43 lm (G-R); (2) With glass inserts, the maximum change in foci for G vs. B was 0.44 lm and for G vs. R was 0.26 lm; and (3) An 11.3 mm thick N-BK7 glass insert shifted the foci 0.9 lm (R), 0.6 lm (G), and 0.35 lm (B), such that the B and R foci were farther apart (2.1 lm vs. 1.7 lm) and the R and G foci were closer together (0.25 lm vs. 0.45 lm). The slopes of the differences in foci were dependent on thickness, index of refraction, and dispersion. The measured differences in foci are comparable to the axial steps of 0