Lattice Lightsheet Microscopy in Life Science Research

During the transition from the naive epiblast to a pluripotent state, over 100 cells are organized into an approximately 60-80 µm diameter pre-implantation mouse embryo. To examine the changes in gene expression during this transition, both live and fixed samples are imaged at different timepoints using spinning disk confocal microscopy. The volumetric analysis of cellular expression patterns provides evidence for molecular mechanisms regulating the development of pre-implantation mouse embryos.

To determine which cells are expressing specific cellular markers and how these cells are organized relative to each other requires volumetric imaging of the pre-implantation embryos by confocal microscopy. The embryos at this stage are sensitive to changes in growth environment, structural integrity and phototoxicity. To prevent any aberrant changes in the developmental phenotype, the embryos could not be could not be forced against the cover glass and needed to be imaged quickly and with low phototoxicity. The only available option was to use spinning disk confocal microscopy. This imaging method satisfied the need for low phototoxicity, and almost provided the speed required, but suffered from the dramatic loss in signal intensity with increased imaging depth into the embryos. Consequently, only halfway through the depth of the samples were typically imaged.

The ZEISS Lattice Lightsheet 7 microscope enabled imaging of the entire depth of the embryos with little to no phototoxicity, at the required speed to prevent any motion artefacts, and with minimal loss of signal intensity. In addition, the volumetric imaging of the embryos using ZEISS Lattice Lightsheet 7 allowed nearly isotropic resolution for a more accurate representation of the cellular organization within the embryo. Importantly, this imaging technology will allow us measurement of gene expression signatures during this crucial stage in embryonic development.

Imaged volume: 230 µm x 124 µm x 94 µm, 30 ms exposure time, 30 x 1000 Lattice Lightsheet, two channels.

Conventional confocal microscopy cannot image the entire 3D cell model with a temporal resolution higher than the beating frequency.

The capability of ZEISS Lattice Lightsheet 7 to perform fast volumetric imaging with subcellular resolution can be used to investigate tissue variations, such as cellular contractility and viability, among others.

One volume every 1.26 sec, 48 vol/min for 1 min, imaged volume: 300 µm x 435 µm x 125 µm, 2 µm step size, 126 planes per volume, 1 ms exposure time, 100 x 1800 Lattice Lightsheet.

Cells are grown on cyclo olefin polymer (COP) with a grid structure to observe how growth cones progress along the grid. Growth cones are extremely light sensitive and start shrinking when exposed to too much excitation light. Additionally, the grid structure on COP poses many challenges to traditional imaging systems. Double image artifacts can be seen when using a traditional laser scanning confocal. COP also has a refractive index of 1.53, whereas glass has a refractive index of 1.52, and the thickness of the COP bottom is 200 µm; these parameters can cause image blurring.

ZEISS Lattice Lightsheet 7 is able to overcome these challenges. By orienting the grid structure to be parallel to the lattice light-sheet and adjusting the Lattice Lightsheet 7-specific free-form optics, the double image can be removed. Additionally, the gentleness of ZEISS Lattice Lightsheet 7 is the perfect tool to investigate the dynamic movement of developing neurons in 4D with high spatiotemporal resolution without disturbing them with too much light.

One volume every 15 sec., 21 volumes in 5 min., imaged volume: 78 µm x 44 µm x 22 µm, 0.3 µm step size, 134 planes per volume, 20 ms exposure time, 30 x 1000 Lattice Lightsheet, two channels.

In previous experiments using confocal microscopy, photobleaching and phototoxicity issues were creating problems with data collection.

ZEISS Lattice Lightsheet 7 enabled volume imaging of the spheres to track cell divisions and rearrangements.

One volume every 10 min for 16 hrs, 97 volumes were recorded, imaged volume: 520 µm x 540 µm x 32 µm, 12ms exposure time, 100 x 1800 Lattice Lightsheet.

Phototoxicity is observed with these samples when using traditional confocal microscopy.

ZEISS Lattice Lightsheet 7 enables imaging of cells for 12 hours with enough resolution to image actin and individual mitochondria, even during mitotic events.

Five positions, one volume every 10 minutes for 12 hours, 73 volumes were recorded, imaged volume: 75 µm x 120 µm x 16 µm per position, 30 ms exposure time, 30 x 1000 Lattice Lightsheet, three channels.

Conventional confocal microscopy requires a compromise on either speed or resolution to image the entire brain organoid in a reasonable amount of time.

ZEISS Lattice Lightsheet 7 images the entire organoid with enough resolution to distinguish individual neurons in under 10 minutes.

Imaged volume: 1.37 mm x 1.18 mm x 76 µm, 5 ms exposure time, 100 x 1800 Lattice Lightsheet, two channels.

Widefield microscopy provided the speed required for live imaging of endothelial cells of blood vessels but only with ZEISS Lattice Lightsheet 7 could higher resolution, particularly in the axial dimension, be achieved.

One volume every 8 sec for 15 min, 115 volumes were recorded, imaged volume: 200 µm x 250 µm x 55 µm, 3ms exposure time, 100 x 1800 Lattice Lightsheet, two channels.

ZEISS Lattice Lightsheet 7 allows for volumetric imaging of ultra-fast membrane dynamics with near-isotropic resolution for detailed characterization of membrane ruffling.

One volume every 1.5 sec for 2 min, 85 volumes were recorded, imaged volume: 58 µm x 60 µm x 9 µm, 3 ms exposure time, 15 x 550 Lattice Lightsheet.

Imaging neurons in an entire C. elegans requires ultra-fast imaging of large volumes – which is challenging for technologies such as traditional confocal microscopy.

ZEISS Lattice Lightsheet 7 not only provides the required temporal resolution but also the spatial resolution to identify and follow individual neurons in the moving worm.

One volume every 2 min for 1 hr 20 min, 41 volumes were recorded, imaged volume: 300 µm x 340 µm x 55 µm, 15 ms exposure time, 100 x 1800 Lattice Lightsheet, two channels.

ZEISS Lattice Lightsheet 7 captures T cells live and in action. Fast, gentle volumetric imaging of cellular interactions with near-isotropic resolution makes it the perfect tool to study T cell behavior and function.

Three positions, one volume every 3 min for 2 hrs 15 min, 507 planes per volume, imaged volume: 300 µm x 130 µm x 16 µm per position, 25 ms exposure time, 30x1000 Lattice Lightsheet, two channels.

Calcium signaling events are known to be ultra-fast events and capturing the full volume of an organoid quickly enough to not miss such events can be challenging.

ZEISS Lattice Lightsheet 7 enables time-lapse imaging of the whole organoid with high temporal resolution to capture Calcium flashes of individual cells.

One volume every 10 sec for 10 min, 376 planes per volume, imaged volume: 145 µm x 350 µm x 75 µm, 5ms exposure time, 100 x 1800 Lattice Lightsheet.