Abstract
Saturated excitation (SAX) microscopy offers high-depth discrimination predominantly due to nonlinearity in the fluorescence response induced by the SAX. Calculation of the optical transfer functions and the edge responses for SAX microscopy revealed the contrast improvement of high-spatial frequency components in the sample structure and the effective reduction of background signals from the out-of-focus planes. Experimental observations of the edge response and x-z cross-sectional images of stained HeLa cells agreed well with theoretical investigations. We applied SAX microscopy to the imaging of three-dimensional cultured cell clusters and confirmed the resolution improvement at a depth of 40 μm. This study shows the potential of SAX microscopy for super-resolution imaging of deep parts of biological specimens.
Highlights
Optical microscopy plays an important role in the observation of microscale structures or molecular activities in biological cells and tissues
We performed fluorescence imaging without a pinhole to determine the contribution of the nonlinear excitation in the Saturated excitation (SAX) microscopy to the background elimination and to the improvement of the spatial resolution, which we examined in the axial direction
We described the depth imaging properties of the SAX microscopy in comparison to confocal microscopy and showed fluorescence images of an approximately 40-μmthick cell cluster with higher spatial resolution than conventional confocal microscopy
Summary
Optical microscopy plays an important role in the observation of microscale structures or molecular activities in biological cells and tissues. To investigate cellular morphology and physiology of a living biological specimen, such as tissue-specific architectures, gene/protein expression, and drug metabolism, it is important to observe volumetric samples such as tissue and cell clusters.[20,21] Multiphoton microscopy is one of the most wellknown techniques used for observing such thick specimens.[22,23,24,25] Multiphoton excitation inherently suppresses the fluorescence emissions from the out-of-focus positions,[22] and the fluorescence detection through a pinhole provides further enhancement of the spatial resolution and allows finer structures to be imaged with high-image contrast.[26,27] A technique to estimate the background fluorescence signal by introducing aberrations in the focus of the excitation laser has been demonstrated to improve. Improved in a manner similar to two-photon excitation microscopy
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