Advances in fluorescence labeling strategies for dynamic cellular imaging

Abstract

Synergistic advances in optical physics, probe design, molecular biology, labeling techniques and computational analysis have propelled fluorescence imaging into new realms of spatiotemporal resolution and sensitivity. This review aims to discuss advances in fluorescent probes and live-cell labeling strategies, two areas that remain pivotal for future advances in imaging technology. Fluorescent protein– and bio-orthogonal–based methods for protein and RNA imaging are discussed as well as emerging bioengineering techniques that enable their expression at specific genomic loci (for example, CRISPR and TALENs). Important attributes that contribute to the success of each technique are emphasized, providing a guideline for future advances in dynamic live-cell imaging.

Cytosolic Delivery of Nanolabels Prevents Their Asymmetric Inheritance and Enables Extended Quantitative in Vivo Cell Imaging

“…This strategy, however, requires a rather time-consuming transfection procedure that may lead to undesired phenotypical alterations and abnormalities in the transplanted cells in comparison to the native cells [11][12][13] . The second strategy is to label the cells with exogenous contrast agents, being either organic probes or inorganic nanoparticles (NPs) 14,15 . Examples are fluorescently labelled dextrans 16,17 and quantum dots (QD) for optical fluorescence imaging [18][19][20] , or superparamagnetic iron oxide NPs and Gadolinium complexes for magnetic resonance imaging (MRI) 4,21,22 .…”

Super-multiplex vibrational imaging

“…Even though existing methods have been used successfully to explore structural-functional relationships in nervous systems, profile RNA in situ , reveal tumor microenvironment heterogeneity or study dynamic macromolecular assembly 1–4 , it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a “color barrier” due to the intrinsically broad (~1500 cm −1 ) and featureless nature of fluorescence spectra 5 that limits the number of resolvable colors to 2 to 5 (or 7-9 if using complicated instrumentation and analysis) 6–8 . Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width ~10 cm −1 ) and thus doesn’t suffer this problem, but its feeble signals make many demanding bio-imaging applications impossible.…”

Nanokit for single-cell electrochemical analyses

“…T he analysis of small molecules and proteins in single cells is critical to understanding the pathways associated with cellular heterogeneity and disease states (1)(2)(3). Fluorescence probes that emit specific spectral signals on binding the target molecules are widely used, present the current state of the art technology, and have provided much of our current understanding of intracellular signaling (4)(5)(6). The challenges for the analysis of additional molecules using standard fluorescence approaches are in the concerns about cytotoxicity and the structural requirements, making the design and synthesis of nonnative probes a bottleneck to new discoveries (7).…”