The authors would like to thank the Biomarker Imaging&#160;Research Laboratory (BIRL) at Sunnybrook Research Institute for their histology services. Support for this work was provided by the Terry Fox Foundation, the Canadian Breast Cancer Foundation&#8208;the Prairie&#8208;NWT as well as a CIHR grant, #MOP-97996.

1. Specimen
<ol>
	<li>Excise the mammary glands surgically from wild-type CDH1 as well as Igfbp7-null mice three days post onset of lactation.</li>
	<li>Spread the glands onto glass slides to help regain native mammary gland morphology.</li>
</ol>
2. Fixation and Tissue Processing
<ol>
	<li>Fix the mammary glands in neutral buffered 4% PFA O/N at 4&#160;oC.</li>
	<li>Store the glands in 70% ethanol prior to tissue processing.</li>
	<li>Transfer the glands to small tissue processing ...

<ol>
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In this study, we have developed an image registration workflow to reconstruct a 3D histology volume from serial 2D histology images, which does not require internal randomly selected landmarks or implanted fiducial markers within the tissue, which might distort the tissue. By the method described, optical blockface images themselves are used as the reference images prior to sectioning. We use external holes drilled in the paraffin block to aid in aligning the blockface images and to correct for the 2D transversal moveme...

IGFBP7 (insulin like growth factor binding protein 7) is a member of the IGF-binding protein family, and has been shown to bind the IGF1 receptor4. Down-regulation of IGFBP7 is known to be correlated with poor prognosis in breast cancer5, while the reintroduction of IGFBP7 in Xenograft tumor models greatly inhibits the tumors growth6 through induction of apoptosis and cellular senescence7. In order to study the effects of IGFPB7, an Igfbp7-null mouse was created5 (unpublished data). While these mice do not develop tumors, they show changes in histology of the ovary, muscle and liver as well as defects in mammary gland developmental patterning (unpublished data). The defective phenotype was first indicated as the null mice have smaller litter sizes and are unable to sustain multiple large litters (unpublished data).
3D histology volumes have the potential to provide useful information for quantitative and comparative analyses and assessment of pathologic findings in volumetric medical images. Three-dimensional confocal, two-photon microscopy can provide high-resolution cell morphological information of the gland at local extent14 , but it has a limited field of view and depth. Histology volume reconstruction provides more information over a much greater spatial extent. Using traditional approaches some distortion is anticipated during the preparation of histological sections, such as shrinkage, expansion, tears, and folds. These distortions make it difficult to register serial histological images into a 3D stack to reconstruct a 3D volume. As the number of consecutive sections with defects increases the similarities between intact sections is reduced and consequently makes the registration process more complicated.
Different methods have been proposed to register histological sections and to create a continuous histology volume. Some techniques depend on intensity variations8, and others are based on the shape of the sections9. For some specimens the anatomical structures can be used as landmarks10,11 along with landmark-based registration methods12,13. But these internal structures might not be detectable throughout the whole volume and for some specimens no reliable anatomical structures can be identified. Some groups have used a pair-wise registration approach and registered consecutive histology images one to another using contours or anatomical structures16-18. Registering serial histology sections to one another without the use of reference images may propagate registration error and change the actual shape of the histology volume. Pair-wise registration approach relies on the consistency of shape of the histology sections and the internal structures throughout the stack of the images; therefore it requires dense sampling of the specimen, which might not always possible, e.g., for clinical specimens.
In this pipeline we use blockface images as a set of reference images for histology volume reconstruction19. Blockface images are taken of the paraffin tissue blocks after mounting on the microtome and before each section is cut. Thus, damage to individual serial sections cut does not interfere with registration of serial sections8,11,15. We capture the blockface images in a different way from the other groups. The optical block face images are obtained by a telecentric lens to eliminate or minimize the barrel and perspective distortion, which usually occurs when using regular lenses in optics. This is one of the advantages of the proposed approach over the other published methods, which perform blockface imaging using regular lenses. The images are taken at a slight oblique angle to use the reflection of the surface of the block for contrast enhancement between the tissue and paraffin surface and to eliminate the shadow of the tissue in depth, under the paraffin surface. A photographic filter is also used to polarize the light coming from the block surface and the tissue to balance the contrast19. To correct for the displacement of the block on the rotary microtome, two to three holes are drilled in the corners of the block, which are easily detectable in the blockface images. The centroids of these holes are used along with landmark-based rigid registration to align the blockface images.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">16% PFA</td>
			<td style="width:179px;">VWR International</td>
			<td style="width:119px;">15710</td>
			<td style="width:295px;">16% Paraformaldehyde solution</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Small tissue processing cassettes</td>
			<td style="width:179px;">VWR International</td>
			<td>CA95029-956</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:260px;">Leica ASP300 Automated Tissue processor</td>
			<td style="width:179px;">Leica</td>
			<td>14047643515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">100% ethanol</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">S25307B</td>
			<td></td>
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			<td height="21" style="height:21px;width:260px;">Xylene</td>
			<td>VWR International&#160;</td>
			<td>CA95057-822</td>
			<td></td>
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			<td height="21" style="height:21px;width:260px;">Paraffin&#160;</td>
			<td style="width:179px;">Thermo Fisher</td>
			<td style="width:119px;">39501006</td>
			<td>Paraplast Tissue Embedding Medium</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Leica EG 1160 Embedding Centre</td>
			<td style="width:179px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Leica rotary microtome</td>
			<td style="width:179px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Milling machine</td>
			<td style="width:179px;">Argo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Microscope slides</td>
			<td style="width:179px;">VWR International&#160;</td>
			<td>CA48312-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">H&#38;E stain</td>
			<td style="width:179px;">VWR International</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Automatic stainer</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Coverslips&#160;</td>
			<td style="width:179px;">VWR International&#160;</td>
			<td>48404-452</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">MEDITE RCM 7000 Glass Coverslipper</td>
			<td style="width:179px;">MEDITE</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Leica SCN400 slide scanner</td>
			<td style="width:179px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">MATLAB</td>
			<td style="width:179px;">MathWorks Inc</td>
			<td>MATLAB 2007b</td>
			<td>Development software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">MeVisLab</td>
			<td style="width:179px;">MeVis Medical Solutions AG</td>
			<td>MeVisLab 2.1</td>
			<td>3D visualization software</td>
		</tr>
	</tbody>
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reconstruction of 3-dimensional histology volume and its application to study mouse mammary glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

A pitfall of traditional microscopy techniques is that the understanding of an organ at the microscopic level is limited to one field-of-view at a time. Even &#8220;total disclosure&#8221; slides, which provide entire slide sections, fail to provide three-dimensional information. With the development of whole slide, dynamic scanning technologies, our ability to see a section in its entirety has increased, however extrapolating structures requires 3D histology volume reconstruction.
To better c...

Watch this Scientific Journal Video about Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands at JoVE.com

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

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14.2K vistas.  University of Toronto. We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands. 