Widefield fluorescence detected photothermal infrared microscopy: high speed chemical analysis and dynamic imaging of biological materials

This presentation covers recent advances in fluorescence-detected photothermal infrared (FL-PTIR) spectroscopy, particularly its use of intrinsic autofluorescence in biological systems to rapidly acquire infrared hyperspectral arrays with ~300 nm spatial resolution. Applications include photosynthetic and mammalian tissues, as well as dynamic imaging of live biological cells in aqueous environments.

FL-PTIR builds on optical photothermal infrared (O-PTIR), a rapidly growing technique for super-resolution infrared chemical imaging and spectroscopy. It leverages the high temperature sensitivity of fluorescence emission—~1%/°C for many fluorophores—compared to ~10⁻⁴/°C for conventional photothermal detection. This results in ~100× higher sensitivity than O-PTIR. FL-PTIR detects localized temperature increases from infrared absorption using either fluorescently labeled or autofluorescent samples.

The FL-PTIR instrument illuminates a sample with infrared pulses from a tunable laser, causing periodic, localized temperature increases that transiently decrease fluorescence emission. A low-cost LED fluorescence excitation source and an sCMOS camera detect these variations over a wide field of view, mapping IR absorption. Hyperspectral arrays of >200 super-resolution IR images spanning the fingerprint region can be acquired in ~15 minutes at 4 cm⁻¹ spectral resolution. Analysis of periodic structures within the silica frustules of marine diatoms has demonstrated FL-PTIR spatial resolution down to 300 nm. FL-PTIR also enables real-time visualization of dynamic events in live biological systems, such as microalgae and yeast cells, with sub-second time resolution. This presentation will showcase FL-PTIR analysis of photosynthetic materials (diatoms, green plant tissue, and microalgae) and mammalian tissue, including collagen.