Impact of Excitation Intensity-Dependent Fluorescence Intensity Ratio of Upconversion Nanoparticles on Wide-Field Thermal Imaging

Erbium ion (Er3+)-doped upconversion nanoparticles (UCNPs) are frequently used for nanothermometry because their fluorescence intensity ratio (FIR) between two green emission bands at ∼525 and ∼545 nm is sensitive to temperature variation. One of the prerequisites for nanothermometry is that the FIR be independent of excitation intensity at constant temperature. In this work, the effect of excitation intensity on the FIR of core–double-shell NaYF4:Yb3+,Er3+@NaYF4:Yb3+,Nd3+@NaYF4 UCNPs was investigated in two environments. The first environment is in aqueous solution, and the second is a monolayer of UCNPs on top of a silica–silicon substrate in air. The experimental results showed that the FIR decreases with the excitation intensity at constant temperature in each case. We further found that the excitation intensity-dependent FIR indeed deteriorated the thermal images acquired by wide-field upconversion fluorescence microscopy, in which a Gaussian laser beam was used to excite UCNPs uniformly coated on a silica–silicon substrate. The nonuniform excitation intensity of the incident laser beam resulted in thermal images that showed nonuniform temperature distributions in a 100 μm range field of view, even though the whole sample was maintained at constant temperature in air. To tackle this problem, we first measured the excitation intensity and temperature dependence of the FIR and the excitation laser intensity distribution on the sample. We then developed a correction scheme to correct the thermal images. With our correction process, the temperature distribution on the sample can be accurately mapped even with nonuniform illumination.