Time-resolved imaging of fluorescent species

In pump-probe (PP) spectroscopy, two short laser pulses — the pump and the probe — excite a sample and reveal its photodynamics after a time delay, respectively. PP spectroscopy enables the characterization of energy transfer processes like those in plant photosynthesis. To achieve a deeper understanding of photochemical and photophysical processes, scientists are proposing new forms of PP spectroscopy, overcoming timescale limitations and, in microscopic forms of PP, introducing spatial resolution.

Garrow et al. combined fluorescence-detected PP (F-PP) — a technique they previously developed to address limitations in timescale and resolution — with fluorescence lifetime imaging microscopy (FLIM) to create PP-FLIM. This technique tracks ultrafast photo-induced dynamics with optical spatial resolution and separates species based on their excitation lifetimes.

To test these qualities, the authors first measured a solution of two highly similar fluorescent dyes with PP-FLIM. The FLIM component effectively disentangled analogous PP spectra by capturing differences in the dyes’ fluorescent decay dynamics.

“Separating the dyes would not be possible without the additional dimension provided by FLIM,” said author Pavel Malý.

The authors also measured single chloroplasts within spinach leaves, obtaining spatial information with three-dimensional optical resolution. Because PP-FLIM uses resonance fluorescence detection, some photo-bleaching can occur without compromising measurement accuracy.

“Light harvesting in photosynthesis proceeds on an ultra-fast timescale that requires high temporal resolution, but sensitive microscopic imaging is necessary as well, to study the complexes individually or in their natural environment,” said Malý.

Next, the authors plan to improve their PP-FLIM setup for studying the finer details of emitting species.

“At the moment, we are implementing PP-FLIM with a new laser in a home-built microscope,” said Malý. “The ultimate goal is measurement of ultrafast dynamics in single molecules and molecular complexes.”

Source: “Pump–probe fluorescence lifetime imaging microscopy,” by Malcolm Garrow, Adil Haboucha, Petr Heřman, and Pavel Malý, Journal of Chemical Physics (2026). The article can be accessed at https://doi.org/10.1063/5.0319029 .

This paper is part of the Festschrift in honor of Minhaeng Cho: Ultrafast Spectroscopy Collection, learn more here .