Fig. 1: Schematic representation of content-enriched fluorescence lifetime fluctuation spectroscopy.

a Schematic representation of the optical setup. Two laser beams are coupled with a dichroic mirror (DM), focused with a 100× objective lens, and scanned across the sample with a pair of galvo-mirrors (GMs) to allow for dual-color experiments. The emitted signal collected by the objective lens is de-scanned by the GMs and split depending on the emitted wavelength and either focused on a SPAD array detector or onto a single-element detector. Every single photon is collected and tagged with a spatial and temporal tag by the BrightEyes-TTM data-acquisition card for time-resolved measurements or by an FPGA-based control unit (DAQ and CU). Our set-up allows for studying both in vitro samples, such as purified proteins, and living cells. b Thanks to the spatiotemporal tags of the SPAD array detector, we can access several temporal scales. The fluorescence lifetime, on the nanosecond time regime, which gives us information about the nano-environment of single molecules, is measured simultaneously with the microseconds intensity fluorescence fluctuation, giving us information about the diffusion. Granted by the single-photon spatial tags, the diffusion times in the different detected areas can be evaluated (diffusion law), providing information about diffusion modality or the micro-environment of the molecules. The temporal and spatial tags can be provided in a multi-color scheme, allowing us to perform dual-color cross-correlation measurements.