Multiview deconvolution approximation multiphoton microscopy of tissues and zebrafish larvae

Abstract

Imaging in three dimensions is necessary for thick tissues and small organisms. This is possible with tomographic optical microscopy techniques such as confocal, multiphoton and light sheet microscopy. All these techniques suffer from anisotropic resolution and limited penetration depth. In the past, Multiview microscopy—imaging the sample from different angles followed by 3D image reconstruction—was developed to address this issue for light sheet microscopy based on fluorescence signal. In this study we applied this methodology to accomplish Multiview imaging with multiphoton microscopy based on fluorescence and additionally second harmonic signal from myosin and collagen. It was shown that isotropic resolution was achieved, the entirety of the sample was visualized, and interference artifacts were suppressed allowing clear visualization of collagen fibrils and myofibrils. This method can be applied to any scanning microscopy technique without microscope modifications. It can be used for imaging tissue and whole mount small organisms such as heart tissue, and zebrafish larva in 3D, label-free or stained, with at least threefold axial resolution improvement which can be significant for the accurate quantification of small 3D structures.

Fluorescence radial fluctuation enables two-photon super-resolution microscopy

“…Previously, combining two-photon microscopy with deconvolution resulted in a high-resolution (approx. 230 nm) observation of thick fixed tissues ( Kapsokalyvas et al, 2021 ). However, the target was a relatively thin area (<200 μm) of the transparent sample.…”

Multimodal imaging of the dynamic brain tumor microenvironment during glioblastoma progression and in response to treatment

“…These applications would also necessitate the combination of multiple fluorophores, and it will therefore be crucial to precisely define the cellular source of the different signals in future studies. Toward this end, the design and implementation of new deconvolution algorithms and spectral unmixing would facilitate the complex analysis pipeline ( Kapsokalyvas et al., 2021 ).…”

Two plus one is almost three: a fast approximation for multi-view deconvolution

“…Several techniques have been devised to address these issues, including sophisticated hardware-based illumination beam shaping and two-sided illumination [ 7 – 10 ]. Furthermore, image-processing methods have been developed such as deconvolution, multi-view image fusion and multi-view deconvolution (MVD) and applied to LSFM [ 11 – 13 ] and, more recently, also to multiphoton microscopy [ 14 ]. For multi-view imaging, samples are typically mounted onto a rotational stage and imaged from different angles swiftly in succession, which ensures that structural changes occurring between exposures are minimized.…”