Systematic and quantitative comparison of lattice and Gaussian light-sheets

Abstract

The axial resolving power of a light-sheet microscope is determined by the thickness of the illumination beam and the numerical aperture of its detection optics. Bessel-beam based optical lattices have generated significant interest owing to their reportedly narrow beam waist and propagation-invariant characteristics. Yet, despite their significant use in lattice light-sheet microscopy and recent incorporation into commercialized systems, there are very few quantitative reports on their physical properties and how they compare to standard Gaussian illumination beams. Here, we measure the beam properties in the transmission of dithered square lattices, which is the most commonly used variant of lattice light-sheet microscopy, and Gaussian-based light-sheets. After a systematic analysis, we find that square lattices are very similar to Gaussian-based light-sheets in terms of thickness, confocal parameter, propagation length and overall imaging performance.

A Versatile Oblique Plane Microscope for Large-Scale and High-Resolution Imaging of Subcellular Dynamics

“…Unlike single objective light-sheet systems that use oil immersion objectives (Maddox et al, 2018), spherical aberrations are not immediately evident ( Figures 2D and E, and Figure 2-Figure Supplement 2) . For an oblique illumination angle of 30 degrees and an excitation NA of 0.16, the raw axial resolution of our system is similar to the raw 666 nm axial resolution reported for the most commonly used square illumination mode of Lattice Light-Sheet Microscopy (Chang et al, 2020; Valm et al, 2017) or a spinning disk microscope using the same NA 1.35 / 100X objective used here ( Figure 2-Figure Supplement 3 ).…”

A versatile oblique plane microscope for large-scale and high-resolution imaging of subcellular dynamics

“…Unlike single-objective light-sheet systems that use oil immersion objectives ( Fadero et al, 2018 ), spherical aberrations are not immediately evident ( Figure 2D and E , and Figure 2—figure supplement 2 ). For an oblique illumination angle of 30 degrees and an excitation NA of 0.16, the raw axial resolution of our system is similar to the raw 666 nm axial resolution reported for the most commonly used square illumination mode of lattice light-sheet microscopy ( Chang et al, 2020 ; Valm et al, 2017 ) or a spinning-disk microscope using the same NA 1.35/100X objective used here ( Figure 2—figure supplement 3 ).…”

“…We chose an annular mask with an outer and inner diameter of 3.76 and 2.98 mm, which corresponds to an outer and inner NA of 0.269 and 0.213, respectively. The selected annular mask and the corresponding pattern on the spatial light modulator results in a square lattice light-sheet with an effective NA of 0.16 ( Chang et al, 2020 ), which matches the illumination NA of the OPM. The resulting lattice was rapidly dithered to create a time-averaged sheet of light during the image acquisition.…”

In vivo profiling of site-specific human cancer cell states in zebrafish

“…Our approach begins to address this very challenge by leveraging the combined advantages of the zebrafish model [6–9] and innovations in high-resolution imaging and computational image analysis, allowing for the throughput and resolution needed to uncover meaningful heterogeneity among noise. First, isotropic high-resolution image acquisition using ASLM enabled the segmenation and morphological characterization of cells in 3D with a sensitivity not achievable with other light-sheet imaging modalities, including the lattice light-sheet microscope [56, 57] (Figure S5a). Simulations of imagery with 3-fold reduced axial resolution yielded segmentation errors and marked shifts in PCA space, demonstrating the importance of high, near-isotropic resolution in 3D measurements (Figure S5b-c).…”

“…Several technological innovations made this workflow possible. First, the improved axial resolution by ASLM compared to other light-sheet imaging modalities, including advanced techniques such as lattice light-sheet microscopy [59,60], enabled high-resolution visualization of cells in 3D which would be otherwise impossible (Figure S5a). This is particularly important in cancer cell biology as opposed to tissue development, where orientation of cells is (black frames, upper part of panels).…”