Cancellation of Bessel beam side lobes for high-contrast light sheet microscopy

Abstract

An ideal illumination for light sheet fluorescence microscopy entails both a localized and a propagation invariant optical field. Bessel beams and Airy beams satisfy these conditions, but their non-diffracting feature comes at the cost of the presence of high-energy side lobes that notably degrade the imaging contrast and induce photobleaching. Here, we demonstrate the use of a light droplet illumination whose side lobes are suppressed by interfering Bessel beams of specific k-vectors. Our droplet illumination readily achieves more than 50% extinction of the light distributed across the Bessel side lobes, providing a more efficient energy localization without loss in transverse resolution. In a standard light sheet fluorescence microscope, we demonstrate a two-fold contrast enhancement imaging micron-scale fluorescent beads. Results pave the way to new opportunities for rapid and deep in vivo observations of large-scale biological systems.

Scattering Assisted Imaging

“…The relative simplicity of the experimental setup makes SAI desirable as it can be applied to basic fluorescence imaging schemes where fluorescent molecules lie on the surface of a strongly scattering material. Since the resolution limit is driven by the size of the speckle grain rather than the system NA, the proposed technique shows its maximum advantage in experiments where high resolution is needed together with a long working distance, a condition that is currently forbidden by the physical NA of the illumination and collection optics 27 , 28 . Involving minimal optical power, SAI can be a powerful tool for the investigation of systems with low damage threshold, and may be easily exported to in-vivo investigation of sensible tissues such as the human retina.…”

Fluorescence imaging with tailored light

“…Due to its thin illumination across large FOV, lattice light-sheet microscopy enables high-resolution long-term live-cell imaging with an extremely low light dose and is regarded as an ideal fluorescence imaging system for many biological studies. Alternatively, it was proposed to use pseudo-nondiffracting beams featuring attenuated side lobes by superimposing two coaxial Bessel beams [171,172] (Figure 8E) or two cosine-Gauss beams [173,174] ( Figure 8F). However, they showed either a rather limited FOV or a relatively thicker beam as contrasted with the lattice light-sheet.…”

Minutes-timescale 3D isotropic imaging of entire organs at subcellular resolution by content-aware compressed-sensing light-sheet microscopy

“…Unlike ASLM, Bessel LSM can retain most of the beam energy in its central lobe at focal plane so that the photobleaching is relatively low. Meanwhile, to minimize the effect from side-lobe excitation 30 , Bessel LSM usually uses a high numerical-aperture (NA) detection objective with a small depth-of-focus. Thus, it is mainly optimized for the live imaging of organelles in a single or a few cells.…”