Multi-dimensional Widefield infrared-encoding spontaneous emission microscopy: distinguishing chromophores by ultrashort infrared pulses

Photoluminescence (PL) imaging has broad application in visualizing biological activities, detecting chemical species, and characterizing materials. Though the spontaneous emission of PL signals can be detected with high sensitivity, it lacks the information on the identity and environment of chromophores. The broad PL bands in the visible wavelength region also limits the total number of simultaneously monitored chemical species. Nonlinear optical microscopies using molecular chromophores have been demonstrated to encode additional information into PL signals. However, such microscopies often operate under the tightly focused confocal configuration due to the high intensity required for nonlinear optical interactions. Herein, we demonstrate a multiplexed widefield imaging method, Multi-Dimensional Widefield Infrared-encoding Spontaneous Emission (MD-WISE) microscopy. Using a pair of femtosecond mid-infrared and visible excitation pulses, MD-WISE can distinguish chromophores, including molecules and quantum dots, that possess nearly identical emission spectra using multiplexed conditions in a three-dimensional space. The space is defined by three independent variables: the temporal delay between the infrared and the visible pulses, and the optical frequencies of the two pulses. The PL emissions from molecules with various functional groups can be distinguished by tuning the infrared pulse to specific vibrational frequencies of functional groups. Quantum dots and molecules can be distinguished in PL images by varying solely the temporal delay. By demonstrating the capacity of registering multi-dimensional information into widefield PL images, MD-WISE microscopy has the potential of expanding the number of species and processes that can be simultaneously tracked in high-speed chemical imaging applications.