Two-color infrared photothermal microscopy

Infrared photothermal microscopy is an infrared (IR) imaging technique that enables non-invasive, non-destructive, and label-free investigations at the sub-micrometer scale. It has been applied in various research areas targeting pharmaceutical and photovoltaic materials as well as biomolecules in living systems. Despite its potency in observing biomolecules in living organisms, its practical application for cytological research has been restricted by the deficiency of molecular information from the IR photothermal signal, due to the narrow spectral width of a quantum cascade laser that is one of the most preferred IR excitation light sources for current IR photothermal imaging (IPI) techniques. Here, we address this issue by bringing modulation-frequency multiplexing into IR photothermal microscopy for developing a two-color IR photothermal microscopy technique. We demonstrate that the two-color IPI technique can be used to obtain the IR microscopic images of two discrete IR absorption bands and to distinguish two different chemical species in live cells with a sub-micrometer spatial resolution. We anticipate that the more general multi-color IPI technique and its use for metabolic studies of live cells could be realized by extending the present modulation-frequency multiplexing method.