Video-rate structured illumination microscopy for high-throughput imaging of large tissue areas.

ABSTRACT

We report the development of a structured illumination microscopy instrument specifically designed for the requirements for high-area-throughput, optically-sectioned imaging of large, fluorescently-stained tissue specimens. The system achieves optical sectioning frame-rates of up to 33 Hz (and pixel sampling rates of up to 138.4 MHz), by combining a fast, ferroelectric spatial light modulator for pattern generation with the latest large-format, high frame-rate scientific CMOS camera technology. Using a 10X 0.45 NA objective and a 7 mm/sec scan stage, we demonstrate 4.4 cm(2)/min area-throughput rates in bright tissue-simulating phantoms, and 2 cm(2)/min area-throughput rates in thick, highly-absorbing, fluorescently-stained muscle tissue, with 1.3 µm lateral resolution. We demonstrate high-contrast, high-resolution imaging of a fluorescently-stained 30.4 cm(2) bovine muscle specimen in 15 minutes comprising 7.55 gigapixels, demonstrating the feasibility of the approach for gigapixel imaging of large tissues in short timeframes, such as would be needed for intraoperative imaging of tumor resection specimens.