Abstract
Light-sheet microscopy is a useful tool for performing biological investigations of thick samples and it has recently been demonstrated that it can also act as a suitable architecture for super-resolution imaging of thick biological samples by means of individual molecule localization. However, imaging in depth is still limited since it suffers from a reduction in image quality caused by scattering effects. This paper sets out to investigate the advantages of non-linear photoactivation implemented in a selective plane illumination configuration when imaging scattering samples. In particular, two-photon excitation is proven to improve imaging capabilities in terms of imaging depth and is expected to reduce light-sample interactions and sample photo-damage. Here, two-photon photoactivation is coupled to individual molecule localization methods based on light-sheet illumination (IML-SPIM), allowing super-resolution imaging of nuclear pH2AX in NB4 cells.
Highlights
The growing interest in large organisms and tissue imaging led to a rapid development in two-photon excitation microscopy [1] and light-sheet based techniques [2] [3]
In order to spatially confine the activation process, approaches based on temporal focusing [29] and selective plane illumination microscopy (IML-SPIM) [30] have been recently developed, allowing the 3D super-resolution imaging of whole cells and thick living cellular spheroids, respectively
The basic idea behind selective plane illumination microscopy (SPIM) relies on the confined illumination within a thin plane orthogonally oriented to the detection axis
Summary
The growing interest in large organisms and tissue imaging led to a rapid development in two-photon excitation microscopy [1] and light-sheet based techniques [2] [3]. In order to spatially confine the activation process, approaches based on temporal focusing [29] and selective plane illumination microscopy (IML-SPIM) [30] have been recently developed, allowing the 3D super-resolution imaging of whole cells and thick living cellular spheroids, respectively All these new techniques extended the 3D imaging capabilities of localization-based methods [31] to thicker and thicker samples, taking a further step towards in vivo super-resolution imaging. After photoactivation, the excitation spectrum shifts maintaining the very same emission spectrum of the single-photon case for the native and photoactivated protein [38]
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