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 equipped with a xyz stage to position the sample in the focal plane of the objective but there is no control of the sample orientation. A positioning method for biological samples needs to fulfill the following criteria: it needs to add a degree of freedom to visualize the sample from
the optimal side. The positioning technique must not interfere with
the imaging. The method should be easy to integrate in any automated microscopy routine. Ultimately, the technique has
to ensure the sample integrity. While optical methods have successfully
been used to position and to orient single cells for light microscopy
Torres-Mapa 2012 Kreysing 2014 Kreysing 2008,
forces are not high enough to position
a millimeter-sized zebrafish embryo.
Introduce the idea and show how you go
about implementing it
We tried whether we could use magnetic
forces to orient a zebrafish embryo within its chorion. We injected magnetic beads into the yolk and we discovered that
we could orient the injected embryo within its chorion by approaching a permanent
magnet close to the embryo (Supplementary
video S1).
Injection of magnetic beads
The super paramagnetic beads were
injected with glass needles into the yolk of the zebrafish embryo to ensure the
integrity of the zebrafish embryo. We
performed the injections at 4 hpf and the injection needle was inserted from
either the vegetal pole or the lateral side and beads were deposited close to
the yolk membrane (Figure 1b). We
found that performing the injections at extremely low pressure (5-10 psi) and
long injection duration (100-150 ms) avoided the dispersion of beads. By
applying a strong constant magnetic field after the injection of the beads with
a permanent magnet the beads were attracted and clump. This aggregation of
beads preserved the single beads from translating through the yolk and eased
the rotation.
Find the right magnetic beads to orient the zebrafish embryo
To ensure the sample integrity no residual force should affect the sample after it has been oriented. Compared to ferromagnetic beads it turned out that super-paramagnetic beads show no residual force once the magnets are removed since they exhibit magnetic properties in the presence of a magnetic field with no residual magnetism
Neuman 2007. The size of these beads needed to be large enough that the beads are stationary and do not translate within the yolk when a magnetic field is applied but they should be small enough that they can be injected without damaging the zebrafish.
Injection of beads does not alter the zebrafish development
Injected embryos were monitored for 4 days and we found no visible delay or defect in development when compared to non-injected wildtype embryos (Supplementary Figure S1). As the embryo developed, the yolk was being used with the beads staying in the remaining yolk, close to the yolk extension.
The injected beads were attracted by the permanent magnet but the zebrafish couldn’t translate towards the electromagnet since it was encapsulated in its chorion. ROTATION We found that by moving the permanent magnet we can orient the zebrafish embryo contactless within its chorion. (Moving magnet too slow) varying magnetic fields
Magnets
The external magnetic field can be
generated by permanent or electromagnets. Although permanent magnets have a
higher magnetic field, electromagnets can be controlled by the applied current
and hence allow a dynamic adaptation of the magnetic field without any movement
of the magnet. The applied force on a super-paramagnetic bead is proportional
to the magnetic field gradient
Neuman 2007. Therefore, we sharpened the core of the
electromagnets to create a sufficiently strong magnetic field gradient, even with
a moderate current (300 mA). Heating of the electromagnets was also negligible.
We designed the electromagnets to be small enough to be oriented around the sample
in any geometry giving the freedom of full 3D sample orientation.
4 Magnets: FIGURE 1
To study
developing zebrafish from the optimal orientation we adapted our magnetic
orientation technique to a SPIM.
We found that a bead size of 2.8 µm allows a rotation of the sample with moderate magnetic fields without a translation of the beads within the zebrafish chorion. The applied force on the fish is proportional to the number of injected beads hence a higher volume of beads is favorable but the injected volume may damage the zebrafish. We found that a volume as low as 1 nl bead solution corresponding to about 15ng of beads or about 1000 beads is sufficient to rotate the embryo at low currents of 300mA.
Why do we need more than one axis of
rotation
SPIM has become the technique of choice
for in vivo imaging in developmental biology
Huisken 2004 Weber 2014. In contrast to most light microscopy techniques, SPIM provides an axis of
rotation about which the sample is rotated to take several views. However, a
single rotational-axis system cannot give the full three-dimensional freedom of
rotation (
Figure 2b). Therefore, not all parts of the sample
can be turned in front of the detection objective and are accessible, e.g. for
photo-manipulation.
Introduce the magnetic manipulation on
a SPIM
To orient the sample in three dimensions
at least four magnets were needed since every magnet acts as an attractor fixed
in space. Here, we included a tetrahedral electromagnet configuration in a SPIM
setup (Figure 2a). We designed a
sample chamber that held the four magnets in a tetrahedral geometry and the
sample tube.
Performance of the system
We measured the performance of the
magnetic rotation and found that a rotation of the zebrafish embryo within its
chorion from one magnet to another one (109.5 degrees) took less than 30s at a current as low
as 300 mA (Figure 1d). This transition time from
one magnet to the next one was anti-proportional to the applied current and could
therefore be tuned according to the application. 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 less than
10s 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. Applying a low
magnetic force could stabilize the orientation of anisotropic samples in a
preferred orientation. This method is especially
suitable to orient samples in an out-of equilibrium orientation
Intermediate angles
To study the sample in the optimal
orientation intermediate orientations between two/three magnets need to be
accessible. We found that these intermediate orientations could be accessed in
two different ways. First, spherical samples with no preferred orientation (as
the zebrafish embryo till the bud stage) could be stopped while rotating from
one magnet to the next one and settled at this intermediate orientation.
Second, by applying a current onto two or more magnets at the same time the
zebrafish oriented to the resulting magnetic field. By changing the ratio of
the applied currents between the different magnets the sample could be
positioned in any orientation in three dimensions.
Tetrahedral geometry gives better coverage
In single sided detection SPIM microscopes the sample is commonly rotated in 90deg steps and four different orientations are acquired to obtain a more homogeneous resolution and better coverage of the sample. These orientations are equally distributed in the plane of rotation. However, due to the 3D nature of the sample this geometry does not cover the whole sample. The four magnets and thereby the four views by switching on only one magnet are homogeneously distributed in 3D (Figure 2b/c). This tetrahedral geometry of the four magnets enables a better coverage of the sample than the single rotational axis SPIM with the same number of stacks.
4 views of the same embryo
We imaged a zebrafish embryo (??) from four different angles in the single rotational axis SPIM mode by manually rotating the tube in 90deg steps and in the multi-view, multi-axes mode by sequentially orienting the sample with one magnet and taking a stack for each magnet. For the registration of the we aligned the data we manually aligned the different angles in the 3d Viewer (????). The single-rotational axis data could be simply aligned
the data the four different views were successfully fused by
The FUSION is still missing!
10 different orientations from the same
embryo
To obtain an almost isotropic resolution
across the whole zebrafish embryo we imaged the embryo from ten different orientations.
We took a stack for the four sample orientations given by the tetrahedral
magnet geometry and the six orientations for all the possible combinations of
two magnets. The resulting data showed no deformations and the embryo could be
imaged from ten different orientations (Figure
2)
Limitations of light microscope as
spinning disk
In contrast to SPIM, common light
microscopes as spinning disk are designed to image the sample from only one side.
To orient the sample in the preferred orientation and to study the sample from
multiples sides we developed an inset consisting of a plate and an arc holding
the two electromagnets. This inset can
easily be adapted to any commercial light microscope. Here, we show the in situ orientation of a zebrafish
larva (5dpf, kdrl?) on a stereoscope. The magnet orientation could be adapted
continuously by sliding the magnets along the arc. The sample was positioned on
the axis of rotation. The zebrafish larvae was embedded in a glass capillary to
prevent it from translating towards the magnets and to ensure the ball bearing mechanism.
2 different orientations from the same
zebrafish larvae
We used two electromagnets to orient the
zebrafish larva about its anterior-posterior axis making use of the elongated
morphology at the larvae stage. We found that the rotation of the zebrafish
larva worked best with an angle of about 35 degrees between the electromagnet
and the plate. By switching on one magnet the zebrafish was slightly translated
towards the wall of the glass capillary and rotated about 180 degrees towards
the magnet. By switching off the magnet the larvae was released from the force
and settled in its resting position (Figure
3). A 180 degrees rotation from the left lateral to the right lateral
resting position about the anterior-posterior axis took not longer than 10 s at a current of 1 A. By rotating the
zebrafish larvae we can image the larvae from the right and the left lateral
side within a single experiment.