Image formation by linear and nonlinear digital scanned light-sheet fluorescence microscopy with Gaussian and Bessel beam profiles

Abstract

We present the implementation of a combined digital scanned light-sheet microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes. A complete characterization of the setup is performed and a comparison of the performance of each DSLM imaging modality is presented using in vivo Caenorhabditis elegans samples. We found that the use of Bessel beam nonlinear excitation results in better image contrast over a wider field of view.

Three dimensional HiLo-based structured illumination for a digital scanned laser sheet microscopy (DSLM) in thick tissue imaging

“…We modified the design of Digital Scanned Light-sheet Microscope (DSLM) [3] as shown in Fig. 1 to provide real time structured illumination to the sample according to the new 3D HiLo principle as well as older, alternative programs of structured illumination [4][5][6][7][8].…”

“…Recently, laser sheet based microscopic techniques [3][4][5][6][7][8] have emerged as a useful alternative for imaging biological tissue specimens of size larger than ~1mm 3 and over long time periods with high speed, good resolution, and significantly reduced photo bleaching and phototoxic effects. Unlike confocal techniques, which use spot-like excitation, laser-sheet based methods attempt to excite an entire plane within the tissue sample; the illuminated plane is either formed at once using cylindrical lens (called selective plane illumination microscopy: SPIM) or by fast-scanning a pencil-like beam (called digital scan laser sheet microscopy: DSLM).…”

Augmented line-scan focal modulation microscopy for multi-dimensional imaging of zebrafish heart in vivo

“…Selective plane illumination microscopy (SPIM) or light sheet microscopy has emerged as a popular choice for in vivo fluorescence imaging of relatively transparent small animal models, including embryonic zebrafish and fruit fly [6][7][8][9][10][11][12][13]. Optical sectioning in SPIM is mainly achieved by illuminating the sample using a light beam propagating within the focal plane of a detection objective, although a confocal slit is employed in some recently reported setups to further enhance the contrast.…”

A compact Airy beam light sheet microscope with a tilted cylindrical lens

“…Dual-side illumination [3,4] or moving the sample along the illumination plane [5] can extend the FOV; however, both methods increase the sample irradiation and may thus induce more photo-bleaching and photo-damage. High axial resolution over an extended FOV has been achieved by digitally scanning a propagation-invariant Bessel or Airy beam [6][7][8][9]. Compared to Gaussian beams, propagation-invariant beams such as Bessel or Airy beams maintain their beam profile over a larger distance, hence a uniformly thin light sheet can be created over a larger FOV.…”