Dynamic, non-contact 3D sample orientation in microscopy

Frederic Berndt1,*, Gopi Shah1,*, Benjamin Schmid1, Jan Brugués1,2, Jan Huisken1

1 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany.
2 Max Planck Institute for the Physics of Complex Systems, Nöthnitzer Str. 38, 01187 Dresden, Germany.

*Equal contribution

Abstract (unreferenced abstract 3 sentences, no more than 70 words)

In vivo imaging of growing and developing samples requires a dynamic adaptation of the sample orientation, which has been impossible in most light microscopes. Here, we present how, after the injection of magnetic beads, a sample can be freely positioned by applying a magnetic field. We demonstrate its performance for zebrafish larvae on a stereoscope and (zebrafish) embryos on a SPIM system.

Main text

Need to image sample from a specific well-defined angle
Inhomogeneous samples have an optimal orientation when studied by light microscopy. For example in developmental biology highly scattering tissues such as pigments, the eyes or the yolk can obscure the organ or tissue of interest. Typically, the sample is carefully embedded in its ideal orientation prior to the experiment. However, after embedding the sample’s orientation is fixed and cannot be adapted to the sample’s development during in vivo experiments or to another region of interest anymore.

Define explicitly what the goal is (with respect to your sample, zebrafish).
To study organisms with optimal resolution during development, a technique to dynamically orient the sample in the microscope in any arbitrary orientation is needed. (While different organisms may require tailored solutions), the popular model organism zebrafish is transparent in the early stages making it the ideal sample for in vivo imaging.

Embedding the work in the literature
To align many zebrafish larvae for high-throughput applications in the preferred orientation microfluidic systems have been developed Lin 2015. For ... neuro  zebrafish larvae have been embedded between two coverslips and imaged from two sides by manually turning the sample Ronneberger 2012. While these techniques allow a precise, static sample orientation they still did not offer an adaptive reorientation of the sample. In light-sheet microscopy (or SPIM Huisken 2004) the sample can be rotated about one axis with a rotation stage.  ...sample orientation...Single-axis insufficient for total control over three-dim. orientation. This may pose a problem in developing organisms when imaging tissues deeply buried in the embryo.


[In addition, multi-view: The entire sample is then reconstructed by fusing views from several angles. In each view the area facing the objective lens is well resolved. Consequently, in such a single rotational–axis system uniform resolution is achieved only along the equator, which faces the detection lens. Therefore the lateral resolution at the polar caps is no better than the axial resolution of the microscope (Figure 2b). ] LATER

{Here, we present a remote positioning technique that after the injection of magnetic beads can freely position the sample by applying a magnetic field and show its excellent performance for a zebrafish larva on a commercial stereoscope and a zebrafish embryo on a custom-built SPIM system.} see above

Requirements of an orientation technique
Typically microscopes are eq