SummaryA major problem in microscopic imaging of ex vivo tissue sections stained with fluorescent agents (e.g. antibodies, peptides) is the confounding presence of background tissue autofluorescence. Autofluorescence limits (1) the accuracy of differentiating background signals from single and multiple fluorescence labels and (2) reliable quantification of fluorescent signals. Advanced techniques such as hyperspectral imaging and spectral unmixing can be applied to essentially remove this autofluorescent signal contribution, and this work attempts to quantify the effectiveness of autofluorescence spectral unmixing in a tumour xenograft model. Whole-specimen single-channel fluorescence images were acquired using excitation wavelengths of 488 nm (producing high autofluorescence) and 568 nm (producing negligible autofluorescence). These single-channel data sets are quantified against hyperspectral images acquired at 488 nm using a prototype whole-slide hyperspectral fluorescence scanner developed in our facility. The development and further refinement of this instrument will improve the quantification of weak fluorescent signals in fluorescence microscopy studies of ex vivo tissues in both preclinical and clinical applications.
SummaryThis paper presents microelectromechanical system micromirrors with sidewall electrodes applied for use as a Confocal MACROscope for biomedical imaging. The MACROscope is a fluorescence and brightfield confocal laser scanning microscope with a very large field of view. In this paper, a microelectromechanical system mirror with sidewall electrodes replaces the galvo-scanner and XYZ-stage to improve the confocal MACROscope design and obtain an image. Two micromirror-based optical configurations are developed and tested to optimize the optical design through scanning angle, field of view and numerical aperture improvement. Meanwhile, the scanning frequency and control waveform of the micromirror are tested. Analysing the scan frequency and waveform becomes a key factor to optimize the micromirror-based confocal MACROscope. When the micromirror is integrated into the MACROscope and works at 40 Hz, the micromirror with open-loop control possesses good repeatability, so that the synchronization among the scanner, XYZ-stage and image acquisition can be realized. A laser scanning microscope system based on the micromirror with 2 μm width torsion bars was built and a 2D image was obtained as well. This work forms the experimental basis for building a practical confocal MACROscope.
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