Shadowless amyloid imaging with quantitative birefringence contrast

In the diagnosis of amyloidosis using Congo red staining, the apple-green birefringence observed under crossed Nicols has been considered the gold standard for over fifty years. However, a variety of colors, including orange and blue, not just apple-green, are observed in clinical fields. Moreover, the issue of polarization shadows under the crossed Nicols also impedes detailed observations of amyloid deposits, such as senile plaques. The introduction of advanced birefringence microscopes into clinical settings is also limited due to equipment-related challenges. In this study, we propose a simple but robust method that equips a standard optical microscope with polarizers to determine the birefringence and dichroism of Congo red-stained amyloid deposits. By employing an innovative image processing technique that averages multiple images, we achieved a correlation of over 95% with the birefringence distribution. This approach not only minimizes commonly encountered polarization shadows but also enhances the clarity and accuracy of amyloidosis diagnoses. Our strategy simplifies the diagnostic process and facilitates the incorporation of quantitative imaging technologies into routine pathological practices. The findings suggest that characteristic birefringence, independent of azimuthal angle, serves as a reliable marker for amyloidosis, potentially revolutionizing diagnostic protocols in the field.