Carolina Araya reveals how light-sheet microscopy is revolutionising 3D imaging
Light-sheet fluorescence microscopy (LSFM) has emerged as a key tool in the study of biological systems – from subcellular processes to entire organisms. The 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. Therefore, 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. This 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. The 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.
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 asymmetric configuration of the illumination and detection objectives (10x/0.3NA and 25x/1.1NA, respectively). Simultaneous two-colour imaging is enabled by spectrally separating two different channels on two sCMOS cameras, both of which can acquire more than 80fps at full frame.
Suited for cell culture applications, this microscope design features a small U-shaped sample compartment (Fig. 2A). This is covered in FEP foil (optically transparent material), which serves as a physical barrier between the sample medium and the immersion medium(Fig. 2B). The foil works as a “curved coverglass” where cells can be cultured or spheroids and small embryos can be placed. The temperature, humidity, CO2 and O2 levels of the incubation chamber can be controlled, allowing for a variety of conditions to be established.
This system is suited to a variety of samples and applications, including: in toto imaging of small animal and embryo models, observing dynamic processes in mammalian cell culture models (e.g. spheroids, organoids), live imaging of plant models, investigating stem cell development and differentiation, in vitro fertilisation research and monitoring, and functional imaging (calcium).
Another recent innovation in design (InVi SPIM Lattice Pro) offers tailorable, interactive adaptability of the beam shape to suit the specific requirements of a sample. This provides flexibility for the type of light-sheets that can be generated, e.g. single or multiple variable Bessel beams, lattice light-sheets for structured illumination, and static or scanned Gaussian beams (Fig. 3A). It enables researchers to combine different types of beams in one experiment and select the setting that gives the best results for their specific 3D high-resolution imaging experiments: large field of view, high speed and optimal spatial resolution (Fig. 3B).
The range of applications includes observing dynamic cellular interactions, cell cycle imaging, studying membrane dynamics, subcellular structure visualisation and long time-lapse imaging.
By only exciting the fluorophores in a single plane, light-sheet microscopy is gentle enough to image specimens over extended periods. Additionally, the technique’s high imaging speed allows researchers to follow the rapid development of organisms not easily observed with traditional scanning microscopes.
Carolina Araya is with Luxendo, a Bruker company
