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.
Highlights
The current clinical reference standard for tissue diagnosis is permanent histopathology, in which excised tissue biopsies or surgical specimens are fixed, dehydrated, embedded in paraffin, sectioned into microscopically thin slices with a microtome, de-paraffinized, stained with histological stains, and mounted on microscope slides for observation by a pathologist
The instrument described here was designed to meet the requirements for high contrast, high resolution images of large, intact surgical specimens within clinically-practical timeframes
Structured illumination microscopy is used to optically-section the surface of the tissue, which serves the purpose of rejecting out of focus light, thereby increasing contrast and effective resolution
Summary
The current clinical reference standard for tissue diagnosis is permanent histopathology, in which excised tissue biopsies or surgical specimens are fixed, dehydrated, embedded in paraffin, sectioned into microscopically thin slices with a microtome, de-paraffinized, stained with histological stains, and mounted on microscope slides for observation by a pathologist This process is the gold-standard by which histological diagnoses are made, the technology is laborious and time-consuming, and has remained little unchanged in the last century. For diagnostic procedures such as prostate core needle biopsy, where false-negative rates can be as high as 50% [3,4], a rapid assessment of the biopsy diagnosis at the point-of-care could allow the clinician to continue to collect biopsies from the organ until the suspicious lesion is successfully sampled, preventing an unnecessary repeat biopsy procedure In both of these cases, high quality microscopic images of the tissue must be obtained in rapid timeframes, in order for a pathologic assessment to be useful for guiding the intervention. With regard to biopsy assessment, even with frozen section analysis, it is not practical to freeze, stain, and section a large number of core needle biopsies (between 6 and 20 for prostate biopsy) within the timeframe of a typical biopsy procedure in the clinic
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