Embedding and injection of magnetic beads

To rotate a sample with magnetic forces, a magnetic handle needs to be inserted into or attached to the sample. We created small magnetic agarose spheres (Supplementary Figure 1 and Supplementary Method 1), which can be embedded together with the sample in an enclosing agarose sphere. To accommodate a variety of sample sizes (500 µm–12 mm), we created hemispherical molds and spherical injection molds with different diameters (Fig. 1b and Supplementary Figure 2). The spherical shape of the embedded sample made later rotation of the sample into the preferred orientation easy.

Alternatively, embryos of some species are encapsulated in a fluid filled chorion and develop on top of the yolk, making it easy to rotate them inside the chorion. In the case of the zebrafish embryo, superparamagnetic beads could be directly injected into the yolk with a microinjection device (Fig. 1c and Supplementary Method 2). We monitored injected embryos with and without magnetic field for 4 days and found no visible delay or defect in development when compared to non-injected wildtype larvae (Supplementary Figure 3). As the fish developed, the beads stayed in the remaining yolk, close to the yolk extension, still permitting magnetic orientation of the larva.

Embedding various specimens in agarose spheres and rotating them was straightforward (Fig. 1d). We oriented 6 mm-sized artemia (Fig. 1e, f) and 12 mm-sized mouse embryos (Fig. 1g, h, Supplementary Movies 1, 2) in a non-contact manner by moving a permanent magnet over the sample sitting in one of the molds. It became apparent that especially for such large samples, the ability to freely rotate the sample is instrumental when multiple areas need to be imaged that cannot be reached in one fixed orientation. Zebrafish embryos rotated smoothly within their chorion upon application of a magnetic field (Fig. 1i, j, Supplementary Movie 3). The rotation results from the attraction of the magnetic beads by the permanent magnet. The force applied led to a translation and a rotation of the sample, thus minimizing the distance between the beads and the magnet.

Microscope insert for magnetic rotation of zebrafish larva

For dynamic control of the magnetic field we then used electromagnets (Supplementary Figure 4). As the force acting on a superparamagnetic bead is proportional to the magnetic field gradient7, the core of the electromagnets were sharpened to create a sufficiently strong magnetic field gradient even with a moderate current (300 mA), keeping the heating of electromagnets to a minimum. With two such electromagnets a sample can be easily rotated around one axis, e.g. in a tube or a capillary.

We developed an insert consisting of a plate and an arc holding two electromagnets that can be easily adapted to any upright or inverted light microscope (Fig. 2a). Two-view imaging of a zebrafish larva (5dpf, Tg(kdrl:GFP)8) on an upright epi-fluorescence microscope is shown. The magnet orientation can be adapted continuously by sliding the magnets along the arc. The rotation of the zebrafish larva worked best with an angle of about 35° between the electromagnet and the plate.

Fig. 2 Insert for multi-view imaging on a single-view microscope. a Schematic showing the insert holding two electromagnets (M1 and M2) on a microscope stage. b–e Bright-field (b, c) and fluorescence (d, e) images of a 5dpf Tg(kdrl:GFP) zebrafish larva rotated about its anterior-posterior axis by providing power to electromagnet M1 and M2, respectively. Scale bar, 1 mm Full size image

The zebrafish larva was embedded in a glass capillary, positioned at the intersection of the magnet axes and oriented about its anterior-posterior axis. When switching on one magnet, the larva was translated towards the wall of the glass capillary and rotated about 180° towards the magnet (Supplementary Movie 4). When the magnet was switched off, the larva was released from the force and settled in its resting position (Fig. 2b–e). A 180° rotation from the left lateral to the right lateral resting position took no longer than 10 s with 1 A current, illustrating that our system can add multi-view capabilities to any conventional microscope.

Tetrahedral electromagnet assembly for 3D sample orientation

To orient the sample in three dimensions, we used four electro-magnets arranged in a tetrahedral geometry with the sample in the center (Fig. 3a). We inserted the magnets such that they neither collide with the objectives nor interfere with imaging. A zebrafish embryo injected with magnetic beads was embedded in an FEP tube. The inner diameter (1.0 mm) was slightly smaller than the chorion (~1.2 mm) to hold the zebrafish in the FEP tube (Fig. 3b). By sequentially switching between four magnets, the embryo was rotated in a non-contact manner and positioned in four different orientations given by the tetrahedral arrangement of electromagnets (Fig. 3b, Supplementary Movie 5). We measured the performance of the system and found that a rotation of the zebrafish embryo from one magnet to another one (109.5°) took <30 s at a current as low as 300 mA. This transition time was inversely proportional to the applied current and could therefore be tuned according to the application (Supplementary Figure 5). A low current led to slow and homogenous rotation whereas a high current led to a rapid reorientation. After releasing the embryo from the magnetic force the zebrafish retracted for <10 s and settled at its final position. We found that the embryo remained stable in its settled position for over a minute, longer than the time it needs to take a three-dimensional stack of the whole embryo on a SPIM system (Supplementary Figure 5).

Fig. 3 Tetrahedral electromagnet geometry for 3D orientation of injected zebrafish embryos. a Four electromagnets (M1, M2, M3, M4) in a tetrahedral geometry were assembled around the sample tube. b Bright-field images of a zebrafish embryo encapsulated in the sample tube. The embryo was oriented by applying a magnetic field by magnet M1, M2, M3, and M4. Scale bar, 500 μm. c The zebrafish embryo was rotated continuously from magnet M1 to magnet M3 by changing the ratio of the applied currents between the two magnets. Scale bar, 200 μm Full size image

Intermediate positions can be accessed by applying currents to two or more magnets simultaneously. The embryo orients along the resulting magnetic field. Hence, by changing the ratio of the currents applied to the two magnets, the embryo can be rotated continuously either to align with the corresponding orientations above or at any intermediate position (Fig. 3c).

Rotation of zebrafish embryos in a multi-axis SPIM setup

To image a developing zebrafish in its optimal orientation with high resolution and low photo-toxicity, we implemented the tetrahedral electromagnet configuration around the sample chamber in a SPIM setup (Fig. 4a, additional SPIM geometries for high-NA imaging can be found in Supplementary Figure 6). Typically, SPIM provides only a single axis of rotation for multi-view imaging6,9 (Fig. 1a) and we considered whether the additional degrees of freedom in our setup could lead to improved coverage of the sample. It is worth noting that SPIM requires special attention to the sample orientations as the perpendicular illumination and detection axes both need obstruction-free access to the area of interest.

Fig. 4 Non-contact 3D orientation technique adapted to a SPIM system. a Schematic of the SPIM setup with the tetrahedral electromagnets arranged around the sample tube. The magnets were held by the sample chamber and the sample was illuminated by a light sheet through two windows. Fluorescence was detected through a third window by a detection objective. The sample chamber and the detection objective were motorized to move the sample through the light sheet and to correct for the different path lengths in air and water. b Schematic representation of the obtained image quality in SPIM imaging, which is influenced by the sample orientation relative to the detection and illumination objectives. c Maximum intensity projection of a single-view stack from a histone labeled zebrafish embryo. Scale bar, 200 μm. Inset: schematic representation of the image quality obtained by a single view. d Maximum intensity projection obtained by multi-view SPIM imaging of four views (0°, 45°, 180°, and 225°). The fusion of the four views shows a homogenous resolution along the equator but a decreasing resolution towards the cap (rotational axis). Scale bar, 200 μm. Inset: schematic representation of the image quality obtained by multi-view fusion of four views. e Maximum intensity projection of a single-view stack obtained with the multi-axis SPIM technique. The view on the animal pole shows the improved image quality of the animal pole (ROI) obtained by orienting the animal pole towards the detection objective by the multi-axis sample orientation technique. Scale bar, 200 μm. Inset: schematic representation of the image quality obtained with the multi-axis SPIM technique. f Local entropy as a measure of the image quality of the single-view, multi-view, and multi-axis data along the animal-vegetal axis. g Stills from a time-lapse of a developing, histone labeled zebrafish taken in different orientations to watch key events in the respective optimal orientation (Supplementary Movie 6). Scale bar, 200 μm Full size image

In SPIM, the view directly facing the detection objective can be captured well, but the image quality suffers from obliquely incident illumination in spherical or ellipsoidal samples (e.g. zebrafish, drosophila embryos). At the same time, the orthogonal views facing the illumination objectives are well illuminated but poorly resolved due to the longer optical path through tissue for detection. Therefore, the best image quality is achieved in the region between the illumination and detection objectives, which can be well illuminated and fluorescence detected with minimal aberrations10 (Fig. 4b). When rotating the sample in a conventional, single rotational-axis system, uniform coverage is achieved only along the equator. The image quality at the poles is always poor owing to its inaccessibility for illumination and detection.

During early development, the embryo is in an orientation with the animal pole on top of the yolk (north pole). The animal pole is therefore only poorly accessible for the conventional SPIM since the rotational axis of the SPIM is parallel to the animal-vegetal axis (A-V-axis) (Fig. 4c). To reconstruct the animal pole, we imaged an injected zebrafish embryo (Tg(h2afva:h2afva-GFP)11) in four different orientations by rotating the sample tube, registering and fusing these non-ideal views. (Fig. 4d). In contrast, in our multi-axis SPIM mode we were able to orient the animal pole facing the detection objective irrespective of its initial orientation and a single stack was sufficient to image the entire region of interest (ROI) (Fig. 4e).

We took the image stack captured after rotation and the result of the multi-view fusion and calculated the local entropy of each plane in the stack as a measure of image quality. The multi-axis rotation delivers superior image quality at the poles of the embryo, regions that are never accessible in the conventional single-axis rotation (Fig. 4f, Supplementary Figure 7). The superior optical coverage obtained in mesoscopic samples via multi-view fusion is well characterized10. As one goes deeper into the stack we observe an increase in image quality in the fused images consistent with this improvement as the combined views provide superior coverage away from the pole. Thus, by positioning the sample in its ideal orientation we achieve superior image quality compared to the multi-view SPIM, with less views needed, reducing light exposure and acquisition time.

To illustrate dynamic sample rotation during sample development, we performed time-lapse imaging of an injected, developing zebrafish embryo (Tg(H2A-GFP)11). To always have the optimal sample orientation for observing the key events during development, we oriented the zebrafish by magnetic forces and rotation of the tube. We captured, e.g. the cellular dynamics from the animal view, collective epiboly movement from the lateral side, the dorsal convergence, and extension movements from dorsal, and head and tail formation from anterior (Fig. 4g, Supplementary Movie 6). During early stages (0–10hpf, corresponding to 1-cell stage to 90% epiboly), the embryo had a nearly spherical shape, making rotation easy. At later stages, with the emergence of pronounced head and tail structures and the convergence of the dorsal side, the sample geometry limited rotations about the anterior-posterior and the dorsal–ventral axis. We adapted the orientation about the left-right axis at these stages by rotating the tube. With both techniques combined, we could adapt the orientation of a single zebrafish embryo in the microscope to watch key events that could otherwise only be captured in several separate experiments.