Abstract
Light sheet microscopy is a powerful approach to construct three-dimensional images of large specimens with minimal photo-damage and photo-bleaching. To date, the specimens are usually mounted in agents such as agarose, potentially restricting the development of live samples, and also highly mobile specimens need to be anaesthetized before imaging. To overcome these problems, here we demonstrate an integrated light sheet microscope which solely uses optical forces to trap and hold the sample using a counter-propagating laser beam geometry. Specifically, tobacco plant cells and living Spirobranchus lamarcki larvae were successfully trapped and sectional images acquired. This novel approach has the potential to significantly expand the range of applications for light sheet imaging.
Highlights
Light sheet fluorescence microscopy (LSFM) or selective plane illumination microscopy (SPIM) uses a thin sheet of light to illuminate a sample, whilst fluorescent images are taken perpendicular to the illuminated plane [1]
We have demonstrated a compact imaging system integrating optical trapping for sample confinement and light sheet imaging. Because this simple geometry allows drugs and compounds of interest to be applied in a continuous manner without a tight physical constraint such as agarose or a coverslip, it could enable the development of samples to be more extensively imaged with less disturbance to the sample than previous approaches
We have shown the feasibility of the system by trapping Bright Yellow 2 (BY-2) tobacco plant cells and wild-type S. lamarcki larvae
Summary
Light sheet fluorescence microscopy (LSFM) or selective plane illumination microscopy (SPIM) uses a thin sheet of light to illuminate a sample, whilst fluorescent images are taken perpendicular to the illuminated plane [1] This geometry gives LSFM multiple advantages over other types of microscopy: Firstly, the unilluminated part of the sample remains unexposed to light and cannot be detected. This enhances the axial resolution and image contrast, but it reduces photo-bleaching and phototoxicity to which the sample is exposed. As the whole plane is simultaneously illuminated and imaged, the imaging speed is dramatically enhanced compared to scanning confocal microscopy These advantages make LSFM suitable for constructing 3D images of large samples and even long term monitoring of a living sample. This modality can been extended by utilizing more advanced beam shapes, such as the Bessel beam or the Airy beam [2,3,4,5]
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