Specimen Selection and Preparation

Whole human embryo specimens from Carnegie stages 13 to 23 were selected from the Carnegie Collection of Human Embryos at the National Museum of Health and Medicine, in the Armed Forces Institute of Pathology (AFIP). These stages approximately represent 32 to 56 days post-ovulation. Formalin-fixed specimens without signs of mechanical damage and without obvious signs of malformations were selected whenever possible. In some cases, embryos with suspected malformations were imaged because more suitable specimens were not available to represent those stages of development. All embryos were photographed with a digital camera on an optical microscope prior to magnetic resonance microscopy (MRM).

The larger and well-fixed embryos (over stage 17) were immersed in Fomblin LC08 (Ausimont USA, Inc., Thorofare, NJ) during MRM imaging to provide a background without signal. Smaller and poorly-fixed specimens were imaged in formalin because they would not have tolerated the density of the Fomblin medium. However, formalin does not provide as much contrast between the embryo and the background medium as the Fomblin does. All embryos were placed in airtight plastic containers and inserted into an imaging coil. Four MR imaging coils were built to accommodate the 10-fold size differences of these embryos: a Helm-Holz coil for the smallest embryos, a solenoid coil for the next sizes, and then two bird-cage coils for the larger specimens.

MRI Imaging

All embryos were imaged in a 9.4 Tesla, super-conducting magnet at Duke University’s Center for In-vivo Microscopy. Each embryo was imaged with predominantly T1-weighted, T2-weighted, and diffusion-weighted pulse sequences to provide 3 perfectly registered three-dimensional data sets. T1-weighted imaging was achieved with a three-dimensional spin-echo GRASS sequence, TR = 95 ms, TE = 3.5 ms, flip angle alpha = 60 degrees, and NEX = 2. T2-weighted imaging was obtained with a three-dimensional spin-echo sequence, TR = 1,000 ms, TE = 50 ms, alpha = 90 degrees, and NEX = 1. Diffusion-weighted imaging was obtained with a three-dimensional spin-echo sequence, TR = 1,000 ms, TE = 15 ms, alpha = 90 degrees, NEX = 1, and diffusion time of 3 ms in the long axis of the embryo. Other details of the pulse sequences can be found below. Images were acquired as 128 sagittal slices with 256 x 256 pixels per slice. The field of view was set to produce isotropic voxels (three-dimensional pixel elements with equal dimensions in the x, y, and z axis). This aspect ratio matched the embryo dimensions that were very close to 2:2:1 for the anterior-posterior, superior-inferior, and left-right axis, respectively. Image resolution ranged from 39 microns per pixel to 156 microns per pixel, depending on the size of the embryo (see table below).

Image Processing

The embryos were separated from background noise in the sagittal images using thresholding, seeding, and masking tools in Adobe Photoshop. These same tools were used to produce the segmented data sets containing single organ systems. The segmented (organ) data sets maintain the original slice numbering system and therefor do not always begin with slice #1 if the organ was not found in slice #1. Coronal and axial images were obtained from the cleaned sagittal images using VoxelView_Ultra 2.5 software (Vital Images, Fairfield, IA) running on a Silicon Graphics workstation. QuickTime animations were created using volume rendering in VoxelView_Ultra, and psuedo-timelapse animations were created using Gryphon Morph 2.5 software.

Magnetic Resonance Imaging Parameters

Carnegie Stage	Post Ov. Days (approx.)	Scan#	TR	TE	Flip Angle	Scan Type	Coil	Resolution*	NEX
13	28	11634	95	3.5	60	T1	HelmHolz	39.1	2
		11635	1000	15	90	Diffusion	HelmHolz	39.1	1
		11636	1000	50	90	T2	HelmHolz	39.1	1
14	32	11627	95	3.5	60	T1	HelmHolz	39.1	2
		11628	1000	15	90	Diffusion	HelmHolz	39.1	1
		11629	1000	50	90	T2	HelmHolz	39.1	1
15	33	11537	95	3.5	60	T1	Solenoid	54.7	2
		11538	1000	15	90	Diffusion	Solenoid	54.7	1
		11539	1000	50	90	T2	Solenoid	54.7	1
16	37	11894	95	3.5	60	T1	Solenoid	62.5	2
		11897	1000	15	90	Diffusion	Solenoid	62.5	1
		11898	1000	50	90	T2	Solenoid	62.5	1
17	41	11422	95	3.5	60	T1	Solenoid	46.9	2
		11424	1000	15	90	Diffusion	Solenoid	46.9	1
		11423	1000	50	90	T2	Solenoid	46.9	1
18	44	11430	95	3.5	60	T1	Bird Cage	78.1	2
		11431	1000	15	90	Diffusion	Bird Cage	78.1	1
		11432	1000	50	90	T2	Bird Cage	78.1	1
19	47	11239	95	3.5	60	T1	Bird Cage	70.3	2
		11237	1000	15	90	Diffusion	Bird Cage	70.3	1
		11238	1000	50	90	T2	Bird Cage	70.3	1
20	50	11906	95	3.5	60	T1	Bird Cage	101.6	2
		11907	1000	15	90	Diffusion	Bird Cage	101.6	1
		11908	1000	50	90	T2	Bird Cage	101.6	1
21	52	Not Available
22	54	11525	95	3.5	60	T1	Bird Cage	140.6	2
		11526	1000	15	90	Diffusion	Bird Cage	140.6	1
		11527	1000	50	90	T2	Bird Cage	140.6	1
23	56	11616	95	3.5	60	T1	Bird Cage	156.3	2
		11617	1000	15	90	Diffusion	Bird Cage	156.3	1
		11618	1000	50	90	T2	Bird Cage	156.3	1
* Resolution is reported in microns per voxel (microns per cubic pixel) with x, y, and z dimensions all being equal.