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.

Vertebrate βγ-crystallins belonging to the βγ-crystallin superfamily lack functional Ca(2+)-binding sites, while their microbial homologues do not; for example, three out of four sites in lens γ-crystallins are disabled. Such loss of Ca(2+)-binding function in non-lens βγ-crystallins from mammals (e.g., AIM1 and Crybg3) raises the possibility of a trade-off in the evolutionary extinction of Ca(2+)-binding. We test this hypothesis by reconstructing ancestral Ca(2+)-binding motifs (transforming disabled motifs into the canonical ones) in the lens γB-crystallin by introducing minimal sets of mutations. Upon incorporation of serine at the fifth position in the N/D-N/D-X-X-S/T(5)-S motif, which endowed a domain with microbial characteristics, a decreased domain stability was observed. Ca(2+) further destabilized the N-terminal domain (NTD) and its serine mutants profoundly, while the incorporation of a C-terminal domain (CTD) nullified this destabilization. On the other hand, Ca(2+)-induced destabilization of the CTD was not rescued by the introduction of an NTD. Of note, only one out of four sites is functional in the NTD of γB-crystallins responsible for weak Ca(2+) binding, but the deleterious effects of Ca(2+) are overcome by introduction of a CTD. The rationale for the onset of cataracts by certain mutations, such as R77S, which have not been clarified by structural means, could be explained by this work. The findings presented here shed light on the evolutionary innovations in terms of the functional loss of Ca(2+)-binding and acquisition of a bilobed domain, besides imparting additional advantages (e.g., protection from light) required for specialized functions.

Fluorescent nanodiamonds (NDs) are strong contenders as bio-labels for life science imaging, diagnostics and therapeutics. Ultimately, for their use in biomedical applications, their size should ideally be less than 10 nm. Yet, even more critical for their specificity and efficient uptake in cellular systems, is their resilience to aggregation, which is dictated by their colloidal stability in complex, physiological environments. To this end, we characterize ultrasmall detonation NDs (~5 nm) by examining their surface chemical profiles and stability in solutions of varying ionic strength and pH. Using dynamic light scattering measurements, we demonstrate that ultra-small ND particles with chemically homogeneous and negatively charged surface profiles are more stable than positive particles under a broad range of simulated biological environments. We show that the colloidal stability of both positive and negative ultra-small NDs is improved by functionalization with bovine serum albumin. Based on these analyses, we propose and describe strategies for enhancing the overall colloidal stability of ultra-small NDs and their resilience to aggregation. Our findings provide a practical framework towards the reduction in size of the bio-conjugates employed to probe complex biological systems, and the advancement of bio-imaging techniques with minimal perturbation of the molecular trafficking in cellular and organelle systems.

The protein corona of nanodiamonds is dominated by low molecular weight proteins and is largely independent of surface chemistry. The pre-incubation of nanodiamonds in serum and the formation of a protein corona decrease the production of reactive oxygen species, increasing the cell viability of macrophages.

Fluorescent nanodiamonds (NDs) are remarkable objects. They possess unique mechanical and optical properties combined with high surface areas and controllable surface reactivity. They are non-toxic and hence suited for use in biological environments. NDs are also readily available and commercially inexpensive. Here, the exceptional capability of controlling and tailoring their surface chemistry is demonstrated. Small, bright diamond nanocrystals (size ˜30 nm) are conjugated to protein filaments of actin (length ˜3-7 µm). The conjugation to actin filaments is extremely selective and highly target-specific. These unique features, together with the relative simplicity of the conjugation-targeting method, make functionalised nanodiamonds a powerful and versatile platform in biomedicine and quantum nanotechnologies. Applications ranging from using NDs as superior biological markers to, potentially, developing novel bottom-up approaches for the fabrication of hybrid quantum devices that would bridge across the bio/solid-state interface are presented and discussed.