Evaluation of few-cycle laser pulses for label-free metabolic imaging on live cells

Abstract Multi-photon (MP) imaging is a well-established optical microscopy technique capable of high-resolution imaging with reduced photodamage and deep tissue penetration. On a single cell level MP imaging enables non-invasive monitoring of cellular states, such as the metabolic activity, which is strongly linked to early detection of abnormal cellular behaviour. In this proof-of-principle study, we present the implementation of sub-10 fs few-cycle laser pulses for label-free MP imaging, representing a potential advancement in the evolution of MP-imaging techniques. Our primary focus is on label free metabolic imaging (MI) of live cells. To assess photoinduced damage from ultrashort laser pulses, we examine their effects on live HeLa cells in vitro . At a power density in the terawatt per square centimetre (TW cm − 2 ) range, the few-cycle laser pulse result in a measurable decrease in NAD(P)H fluorescence in HeLa cells- an early indicator of photoinduced cellular damage. We further demonstrate the application of few-cycle MI in live HeLa cells treated with the therapeutic agent Doxorubicin (DOX). Remarkably, a metabolic shift towards oxidative phosphorylation is observed within just 15 min of DOX incubation. In these therapeutic experiments prolonged exposure to the few-cycle laser also leads to a significant increase in NAD(P)H autofluorescence, particular in nuclear and mitochondrial regions-sites rich in DNA-suggesting a photoactivation effect of DOX that enhances NAD(P)H fluorescence emission. Overall, this study demonstrates the feasibility of using a few-cycle broadband laser excitation source for label-free bioimaging. The results reveal complex interaction dynamics between the laser´s spectral components and cellular targets. These findings highlight the need for careful optimization to minimize phototoxic while also suggesting new opportunities therapy-triggering and real-time metabolic monitoring through spectral engineering.