Wide field light-sheet microscopy with lens-axicon controlled two-photon Bessel beam illumination

Abstract

Two-photon excitation can lower phototoxicity and improve penetration depth, but its narrow excitation range restricts its applications in light-sheet microscopy. Here, we propose simple illumination optics, a lens-axicon triplet composed of an axicon and two convex lenses, to generate longer extent Bessel beams. This unit can stretch the beam full width at half maximum of 600–1000 μm with less than a 4-μm waist when using a 10× illumination lens. A two-photon excitation digital scanned light-sheet microscope possessing this range of field of view and ~2–3-μm axial resolution is constructed and used to analyze the cellular dynamics over the whole body of medaka fish. We demonstrate long-term time-lapse observations over several days and high-speed recording with ~3 mm3 volume per 4 s of the embryos. Our system is minimal and suppresses laser power loss, which can broaden applications of two-photon excitation in light-sheet microscopy.

Non-Diffracting Light Wave: Fundamentals and Biomedical Applications

“…Therefore, a uniform magnitude of size upon propagation is ideal for lightsheet illumination. Moreover, the non-diffracting beam features shape-preserving for a distance much longer than the Rayleigh distance, offering the ability to extend the illumination FOV in the LSFM application [11,136]. The Bessel focus TPLSM allows volumetric imaging of neurovascular dynamics while preserving the high lateral resolution and multi-color capability [25].…”

“…LSFM in single-and two-photon modalities has been a powerful, low-photodamage, widefield microscope for fast volumetric imaging of biological tissue. The 2-week posthatching (wph) larva of the FLT4-EGFP zebrafish strain expresses EGFP in the lymphatic endothelial cells, with angiography labeled by dextranconjugated tetramethylrhodamine B being imaged with LSFM (Figure 12A) [136]. The 3D reconstruction of the entire fish clearly shows the vascular system of the zebrafish larva.…”

“…Green represents the EGFP signals from the FLT4-EGFP transgenic strain, and red demonstrates the dextran-conjugated rhodamine signals from the blood vessel. Yellow rectangular illustrates a magnified view at the body trunk [136]. (B) Maximum intensity projection of Thy-1 GFP-expressing mouse brain tissue, using the two-photon planar Airy light-sheet [127].…”

“…Reprinted with permission of OSA [127]. Reproduced from Springer Nature [26,136]. repetition rate of 80 MHz served as the pump and Stokes beam.…”

Comparative Study in Zebrafish and Medaka Unravels the Mechanisms of Tissue Regeneration

“…In addition, the Fli1 antibody (ab133485) can be used to mark endothelial cell nuclei in zebrafish by immunostaining (Bensimon-Brito et al, 2020). Aside from post-fixation staining, angiography can be achieved by fluorescent dextran injection into the circulation of larval (Hoeppner et al, 2015;Takanezawa et al, 2021) and adult fish (Pugach et al, 2009).…”