High-throughput high-dynamic range imaging by spatiotemporally structured illumination

Abstract

Recent advances in biochemistry and optics have enabled observation of the faintest signals from even single molecules. However, although biological samples can have varying degrees of fluorescence expression ranging from a single to thousands of fluorescent molecules in an observation volume, the detection range is fundamentally limited by the dynamic range (DR) of current detectors. In other words, for many biological systems where faint and strong signal sources coexist, traditional imaging methods make a compromise and end up choosing a limited target signal range to be quantitatively measured while other signal levels are either lost beneath the background noise or saturated. The DR can be extended by taking multiple images with varying exposures, which, however, severely restricts data throughput. To overcome this limitation, we introduce structured illumination high dynamic range (SI-HDR) imaging, which enables real-time HDR imaging with a single measurement. We demonstrate the wide and easy applicability of the method by realizing various applications, such as high throughput gigapixel imaging of mouse brain slices, quantitative analysis of neuronal mitochondria structures, and fast 3D volumetric HDR imaging.