recording light-microscopy images of large, nontransparent specimens, such as developing multicellular organisms, is complicated by decreased contrast resulting from light scattering. early zebrafish development can be captured by standard light-sheet microscopy, but new imaging strategies are required to obtain high-quality data of late development or of less transparent organisms. We combined digital scanned laser light-sheet fluorescence microscopy with incoherent structuredillumination microscopy (dslm-si) and created structuredillumination patterns with continuously adjustable frequencies. our method discriminates the specimen-related scattered background from signal fluorescence, thereby removing out-offocus light and optimizing the contrast of in-focus structures. dslm-si provides rapid control of the illumination pattern, exceptional imaging quality and high imaging speeds. We performed long-term imaging of zebrafish development for 58 h and fast multiple-view imaging of early Drosophila melanogaster development. We reconstructed cell positions over time from the Drosophila dslm-si data and created a fly digital embryo. Powerful new quantitative approaches to developmental biology have emerged that aim at understanding morphogenesis, develop mental genetics and their relationship systemwide 1-9 . It is now possible to comprehensively follow cell behavior during the first day of zebrafish development 6 or to perform largescale mapping of gene expression profiles in nematodes 9 . Such largescale and highthroughput studies rely on advanced fluorescence micro scopy and automated approaches to image processing and data analysis. The robustness of these approaches, however, depends substantially on the performance of the light microscope. Recently, we developed digital scanned laser light sheet fluores cence microscopy (DSLM), in which a single plane in a specimen is quickly scanned with a thin laser beam while a camerabased detection arm records a fluorescence image at a right angle to the illumination axis. This approach provides a combination of crucial properties: imaging speed and signaltonoise ratio are high, and the energy load on the specimen, and thus photobleaching and phototoxicity, are kept at a minimum 6,7 . Early zebrafish embryos can be recorded for up to 24 h and yield image data, from which cell movements can be reconstructed with high fidelity. However, similar analyses at later stages of embryogenesis or for less trans parent specimens, such as Drosophila melanogaster embryos, are hampered by substantially increased light scattering. Image qual ity and contrast suffer when imaging highly complex multicel lular specimens with many cell membranes that act as refractive interfaces or in embryos, in which somatic cells are engulfed by a lipidrich yolk (for example, early Drosophila embryos). To address this limitation, we previously introduced incoher ent structuredillumination microscopy 10 to light sheet-based imaging 11 by placing a mechanical ruling in a conjugate image plane in the illumination arm of a selective plane illumination microscope 12 (a method named selective plane illumination microscopy with structured illumination; SPIMSI) to illumi nate the specimen with a fixed pattern of stripes rather than a homogeneous light sheet. Acquiring a shift series of images at different spatial phases of this pattern allowed us to discriminate the scattered background light against signal fluorescence using a simple processing algorithm. Unfortunately, the entire ruling had to be shifted to change the phase of the profile, and different rul ings were required for different frequencies. SPIMSI, therefore, suffered from low image quality, low imaging speed and the lack of flexibility regarding the shape and frequency of the illumina tion pattern 12 . Here we present a DSLM implementation of structured illumi nation (DSLMSI) that addresses all three issues. In DSLMSI, the intensity of the illuminating laser beam is electronically modulated in synchrony with the scanning process. This allowed us to create highquality sinusoidal intensity profiles. Harmonic components, phase and frequency of the structuredillumination pattern are flexible and can be quickly adjusted to address spatiotemporal
Digital scanned laser lightsheet fluorescence microscopy is then combined with incoherent structuredillumination microscopy to produce the 3D locations of stained nuclei in Drosophila embryos REF .
Fast, high-contrast imaging of animal development with scanned light sheet–based structured-illumination microscopy
Nature Methods
Nature Methods
Biology Medicine