3D reconstruction of dual color acquistion of Her2/neu and Her 3. A) conventional wide-field fluorescence image of an AG11132 mammary epithelial cells. Her3 is labelled with Alexa488 (green) and Her2/neu with Alexa568 (red). B) magnified image of a small area of A. C) localization microscopy SPDM of the same region of interest as in B. D) and E) show a 3D reconstruction of the protein clusters using the combination of SPDM and SMI microscopy (LIMON technology). Scale bars in D) and E) are 500 nm in each direction.
By combining SPDMphymod with SMI (both invented in Christoph Cremer´s lab in 1996) a 3D dual colour reconstruction of the spatial arrangements of Her2/neu and Her3 clusters was achieved. The positions in all three directions of the protein clusters could be determined with an accuracy of about 25 nm.
Her2/neu and Her3 are tyrosine kinase receptors and their overexpression is related to certain types breast cancer. The paper analyses the Her2 distribution pattern in breast cancer cells and counts the clusters (20 637 clusters with a mean diameter of 67 nm) with a specifically developed clusters finding algorithm for super resolution microscopy. This methods could help for the exploration of the resistance mechanism in Trastuzumab/Herceptin treatment.