Expanding the capabilities of multifocus microscopy (MFM) for biological fluorescence imaging

We have implemented a highly light-efficient and aberration-corrected MFM system (Fig 1a) which enables biological researchers to study quickly moving, living samples over extended 3D volumes with single molecule sensitivity at acquisition speeds limited by the readout-time of a single camera frame [1]. The multi-focus image consists of a focal series of 2D wide-field images simultaneously formed on the camera. The multi-focus grating (Fig 1b) refocuses and distributes the fluorescence emission light from the sample evenly and efficiently (67%) between the planes in the multi-focus image. To allow imaging across the visible spectrum, the chromatic dispersion (inherent to any diffractive optical component) that is introduced by the multi-focus grating is corrected by a highly efficient multi-paneled blazed grating combined with a multi-faceted prism.

Figure 1. MFM layout. (a) The multi-focus grating is placed in the microscope Fourier plane, and splits the fluorescence emission light from the sample into a set of diffractive orders which each form a 2D image of the sample. (b) A geometrical distortion of the grating introduces a phase-shift to the wavefront of each non-zero diffractive order. This phase-shift is adjusted to compensate for the out-of-focus wavefront error of each focal plane according to the Abbe sine condition to minimize depth-induced spherical aberration and allow refocusing deep into the sample.

[1] Abrahamsson et al., "Fast multicolor 3D imaging using aberration-corrected multifocus microscopy," Nat. Methods, 10, 60-63 (2013).