We present a combination of light-sheet excitation and two-dimensional fluorescence intensity ratio (FIR) measurements as a simple and promising technique for three-dimensional temperature mapping. The feasibility of this approach is demonstrated with samples fabricated with sodium yttrium fluoride nanoparticles co-doped with rare-earth ytterbium and erbium ions (NaYF₄:Yb³⁺/Er³⁺) incorporated into polydimethylsiloxane (PDMS) as a host material. In addition, we also evaluate the technique using lipid-coated NaYF₄:Yb³⁺/Er³⁺ nanoparticles immersed in agar. The composite materials show upconverted (UC) fluorescence bands when excited by a 980 nm near-infrared laser light-sheet. Using a single CMOS camera and a pair of interferometric optical filters to specifically image the two thermally-coupled bands (at 525 and 550 nm), the two-dimensional FIR and, hence, the temperature map can be readily obtained. The proposed method can take optically sectioned (confocal-like) images with good optical resolution over relatively large samples (up to the millimetric scale) for further 3D temperature reconstruction.

Upconversion nanoparticles (UCNPs) are well-known for their high efficiency, photostability, near-infrared excitation, and ability to estimate temperature through ratiometric imaging of two thermally coupled fluorescence bands. This work demonstrates the feasibility of volumetric temperature mapping in internal biological systems using light-sheet fluorescence microscopy and lipid-coated UCNPs as nanothermometry markers. This approach enables real-time thermal mapping with both high spatial and temporal resolution at the cellular and subcellular levels. To validate the method, we performed 3D temperature imaging on fixed Caenorhabditis elegans ( C. elegans ) after UCNP ingestion. The proposed technique represents a cutting-edge method for accurate 3D analysis of temperature-driven biological processes. It holds significant potential for applications in living organisms, offering a non-invasive tool to monitor intracellular and organ-specific temperature dynamics.