Strategies for contrast improvement in optical microscopy
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Optical microscopy is an indispensable tool in biomedical laboratories since it is minimum invasive and can provide high spatiotemporal resolution when imaging biological tissues and organisms. However, recent advancements in biological and neurological sciences impose new challenges on optical microscopes, where one wants to image over extended volumes at high speeds, or deep inside scattering tissues. Conventional wide-field fluorescence microscopy (WFM) is able to image a 2D field-of-view at high speeds, but suffers from degrading contrast due to out-of-focus fluorescence. Two-photon microscopy can image deep inside scattering tissue compared to WFM, but the maximum attainable penetration depth is still limited by signal contrast. The presence of tissue scattering also incurs inaccuracy in point spread function (PSF) estimation, which is critical in deconvolution microscopy for contrast enhancement. In chapter 2 we describe a widefield-based extended depth-of-field (EDOF) fluorescence microscopy for high-speed high-contrast volumetric imaging. The system makes use of a digital micromirror device (DMD) to target illumination only on in-focus sample features within the imaging volume, significantly reducing the out-of-focus fluorescent background that plagues the typical widefield, and particularly EDOF, microscopes. This technique greatly enhances the image contrast and signal-to-noise ratio, while reducing the light dosage delivered to the sample. Image quality is further improved by the application of a robust deconvolution algorithm. These advantages are demonstrated for in vivo calcium imaging in the mouse brain. In chapter 3 we describe a variant of two-photon microscopy for high contrast imaging in deep tissue. The technique is based on the previously proposed differential aberration imaging (DAI) strategy where image contrast is enhanced by subtracting an aberrated image from an unaberrated one. This technique, though simple and effective, compromises imaging speed because two images must be taken sequentially. A new strategy for two-photon DAI based on near-instantaneous temporal multiplexing is proposed here, enabling high-speed imaging with pixel rates limited only by fluorescence lifetime and laser repetition rate. It can be implemented with standard two-photon microscopes since it does not require active optical elements and it is based on a synchronized sampling strategy that does not require specialized hardware. The resultant contrast improvement is demonstrated by imaging fluorescently-labeled mouse brain at video-rate. In chapter 4 we describe a theoretical model for imaging fluorescent objects embedded inside scattering medium. The model provides a simple analytical solution for estimating PSF within the forward scattering limit, which can be used for contrast improvement in deconvolution microscopy. We verify the results using Monte Carlo simulation. We also apply the model to a partitioned aperture detection system, demonstrate both theoretically and experimentally that one can use strongly scattered light for quantitative 3D localization of fluorescent objects in scattering medium with micrometer-level precision, up to the depth approaching transport mean free path.
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