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.

Sub-diffraction microscopy enables bio-imaging with unprecedented clarity. However, most super-resolution methods require complex, costly purpose-built systems, involve image post-processing and struggle with sub-diffraction imaging in 3D. Here, we realize a conceptually different super-resolution approach which circumvents these limitations and enables 3D sub-diffraction imaging on conventional confocal microscopes. We refer to it as super-linear excitation-emission (SEE) microscopy, as it relies on markers with super-linear dependence of the emission on the excitation power. Super-linear markers proposed here are upconversion nanoparticles of NaYF 4 , doped with 20% Yb and unconventionally high 8% Tm, which are conveniently excited in the near-infrared biological window. We develop a computational framework calculating the 3D resolution for any viable scanning beam shape and excitation-emission probe profile. Imaging of colominic acid-coated upconversion nanoparticles endocytosed by neuronal cells, at resolutions twice better than the diffraction limit both in lateral and axial directions, illustrates the applicability of SEE microscopy for sub-cellular biology.

Fluorescent nanodiamonds (FNDs) are vital to many emerging nanotechnological applications, from bioimaging and sensing to quantum nanophotonics. Yet, understanding and engineering the properties of fluorescent defects in nanodiamonds remain challenging. The most comprehensive study to date is presented, of the optical and physical properties of five different nanodiamond samples, in which fluorescent nitrogen‐vacancy (NV) centers are created using different fabrication techniques. The FNDs' fluorescence spectra, lifetime, and spin relaxation time (T1) are investigated via single‐particle confocal fluorescence microscopy and in ensemble measurements in solution (T1 excepted). Particle sizes and shapes are determined using scanning electron microscopy and correlated with the optical results. Statistical tests are used to explore correlations between the properties of individual particles and also analyze average results to directly compare different fabrication techniques. Spectral unmixing is used to quantify the relative NV charge‐state (NV− and NV0) contributions to the overall fluorescence. A strong variation is found and quantified in the properties of individual particles within all analyzed samples and significant differences between the different particle types. This study is an important contribution toward understanding the properties of NV centers in nanodiamonds. It motivates new approaches to the improved engineering of NV‐containing nanodiamonds for future applications.

Mobile phone microscopes are a natural platform for point-of-care imaging, but current solutions require an externally powered illumination source, thereby adding bulk and cost. We present a mobile phone microscope that uses the internal flash or sunlight as the illumination source, thereby reducing complexity whilst maintaining functionality and performance. The microscope is capable of both brightfield and darkfield imaging modes, enabling microscopic visualisation of samples ranging from plant to mammalian cells. We describe the microscope design principles, assembly process, and demonstrate its imaging capabilities through the visualisation of unlabelled cell nuclei to observing the motility of cattle sperm and zooplankton.