ZEISS Lattice Lightsheet 7

Light sheet microscopy in general (also called Gaussian light sheet microscopy) is well known for its gentle imaging conditions at superior imaging speed. The groundbreaking concept of decoupling excitation and detection allows illumination of only the part of the specimen that is in the focal plane of the detection objective lens. By moving the sheet with respect to the sample and recording one image per focal plane, you can acquire volumetric data without exposing the out-of-focus sample areas.

Lattice light sheet microscopy combines the advantages of light sheet microscopy with near-isotropic resolution in the confocal range. Advanced beam shaping technology creates lattice-shaped light sheets that are significantly thinner than standard Gaussian light sheets and thus provide increased resolution at comparable imaging speeds. The lattice structure of the light sheet is created using a Spatial Light Modulator (SLM), then projected onto the sample after passing scanners that dither the lattice structure to create a smooth light sheet.

Cos 7 cells transiently transfected with CalnexinmEmerald and EB3-tdTomato. EB3 labels the growing ends of microtubules and is necessary for the regulation of microtubule dynamics. Calnexin is a protein of the ER where proteins are synthesized. Cells were imaged for 1.5 hrs every 80 secs, imaged volume: 175 × 120 × 70 μm³.

Time lapse movie showing dynamics of a U2OS cell stably expressing Actin-GFP (cytoskeleton, cyan). Cells were also labeled with MitoTracker™ Red CMXRos (Mitochondria, green) and Draq 5 (Nucleus, magenta).

Cos 7 cell transiently transfected with Tomm20-mEmerald and Calreticulin-tdTomato. Tomm20 labels the outer membrane of mitochondria, Calreticulin is a protein of the ER where proteins are synthesized. Both are extremely delicate and light-sensitive organelles that are difficult to image with conventional methods. One volume every 30 secs, imaged for 1 hr 15 mins, imaged volume: 175 × 210 × 20 μm³. A total of 85,800 images was recorded; 572 volume planes for 150 time points.

Cos 7 cell expressing Lifeact-GFP. Maximum intensity projection. The cell was imaged constantly for 9 hrs; one volume (115 × 60 × 25 μm³) every 10 secs. A total of 1,005,000 images was recorded; 201 volume planes for 5,000 time points.

Cos 7 cells transiently transfected with mEmerald-Rab5a and Golgi7-tdTomato. Golgi7 is a protein associated to the Golgi and Golgi vesicles. Rab5a is an early endosome marker. Tracking of vesicles in 3D with near-isotropic resolution becomes reality. Tracking was performed in arivis Vision4D^®.

T cell expressing Lifeact-GFP. Color-coded depth projection and maximum intensity projection side- by-side. The T cell was imaged constantly for over 1 hr; one volume every 2.5 secs. Sample: courtesy of M. Fritzsche, University of Oxford, UK.

Fixed mouse germinal vesicle oocytes stained for the nuclear envelope (anti-lamin, cyan), actin (phalloidin, magenta), and microtubules (anti-tubulin, yellow). The Sinc3 15 × 650 lattice light sheet was used for high-resolution imaging of microtubule and actin structures. Follow the 3D structure of the microtubules in the movie. Sample: courtesy of C. So, MPI Göttingen, Germany.

Fixed mouse germinal vesicle oocytes stained for the nuclear envelope (anti-lamin, cyan), actin (phalloidin, magenta), and microtubules (anti-tubulin, yellow). The Sinc3 100 × 1,800 lattice light sheet was used for imaging of the whole oocyte. Sample: courtesy of C. So, MPI Göttingen, Germany.

Live mouse oocytes arrested in metaphase II and stained for mitochondria (cyan), microtubules (magenta) and chromosomes (yellow). Sample: courtesy of C. So, MPI Göttingen, Germany.

DeltaD-YFP transgenic zebrafish embryo (Liao et al. 2016, Nature Communications). Fusion protein driven by a transgene containing the endogenous regulatory regions, expression in the tailbud and pre-somitic mesoderm. Signal visible in the cell cortex, and in puncta corresponding to trafficking vesicles (green). Nuclei in magenta. The embryo was imaged for 5 minutes constantly; one volume (150 × 50 × 90 μm³) every 8 sec. Sample: courtesy of Prof. Andrew Oates, EPFL, Switzerland.

High-speed movie of zebrafish embryo. Volumetric imaging of trafficking mRNA molecules (green). Nuclei are shown in magenta. Data is displayed as maximum intensity projection. One volume (86 × 80 × 12 μm³) was recorded every 2.5 sec. Sample: courtesy of Prof. Andrew Oates, EPFL, Switzerland.

Trafficking mRNA molecules were tracked in arivis Vision4D^®. The movement of the zebrafish embryo was first corrected using a nucleus reference track. Then individual mRNA molecules were tracked over time to result statistics such as speed and directionality. Sample: courtesy of Prof. Andrew Oates, EPFL, Switzerland.

Drosophila melanogaster is a model organism in many research fields such as biomedical research. Many genetically modified variants are available to researchers. This video shows a drosophila embryo with GFP labeling as it moves over time. A total of 91,100 i mages were taken, 911 volume planes, 100 time points. One volume, every 15 secs; imaging duration 25 mins, imaging volume: 300 × 455 × 145 μm³.

Spheroids and organoids are in vitro models of organs – much smaller and simpler but easy to produce and thus for developmental biologists an invaluable tool to study organ development. Unlike cell cultures, which usually consist of a monolayer of cells only, cells in spheroids / organoids form three-dimensional structures, allowing for the investigation of cell migration and differentiation inside 3D cell models. With lattice light sheet microscopy, imaging the development and self-organization of organoids becomes reality. Here, we can see a 3D rendering of a spheroid consisting of cells expressing H2B-mCherry (cyan) and α-Tubulin-mEGFP (magenta). Not every cell is labelled.