“Dry-immersion” LSFM chambers for serial imaging of organoids and spheroids

Session

Live and Functional Imaging Technologies Part 2

Authors

Francesco Pampaloni (1), Julien Colombelli (2)

Affiliations

1. Goethe University Frankfurt - BMLS, Frankfurt am Main, Germany
2. Institute for Research in Biomedicine (IRB), Barcelona, Spain

Keywords

Light sheet microscopy, live imaging, organoids, spheroids.

Abstract text

Light sheet fluorescence microscopy (LSFM) is the technology of choice for imaging large organs and three-dimensional cell cultures. However, the geometry of conventional LSFM set-ups does not allow operations that are common in microscopy, such as exchanging the objective lens, or quickly remove a substrate plate and serially replacing it with other ones. Although Oblique Plane Microscopy (OPM) solves many of these issues by integrating light sheet illumination in an inverted microscope, it requires a very precise alignment to not compromise the overall NA. Moreover, OPM does not allow for straightforward multiangle imaging, which is quite important for the analysis of large specimens in toto. Thus, a new generation of LSFM chambers allowing for serial imaging of multiple specimens is a highly desirable advancement for biologists. We present LSFM chambers that implement a novel technology for “dry immersion” of water-dipping objective lenses. Due to the “dry-immersion” approach, the chambers are easily detached from the set-up without removing neither the immersion water nor the specimen inside. In one of the possible experimental setups, the chamber with its specimen can be placed back in the incubator and replaced in the microscope with another one. This allows serial imaging of different specimens during the same experiment. In addition to the “dry-immersion” technology, the construction of the chambers itself exploits several innovative approaches: fully 3D printed as a block, “minimally assembled” (meaning that no screws, O-ring, or other parts are used to assemble and seal the chamber), “panoramic” observation window. We demonstrate the application of the new chambers for the screening of organoids and spheroids.