Multidirectional digital scanned light-sheet microscopy enables uniform fluorescence excitation and contrast-enhanced imaging

Abstract

Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful method for rapid and optically efficient 3D microscopy. Initial LSFM designs utilized a static sheet of light, termed selective plane illumination microscopy (SPIM), which exhibited shadowing artifacts and deteriorated contrast due to light scattering. These issues have been addressed, in part, by multidirectional selective plane illumination microscopy (mSPIM), in which rotation of the light sheet is used to mitigate shadowing artifacts, and digital scanned light-sheet microscopy (DSLM), in which confocal line detection is used to reject scattered light. Here we present a simple and passive multidirectional digital scanned light-sheet microscopy (mDSLM) architecture that combines the benefits of mSPIM and DSLM. By utilizing an elliptical Gaussian beam with increased angular diversity in the imaging plane, mDSLM provides mitigation of shadowing artifacts and contrast-enhanced imaging of fluorescently labeled samples.

Imaging neural events in zebrafish larvae with linear structured illumination light sheet fluorescence microscopy

“…Various computational methods 41,43 have been developed to mitigate the stripe artifacts, however, the large amount of data in LSFM images exponentially increases the computational cost of these methods. 44 Multidirectional SPIM 42 is a promising method for alleviating stripe artifacts; however, it is not compatible with SI. Self-reconstructing beams have been shown to mitigate stripe artifacts and are compatible with DSLM-SI and, therefore, could be used in combination with DSLM-SI and LR-SI reconstruction.…”

“…Self-reconstructing beams have been shown to mitigate stripe artifacts and are compatible with DSLM-SI and, therefore, could be used in combination with DSLM-SI and LR-SI reconstruction. 45,46 Recently, two approaches 44,47 that are also compatible with DSLM-SI have been developed to address this issue. We are also working on an approach to mitigating stripe artifacts that is compatible with DSLM-SI.…”

Fast multi-directional DSLM for confocal detection without striping artifacts

“…The resulting waist along the detection direction is w z /2 = 8 µm, which leads to a Rayleigh length of the virtual light-sheet of 530 µm along the illumination propagation direction, providing, thus, an almost uniform illumination over the FOV. The waist along the pivoting direction is w y /2 = 122 µm, which is very close to the desired optimum value and provides for a small beam divergence, differently from the configuration presented in [34]. The high degree of angular diversity required for efficient shadowing suppression is brought by beam pivoting, that results in an effective numerical aperture of NA eff y = NA y + n r e f r sin α = 0.0635.…”

“…Another way to solve the striping problem leverages a multi-directional illumination approach, where the sample is illuminated from one or two opposite directions by a beam pivoting relative to the focal plane [28,29]. To realize such multi-directional selective plane illumination microscopy, the beam must rotate faster than the image acquisition rate, i.e.…”

Spatially modulated illumination allows for light sheet fluorescence microscopy with an incoherent source and compressive sensing

“…However, they are not well suited for SPIM microscopy, because their low spatial coherence limits the possibility to focus the light into a single plane. Here we show not only that LEDs can be used to optically section the sample, but also that the use of incoherent light can reduce the unwanted speckle pattern and shadowing effects typical of SPIM [5], being an alternative to methods shown in [6][7][8].…”