In this article an application of the commercially available high-resolution light sheet-based microscope, Lightsheet Z.1 from ZEISS, to the investigation of large cultured organotypic tissues, so called cellular spheroids is presented.
A light sheet-based microscope obtains an optical section of a fluorescent specimen by illuminating only a section of the sample with a thin sheet of light. The emitted fluorescence is recorded with a camera through a wide-field microscope that is oriented perpendicularly to the light sheet. This microscopy technique provides much faster image acquisition while exposing the specimen to relatively low amounts of light, thereby reducing photobleaching and photoxicity. The improvement is especially significant with
large specimens that require many sections in order to generate a three-dimensional image, such as entire organisms, organoids mimicking tissues or three dimensional cell cultures. Finally, mounting of specimens in transparent aqueous gels allows them to be imaged from multiple directions (Multiview imaging), generating high contrast images of highly scattering specimens.
Experimental Procedures and Results
Human bronchial epithelial cells (HBE cells) were cultured as scaffold-free organotypic spheroids using a hanging drop method and infected with a transgenic human adenovirus type C2 with E3B region substituted by an eGFP cassette expressed under the major CMV promoter (HAdV-E3BGFP).
Spheroid samples were imaged using Lightsheet Z.1 with 20x/1.0 detection optics and two-sided 10x/0.2 illumination optics. To counteract the degradation of the light sheet by the highly scattering spheroid, the specimen was sequentially illuminated through each of the two opposing illumination objectives, generating pairs of single-side illumination images, which were instantaneously combined into optical sections with considerably improved penetration depth.
Multiview imaging not only provided good quality data over a larger part of the spheroid, but it also increased axial resolution of the resulting images. This could be easily shown on the images of the fluorescent beads, which in a single-view image appeared elongated along the direction of the microscope’s
Light sheet fluorescence microscopy enables scientists to image large spheroidal cell cultures with high frame rate and low light exposure. Additionally, the light-scattering characteristic of large spheroid cell cultures can be efficiently circumvented by dual side illumination and Multiview imaging. Lightsheet Z.1 combines the advantages of a light sheet microscope with easy specimen mounting, full experimental control and robust
image processing tools provided by ZEN imaging software. This makes Lightsheet Z.1 a perfect tool for imaging of large and highly scattering specimens. The high acquisition rate and simple specimen preparation enable imaging of many samples in a short amount of time. Furthermore, the Lightsheet Z.1 is shown to be a tool for the development of novel assays in infectious disease biology.