Multi-color optical microscopy using modulated excitation lasers and single-frequency demux

Abstract

Multi-color microscopy has been a powerful tool for industrial and biomedical applications. Traditionally, image acquisition through different color channels has to be carried out separately using a single detector for sequential detection or multi-detectors for simultaneous detection. This costly and complex process may introduce colocalization errors due to the alignment. This paper describes a compact multi-color microscope using modulated excitation lasers and a single electronic frequency demux for signal detection. To mark fluorescence from different labels in the sample separately, the excitation lasers are intensity modulated with different frequencies. After fluorescence is detected, the electronic signals are demultiplexed with a single spectrum analyzer. This method allows the realization of multi-color imaging with minimal amount of detection elements. Multi-color imaging of fluorescent spheres, gold nanoparticles, and biological tissues are carried out. A fiber-coupling system has also been used to simplify the optical alignments, while its focal chromatic aberration has been characterized.