About MR Microscopy, Techniques

Preparation of Mouse Embryos

A readily available strain of mice were mated overnight, and females with a vaginal plug that next morning (indicating successful mating) were used to provide the embryos. The morning after mating was considered day .5 for determining embryonic age. At the desired age, the pregnant female mouse was euthanized, the uterus was exposed, and the embryos were carefully removed one at a time. Most of the embryos were then placed in fixative to preserve the tissues. In some embryos, the umbilical artery and vein were injected

with fixative and a contrast material to highlight the developing arteries and veins. The contrast material provides an intense magnetic resonance signal in the blood vessels where it is retained.

After fixation, the embryos were embedded in agarose and enclosed in an airtight capsule

. The specimens were then placed into radio-frequency coils, custom built to match the size of the embryos. The coil and embryos were placed into a 9.4 Tesla magnetic resonance scanner to acquire three-dimensional image data

. The three dimensional volumetric images were captured as 256 x 256 x 128 voxel arrays (128 slices with 256 by 256 pixels in each slice representing a field of view of approx. 14 x 14 x 7 mm). The field of view varied depending on the size of the embryo. Because magnetic resonance microscopy is non-destructive, the embryos remained intact throughout the imaging process.

How the Images Were Produced

Embryos were typically scanned using three-dimensional spin warp encoding with a repetition time (TR) of 200 ms, an echo time (TE) of 7 ms, and two excitations per view (NEX = 2), leading to a scan time of approx. 3.5 hours per embryo. During the magnetic resonance scan and subsequent data processing, 8.4 million voxels (256 x 256 x 128 voxels) of the embryos were sampled and then characterized by a 16-bit number representing the strength of the signal emanating from each voxel of the specimen. Each voxel of data represents a cube with equal x, y, and z dimensions

. This permits the data to be viewed from any side and electronically sliced in any plane without spatial distortions

.

The three-dimensional data were ¨volume-rendered¨ on a Silicon Graphics¤ workstation using Vital Image's VoxelView_ULTRA® to provide the whole embryo views and slice views. Volume rendering is accomplished by 1) selecting either the whole three-dimensional data set or some subset of it (slices or slabs), 2) assigning a brightness value and opacity value to each voxel based on the voxel's signal level, 3) passing "rays" through the data from back to front and allowing the rays to be attenuated according to the opacity and brightness values of the voxels encountered, and 4) displaying the

as a collection of pixels that represent the degree of attenuation of the "rays" as they passed through the data.

Rotational movies were made by volume rendering each embryo 36 times with 10 degrees of rotation between views

. The slice images were rendered as one-voxel thick slabs in each plane. The rendered images were copied to a Macintosh computer and organized into QuickTime¤ movies, one movie containing the sagittal, one movie the coronal, and one movie the transverse sections of each embryo

. The "time-lapse" movies (see movie) were created on a Macintosh computer using morphing software.

Magnetic Resonance Microscopy

Magnetic resonance microscopy, as used to acquire these embryo images, is similar to clinical magnetic resonance imaging in its use of radio-frequency energy to non-destructively probe the specimen. However, in order to achieve the very high spatial resolution necessary to image embryos, magnetic resonance microscopy employs higher magnetic fields, stronger gradients to perturb the magnetic field, and specialized receiver coils matched to the small specimen sizes. The imaging techniques used in this work characterizes the water in the tissues of the embryo. The magnetic resonance signal is affected by factors such as the amount of water present, the rate of diffusion of water through tissues, the degree of bulk flow of water through vascular channels, and the nature of binding interactions between water and its neighboring molecules.

To understand the generation of a proton-based magnetic resonance signal, it is helpful to consider the hydrogen protons as spinning tops

. The orientation of these spinning protons can be considered to be random under normal conditions. However, the spinning protons behave like bar magnets and align themselves when placed in a magnetic field. Like a spinning top, the protons precess (wobble in a circular pattern). The rate at which they precess is related to the strength of the magnetic field they are experiencing. If the magnetic field varies across the specimen (a magnetic gradient), then the protons will precess at different rates according to their position along this gradient

. The spinning protons will absorb radio-frequency energy at the same frequency as their precession, thus only those protons at a given location along a magnetic gradient will absorb radio-energy of a single frequency.

When the protons absorb the radio-frequency energy, their precession is knocked out of alignment with the magnetic field

. The degree to which they are knocked out of alignment depends upon the amount of energy they absorb which can be controlled by the magnitude and duration of the radio energy they receive. After the radio-frequency pulse is discontinued, the protons realign themselves with the magnetic field. The rate at which they realign (e.g. T1 relaxation rates) depends on properties that are intrinsic to the tissues surrounding the water molecules. These properties influence how quickly or how slowly the protons realign themselves with the magnetic field and become available to emit their magnetic resonance signal. By changing the magnetic field gradients, the radio frequency energies, and the timing of detection, distinct images can be obtained from a single specimen, each image reflecting a different characteristic of the water within its tissue