Multifocal structured illumination fluorescence microscopy with large field-of-view and high spatio-temporal resolution

Fluorescence imaging is widely employed in biological discovery due to its excellent molecular sensitivity and contrast. However, due to light scattering wide-field fluorescence images are blurred resulting in very low spatial resolution and low image contrast. The existing scanning optical microscopy techniques are commonly restricted to sub-millimeter field-of-view or otherwise slow imaging speeds, limiting their applicability for imaging of fast biological dynamics occurring on larger spatial scales. Herein, we developed a rapid scanning wide-field multifocal structured illumination microscopy method based on a beam-splitting grating and an acousto-optic deflector synchronized with a high speed camera. The multi-beam pattern is focused by a condensing lens and a macroscopic objective to generate multifocal structured illumination profile on the imaged sample that is rapidly scanned at kHz rates. Experimental results show that the proposed method can achieve real-time fluorescence microscopy over a centimeter-scale field of view. Owing to the low numerical aperture of the diffracted beams, the illumination has a large depth of focus and hence is generally not affected by the sample's curvature, which allowed here imaging of perfusion in the entire mouse cerebral cortex noninvasively. The new approach can be readily incorporated into traditional wide-field microscopes to attain optimal tradeoff between spatial resolution and field of view. It further establishes a bridge between conventional wide-field macroscopy and laser scanning confocal microscopy, thus anticipated to find broad applicability in a variety of applications looking at large-scale fluorescent-based biodynamics.