Background-Free Mid-Infrared Photothermal Microscopy via Single-Shot Measurement of Thermal Decay

Mid-infrared photothermal (MIP) microscopy is an emerging tool for biological imaging, offering high sensitivity, subcellular resolution, and rapid image acquisition. However, the MIP signal of low concentration molecules in biological systems is often hindered or masked by background absorption, largely contributed by water, resulting from the H-O-H scissors-bending band in the fingerprint window or the bend-libration combination band in the cell-silent window. To preserve all desired signals while suppressing the background, we report a single-shot time-resolved MIP measurement that allows differentiation between the background and analyte signal based on their distinct photothermal dynamics. The results show that the thermal decay of the background is significantly longer than that of the desired intracellular signal, mainly due to the larger mass and heat capacity of water compared to those of intracellular features. Through two-component exponential fitting, we successfully differentiated and suppressed the background, while preserving the desired intracellular signal in both the fingerprint and cell-silent windows. By leveraging the thermal dynamics differences obtained from a single-shot measurement, we effectively remove the background and enhance the detection of small signals in a biological system.