Brightness and Photostability of Emerging Red and Near-IR Fluorescent Nanomaterials for Bioimaging

Many novel fluorescent nanomaterials exhibit radically different optical properties compared to organic fluorophores that are still the most extensively used class of fluorophores in biology today. Assessing the practical impact of these optical differences for bioimaging experiments is challenging due to a lack of published quantitative benchmarking data. This study therefore directly and quantitatively compares the brightness and photostability of representatives from seven classes of fluorescent materials in spectroscopy and fluorescence microscopy experiments for the first time. These material classes are: organic dyes, semiconductor quantum dots, fluorescent beads, carbon dots, gold nanoclusters, nanodiamonds, and nanorubies. The relative brightness of each material is determined and the minimum material concentrations required to generate sufficient contrast in a fluorescence microscopy image are assessed. The influence of optical filters used for imaging is also discussed and suitable filter combinations are identified. The photostability of all materials is determined under typical imaging conditions and the number of images that can be acquired is inferred. The results are expected to facilitate the transition of novel fluorescent materials from physics and chemistry into biology laboratories. Organic fluorophores show high fluorescence brightness and poor photostability. Diamond and ruby nanoparticles are extremely photostable, but are not as bright as organic fluorophores or semiconductor quantum dots. While most fluorescent materials investigated in this study photobleach within seconds or a few minutes, diamond and ruby nano­particles can be imaged indefinitely in biological systems.