Project: 3-D imaging cryomicrotome with adjustable resolution and micro sampling capabilities

3-D imaging cryomicrotome with adjustable resolution and micro sampling capabilities

This is a summary of the request.

The imaging cryomicrotome is a unique instrument that allows 3-dimensional imaging of the distribution of fluorescent markers, autofluoresence, bright field contrast or other spectroscopic properties (e.g. related to hemoglobine oxygenation) that help define structure and function in larger and smaller tissue areas with an adjustable resolution.This application aims at building and improving a cryomicrotome allowing 1) 3D reconstruction of fluorescence images with a spatial resolution of 30 microns (30M image), 2) 3D reconstructions of vascular subsystems with a resolution of 1 micron selected via online analysis of 30M images, 3) tissue sampling (microgram range) at positions determined from the 30M images. The trust of the program is on the heart and wrist joint. This technique will enable us to link microscopic (capillary bed) and macroscopic structures (arterial and venous system) in the heart, and to measure differentially biochemical parameters in the heart whereby sampling positions are determined by local differences in vessel structure and distribution.

Clinical relevance of the application: 1) Malperfusion is the major cause for failing heart function. The detailed structure of the vascular bed as the backbone should be better defined in order to better understand the cause of local and borderline ischemia. 2) Drug induced vascular growth in diseased hearts is the solution of last resort for many patients in which balloon angioplasty or bypass surgery is no further option. Improved insights in processes such as collateralization and remodelling will be crucial in designing new therapeutic modalities. 3) In areas like orthopedics there is a need for a reference image modality for e.g. CT and MRI.

Recent results with a prototype machine with limited possibilities revealed the organization of the coronary vascular beds into subunits clearly separated by regions where vessels of diameters above the resolution (40 microns) are absent. These distinct differences in vascular structure generate questions with respect to the regional organisation of the very small vessels (<40 micron), as well as regional differences in biochemical signalling related to localized vascular growth and vascular tree stabilization. Although our preliminary study focused on the intramural coronary circulation, similar application to other organs is to be expected.

The specific aims for which the machine will be used:

1) To reconstruct the coronary arterial and venous tree structure in hearts obtained from large and small experimental animals but also in human hearts obtained through the Dutch transplant program.

2) To measure blood flow distribution in relation to the coronary tree structure

3) To measure capillary network properties in relation to position relative to the arterial and venous tree.

4) To measure the larger tree as well as microvascular morphometric data in hearts with induced

vascular growth either in response to partial coronary occlusions or induced by growth factors.

5) To investigate the correlation between coronary tree structure and muscle fibre orientation asdetermined with MRI-diffusion tensor imaging in the same heart.

6) To apply genomics and proteomics to small tissue samples (micrograms) taken from

different areas in the heart directed by online image analysis in the cryomicrotome. Tissue around larger arteries will be compared to tissue located peripheral in the tree.

7) To determine different anatomical structures (wrist joint) as reference for other imaging modalities.

This list of projects is limited to the projects that are under way (1,2,4,7) or planned either with staff within the AMC (3) or with colleagues in this country or abroad (5).

With the cryomicrotome as proposed we will develop toward an expert center for this technique that will be beneficial for researchers in the Netherlands and abroad. The technique can be applied in research related to normal function and disease induced functional changes in the heart and other organs and used as reference for other imaging modalities such as CT, MRI or ultrasound. The technique is certainly not limited to the heart or the wrist joint but will be applied to other organs as well.