Light-Sheet Fluorescence Microscopy with Scanning Non-diffracting Beams

Abstract

Light-sheet fluorescence microscopy (LSFM) has now become a unique tool in different fields ranging from three-dimensional (3D) tissue imaging to real-time functional imaging of neuronal activities. Nevertheless, obtaining high-quality artifact-free images from large, dense and inhomogeneous samples is the main challenge of the method that still needs to be adequately addressed. Here, we demonstrate significant enhancement of LSFM image qualities by using scanning non-diffracting illuminating beams, both through experimental and numerical investigations. The effect of static and scanning illumination with several beams are analyzed and compared, and it is shown that scanning 2D Airy light-sheet is minimally affected by the inhomogeneities in the samples, and provides higher contrasts and uniform resolution over a wide field-of-view, due to its reduced spatial coherence, self-healing feature and longer penetration depth. Further, the capabilities of the illumination scheme is utilized for both single-and double-wavelength 3D imaging of large and dense mammospheres of cancer tumor cells as complex inhomogeneous biological samples.

Multicolor light‐sheet microscopy for a large field of view imaging: A comparative study between Bessel and Gaussian light‐sheets configurations

“…Even though the lateral resolution is approximately the same for the three configurations (see Table 3), the images acquired with SPIM (Figure 5G‐I) present the lowest quality. Therefore, the dotted black trace in Figure 5J results in a noisy and unresolved nucleus, attributed to the beam scattering across the sample and the SPIM's low optical sectioning capability [22]. Overall, Figure 5 demonstrates that DSLM‐Bessel competes fairly with DSLM‐Gaussian configuration for short samples with dimensions comparable with the latter configuration FOV size for multicolor imaging purposes.…”

“…Moreover, using higher numerical aperture microscope objectives to create the multiple excitation light sheets for wide-field imaging over large and volumetric samples was worthless because of its shorter DoF. It also conveyed the inherent chromatic defocusing [21] and scattering problems of Gaussian beams for LSFM imaging [4,18,22].…”

Lightsheet Microscopy

“…Further improvement occurred with digital scanning (Kafian, Lalenejad, Moradi‐Mehr, Birgani, & Abdollahpour, 2020; Keller & Stelzer, 2008; Keller et al., 2010) using galvanomic mirrors to scan the orthogonal focal plane. Consequently, dynamic lightsheets are more homogenous than static ones.…”

Light-sheet mesoscopy with the Mesolens provides fast sub-cellular resolution imaging throughout large tissue volumes

“…An evolution of this method is the confocal line detection light-sheet in which only one line of pixels is recorded by synchronizing detection and illumination ( Fahrbach et al., 2013 ). These methods are known to reduce stripe artifacts and increase the axial resolution if used in combination with non-diffractive beams ( Kafian et al., 2020 ; Ricci et al., 2022 ).…”

“…The limits of Gaussian optics in forming a light-sheet have been circumvented using non-diffractive beams such as Bessel beams and Airy beams (( Fahrbach et al., 2010 ; Vettenburg et al., 2014 ; Kafian et al., 2020 ). In particular, the Airy electric field envelope can be described as the function ( Siviloglou and Christodoulides, 2007 ): where describes the Airy electric field envelope, which depends on and s, respectively, a normalized propagation distance and a transverse coordinate; is a positive parameter, typically <<1.…”