Comprehensive approach to coregistration of autoradiography and microscopy images acquired from a set of sequential tissue sections

Histopathologic validation of a PET tracer requires assessment of colocalization of the tracer with its intended biologic target. Using thin tissue section autoradiography, it is possible to visualize the spatial distribution of the PET tracer uptake and compare it with the distribution of the intended biologic target (as visualized with immunohistochemistry). The purpose of this study was to develop and evaluate an objective methodology for deformable coregistration of autoradiography and microscopy images acquired from a set of sequential tissue sections. Methods: Tumor-bearing animals were injected with 3′-deoxy- 3′- ¹⁸F-fluorothymidine ( ¹⁸F-FLT), ¹⁴C-FDG, and other markers of tumor microenvironment including Hoechst 33342 (bloodflow surrogate). After sacrifice, tumors were excised, frozen, and sectioned. Multiple stacks of sequential 8 mm sections were collected from each tumor. From each stack, the middle (reference) sections were used to obtain images of ¹⁸F-FLT and ¹⁴CFDG uptake distributions using dual-tracer autoradiography. Sections adjacent to the reference were used to acquire all histopathologic data (e.g., images of cell proliferation, hematoxylin and eosin). Hoechst images were acquired from all sections. To correct for deformations and misalignments induced by tissue processing and image acquisition, the Hoechst image of each nonreference section was deformably registered to the reference Hoechst image. This transformation was then applied to all images acquired from the same tissue section. In this way, all microscopy images were registered to the reference Hoechst image. The Hoechst-to-autoradiography image registration was done using rigid point-set registration based on external markers visible in both images. Results: The mean error of Hoechst to ¹⁸F-FLT autoradiography registration (both images acquired from the same section) was 30.8 ± 20.1 μm. The error of Hoechst-based deformable registration of histopathologic images (acquired from sequential tissue sections) was 23.1 ± 17.9 μm. Total error of registration of autoradiography images to the histopathologic images acquired from adjacent sections was evaluated at 44.9 μm. This coregistration precision supersedes current rigid registration methods with reported errors of 100-200 μm. Conclusion: Deformable registration of autoradiography and histopathology images acquired from sequential sections is feasible and accurate when performed using corresponding Hoechst images.