MICROSCOPY & IMAGING
Fig.1. Comparison of confocal laser-scanning and light-sheet fluorescence microscopy
INNOVATION IN IMAGING Carolina Araya reveals how light-sheet microscopy is revolutionising 3D imaging L
ight-sheet fluorescence microscopy (LSFM) has emerged as a key tool in the study of biological systems – from subcellular processes to entire organisms. Te superior suitability of in vivo 3D imaging of large samples over extended time periods and the gentle sample handling in these microscopes has led to the increased use of this technique throughout the biological sciences, including in emerging applications in developmental biology, cell biology, plant research and neuroscience. In contrast to conventional
epifluorescence microscopes, light- sheet microscopy places two objectives orthogonally to each other: one for illumination and one for detection. Terefore, this optical method allows
illuminating just a thin section of the sample with a narrow sheet of light lying in the focal plane of the detection objective. Only the currently imaged plane of the sample is excited, and the rest remains unexposed to light. Scanning the light-sheet through the sample allows researchers to acquire complete volume data with high resolution in 3D. In conventional microscopy methods, the entire sample volume is illuminated at each individual imaging plane, resulting in a much larger amount of light deposited on the sample (Fig. 1). An additional advantage of light-sheet
microscopy is its camera-based, fully parallelised image acquisition. In standard imaging techniques, one or several cones of light converge on the focal plane of the
detection lens and are scanned across the plane (point by point) to build a complete image. Tis sequential approach limits the acquisition speed and requires higher light intensities, which, together with repeated light exposure, yields more photobleaching and phototoxicity. In biological microscopy, it is necessary to balance between the limits of time resolution, sample size/field-of-view and the duration of experiments. Light-sheet microscopy allows researchers to increase speed, decrease phototoxicity and obtain high-resolution, low-noise images of larger samples. Te minimal phototoxicity and photobleaching in light-sheet microscopy opens important new avenues of research. Experiments that are difficult to manage with traditional microscopy techniques are now possible.
Fig.2. Schematic of the illumination and detection optical concept in an inverted light- sheet microscope (A) and sample chamber (B)
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DEVELOPMENTS IN MICROSCOPE DESIGN Light-sheet microscopes are designed to maximise photon efficiency and enable long-term imaging under precisely controlled environmental conditions. In particular, inverted configurations are ideal for imaging 2D and 3D cell cultures as well as small embryos. One recent system (InVi SPIM, Bruker/Luxendo) features an inverted microscope with
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