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

Abstract

Abstract:We report the development of a structured illumination microscopy instrument specifically designed for the requirements for higharea-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, fluorescentlystained muscle tissue, with 1.3 μm lateral resolution. We demonstrate highcontrast, 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.

DRAQ5 and Eosin (‘D&E’) as an Analog to Hematoxylin and Eosin for Rapid Fluorescence Histology of Fresh Tissues

“…Eosin intensity was not significantly affected by subsequent application of DRAQ5. Samples were imaged with a custom epi-fluorescence microscope with optical sectioning structured illumination microscopy (SIM) capability based on an automated modular platform (RAMM, Applied Scientific Instrumentation) and described in detail in our previous publications [13, 14]. Briefly, the system consists of a 470 nm LED for eosin excitation (M470L2, Thorlabs), and was modified in this work to include a 630 nm LED for DRAQ5 excitation (UHP-Mic-LED-630, Prizmatix).…”

“…The normalized spatial pattern frequency (ν) for each excitation channel was determined using the relationship v = f λ ex / NA, where λ ex is the center excitation wavelength of the respective LED and NA is the numerical aperture of the objective lens [13]. Using ν and the center emission wavelengths of the eosin and DRAQ5 channels, theoretical calculations for the axial response of the system in terms of half-width-half-maximum (HWHM) of each channel was made following Karadaglic and Wilson [27].…”

“…This nonselective staining makes it useful as a general comprehensive fluorescence histology stain, as we and others have demonstrated [13, 14, 23], but it also limits its utility as a fluorescent H&E analog since it demonstrates a staining mechanism that mixes features of both hematoxylin and eosin. Also, since AO binds cytoplasmic RNA, it does not specifically label the nucleus like hematoxylin.…”

Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease

“…These advantages of SIM may enable optical resolution of podocyte substructure, and differentiation of its normal and abnormal forms, thereby allowing accurate and rapid diagnosis of nephrotic disease. We remark that SIM is being tested in other areas of medical diagnosis, for example, the high-throughput determination of tumor margins in tissue sections [11], but none so far for the purpose of resolving structures of biological and medical importance that are otherwise visible only by EM.…”

Rapid imaging of large tissues using high-resolution stage-scanning microscopy

“…Especially when high numerical aperture objective was used to obtain high resolution and the field of view was reduced to less than 500 × 500 μm 2 [7]. It was indispensable to image numbers of mosaics in order to cover the entire specimen area [8][9][10][11]. Repetitive imaging is cost-ineffective since the stage will be controlled to go through an acceleration-constant speed-deceleration process, which is inefficient.…”