2.2 Experimental label-free NLO data from cell cultures

We employ a lab-built multimodal NLO scanning microscope to collect morphological, metabolic, and chemical information from HepG2 cells. The microscope has been described in detail in (Crisafi et al., 2018). Briefly, our system is based on a multi-branch Erbium-doped amplified fiber laser that yields a pump beam at 780 nm and a Stokes beam, tunable in the range between 950 and 1050 nm, with 40 MHz repetition rate. The pump beam is modulated with an acousto-optic modulator at 1 MHz. An in-line balanced detection scheme was employed for the SRS measurements, based on what is described in Crisafi et al. The temporal overlap of the two trains of pulses is achieved by operating a manual delay line, positioned on the path of the Stokes beam. The two beams are spatially combined with a dichroic mirror and sent into our homebuilt vertical microscope. The beams are focused via a water-immersion 100X 1.25NA 0.25 mm working distance (WD) objective (C-Apochromat, Carl Zeiss, Germany) and collected by an oil-immersion 40X 1.30NA 0.19 mm WD (CFI Super Fluor, Nikon, Japan) objective. The average laser powers were kept constant on the sample plane at 7.5 mW for the pump and 0.5 mW for the Stokes for all measurements. The Stokes power is limited by the laser source and by the in-line balanced detection scheme, which is fundamental to achieve almost shot-noise-limited performances. Several nonlinear processes can stem from synchronized dual-beam excitation, such as SRS and TPEF. Multispectral detection was performed via a photomultiplier, for the TPEF modality, and a balanced photodiode, serving both for the SRS modality and transmission light modality, on the same field of view. Following two-photon excitation via the 780 nm pump beam, fluorescence signal was epi-detected in the 400–600 nm range using a short-pass filter (FESH0600, Thorlabs), to cover the complete emission spectra of Flavin Adenine Dinucleotide (FAD) and Nicotinamide Adenine Dinucleotide (NADH). These are important coenzymes which act as electron acceptor and donor, respectively, in key metabolic pathways, such as glycolysis, Krebs cycle, and oxidative phosphorylation (Heikal, 2010). Compared to their reduced (FADH₂) and oxidized (NAD⁺) forms, these molecules are also autofluorescent and represent excellent endogenous sources of optical contrast, offering a way to monitor subtle changes in cellular metabolism (Georgakoudi et al., 2012). Single-channel SRS was detected at 2850 cm⁻¹, corresponding to the strong CH₂ stretching mode of lipids. SRS microscopy at this Raman mode has been proven to provide reliable measurements of lipid droplets in different cell lines, allowing both for visualizing their distribution and quantifying their cellular concentration in a non-invasive, label-free fashion (Gupta et al., 2019). Therefore, the acquisition of these three imaging channels is particularly convenient and efficient for our multimodal NLO system, allowing us to seamlessly obtain a co-registered three-channel image in a single measurement. At the same time, these channels comprise informative but not strongly correlated features, which is extremely advantageous to improve the training process and achieve the best classification performances of a neural network (Toloşi and Lengauer, 2011).