Fast 5D imaging using remote focusing in the detection arm of light sheet microscopy

Real-time functional imaging of live cells is fundamental to biomedical imaging. However, due to the agility of biological specimens, carrying out high-resolution volumetric imaging without agitating the sample has been a really difficult problem. Several attempts, employing vari-focus lenses, mechanical mirrors, and acousto-optics modulators have been proposed to do the refocusing of light required for such 3D imaging. Although, they all suffer from massive aberrations introduced by the focusing elements. Matching-objective-based remote-focusing has been promising for high NA imaging systems since they do not introduce significant spherical aberrations. However, owing to the incoherent and unpolarized nature of the emitted fluorescence signal, manipulating this light to be employed by remote focusing is very challenging. Therefore, remote-focusing has been primarily limited to the illumination arm, which uses polarized laser light. Here we show for the first time a novel methodology to perform aberration-free remote focusing without losing the expensive fluorescent signal to acquire fast 5D (3D space + time + wavelength) imaging, 70mm range in the z direction with the maximum frame rate of 5000 frames/s. Although, we show the usefulness of this approach using light sheet microscopy, because of the fundamental nature of the problem we envision that this technique will have a high impact on the broader microscopy community.