Tumor hypoxia is a major indicator of treatment resistance to chemotherapeutic drugs and fluorescence optical tomography has tremendous potential to provide clinically useful functional information by targeting tumor hypoxia. The aim of this work is to design a cheap and non-invasive method to image hypoxia in tumor cells. In this work, synthesis, photophysical characterization and hypoxia evaluation of a series of 2-nitroimidazole coupled indocyanine green dyes (first, second and third generation) is presented. Among the three generations, the third generation dyes are effective in providing a strong fluorescence signal and stayed much longer (up to 24h) in the hypoxic regions of tumor cells. We have observed that incorporation of a methyl group distal to a terminal long-chain alkyne leads to an increased rate of isomerization to the corresponding internal alkyne. Compared to a long-straight chain terminal alkyne, the isobranch analog isomerizes about three times faster under identical conditions. In both cases, equilibration to a 95-97:5-3 mixture of internal:terminal alkyne follows isomerization.