Two-photon microscopy plays a significant role in biological applications, as it enables non-invasive, high-resolution imaging of thick tissue samples. This paper begins by elucidating fundamental concepts of two-photon microscopy to facilitate a deeper understanding of its luminescent mechanism. Subsequently, we provide a comprehensive overview of the imaging system, encompassing the light source, scanning unit, fluorescence collection, and detection components. As two-photon microscopy continues to evolve, We undertake assessments of cutting-edge optical instrumentation, emphasizing novel approaches for imaging across wider fields of view, at higher speeds, and with increased volumetric imaging capabilities. Furthermore, we summarize and compare the technical specifications of advanced two-photon imaging. Following this, the manuscript showcases the transformative applications of these innovative technologies in biological imaging, providing further evidence of the indispensable role of two-photon microscopy in biomedical imaging research.
SignificanceTwo-photon fluorescence microscopy (TPFM) excited by Gaussian beams requires axial tomographic scanning for three-dimensional (3D) volumetric imaging, which is a time-consuming process, and the slow imaging speed hinders its application for in vivo brain imaging. The Bessel focus, characterized by an extended depth of focus and constant resolution, facilitates the projection of a 3D volume onto a two-dimensional image, which significantly enhances the speed of volumetric imaging.AimWe aimed to demonstrate the ability of a TPFM with a sidelobe-free Bessel beam to provide a promising tool for research in live biological specimens.ApproachComparative in vivo imaging was conducted in live mouse brains and transgenic zebrafish to evaluate the performance of TPFM and Bessel-beam-based TPFM. Additionally, an image-difference method utilizing zeroth-order and third-order Bessel beams was introduced to effectively suppress background interference introduced by sidelobes.ResultsIn comparison with traditional TPFM, the Bessel-beams-based TPFM demonstrated a 30-fold increase in imaging throughput and speed. Furthermore, the effectiveness of the image-difference method was validated in live biological specimens, resulting in a substantial enhancement of image contrast. Importantly, our TPFM with a sidelobe-free Bessel beam exhibited robustness against axial displacements, a feature of considerable value for in vivo experiments.ConclusionsWe achieved rapid, high-contrast, and robust volumetric imaging of the vasculature in live mouse brains and transgenic zebrafish using our TPFM with a sidelobe-free Bessel beam.
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