Molecular behavior can change dramatically at interfaces, and these changes become even more pronounced when molecules are confined within nanoscale dimensions. In living organisms, nanoscale channels play a crucial role in selectively transporting molecules and processing information. This characteristic of biological channels has inspired researchers to design and study artificial systems that emulate these transport abilities. The field of nanofluidics aims to understand the fundamental principles of nanoscale molecular transport and develop scalable artificial systems for applications in energy, molecular sieving and more. Traditional nanofluidic studies often rely on measuring minute ion and flow characteristics, averaging the behavior of many molecules and potentially overlooking critical details. However, advancements in modern optics now allow selective imaging of interfacial molecules and even enable tracking of individual molecules. These advanced techniques offer a richer dataset for nanofluidic measurements through correlative or enhanced microscopy approaches. Despite their potential, the synergies between these methods remain largely unexplored.

In this thesis, we investigate innovative materials, methodologies, and instruments for the optical investigation of interfacial molecular processes and nanofluidic transport.
First, we leverage a newly found property of a two-dimensional material, hexagonal boron nitride: liquid-activated fluorescence. The 2D crystal, when immersed in suitable liquids, reveals interfacial dynamics of single molecules. We show that the fluorescence characteristics of the emitters can be used as in-liquid sensors for nanofluidic systems. We discuss further sensing applications of the phenomenon and its probable mechanism.
Second, we introduce a new measurement technique: nanofluidic operando imaging, consisting in real-time observation of nanochannel devices in action during ion transport measurements. We apply this methodology to nanofluidic memristors (resistors with memory): highly asymmetric nanochannels. We identify the memory mechanism as arising from controllable voltage-induced channel deformations. This enabled performing logic operations with these nanofluidic systems, a step towards neuromorphic nanofluidics.
Third, we explore a novel tool to image molecules with enhanced information: a gated photon-counting camera. This single-photon avalanche diode (SPAD) camera enables binary imaging to bypass the temporal averaging operated by standard cameras. Using its ability to time photon arrivals relative to the excitation, we introduce a massively multiplexed framework for fluorescence lifetime imaging at the single-molecule level. We apply these advancements to the investigation of biological nanopores and DNA origami.
Overall, this thesis highlights the capability of optical techniques to enhance our understanding of nanofluidic systems and opens new avenues for the development of single-photon imaging.