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Single‐molecule localization microscopy has proved promising to unravel the dynamics and molecular architecture of thin biological samples down to nanoscales. For applications in complex, thick biological tissues shifting single‐particle emission wavelengths to the shortwave infrared (SWIR also called NIR II) region between 900 to 2100 nm, where biological tissues are more transparent is key. To date, mainly single‐walled carbon nanotubes (SWCNTs) enable such applications, but they are inherently 1D objects. Here, 0D ultra‐small luminescent gold nanoclusters (AuNCs, <3 nm) and ≈25 nm AuNC‐loaded‐polymeric particles that can be detected at the single‐particle level in the SWIR are presented. Thanks to high brightness and excellent photostability, it is shown that the dynamics of the spherical polymeric particles can be followed at the single‐particle level in solution at video rates for minutes. We compared single particle tracking of AuNC‐loaded‐polymeric particles with that of SWCNT diffusing in agarose gels demonstrating the specificity and complementarity of diffusion properties of these SWIR‐emitting nano‐objects when exploring a complex environment. This extends the library of photostable SWIR emitting nanomaterials to 0D nano‐objects of variable size for single‐molecule localization microscopy in the second biological window, opening unprecedented possibilities for mapping the structure and dynamics of complex biological systems. [ABSTRACT FROM AUTHOR]

Reduced scattering and autofluorescence in the second near‐infrared window (NIR‐II, 1000–1700 nm) hold the promise of high‐resolution photoluminescence (PL) in vivo imaging at depths well beyond the µm scale. Yet, contrast agents with bright emission in this spectral region remain limited. Ultrasmall gold nanoclusters (AuNCs) are of particular interest for NIR‐II PL imaging. However, they exhibit limited brightness. Here, high amounts of NIR‐II emitting AuNCs are encapsulated in small polymer nanoparticles (NPs) made of poly(ethyl methacrylates). Using nanoprecipitation, loadings of up to 50 wt% can be reached, corresponding to over 10 000 AuNCs per 60 nm NP. PL quantum yields up to 1.3% and NP brightnesses up to 3.8 × 106 M−1 cm−1 are accomplished. Increasing inter‐AuNC interactions at higher loading result in a bathochromic shift of the PL emission. According to time‐resolved PL measurements, this can be attributed to distinct inter‐AuNC energy transfer pathways between the core and surface‐centered PL modes. Evaluation of the nanohybrids in tissue‐mimicking models for in vivo blood vessel imaging shows that the ultrabright and red‐shifted NIR‐II PL results in significantly improved detection sensitivity and resolution, which makes AuNC‐loaded polymer NPs highly promising candidates for advanced in vivo imaging. [ABSTRACT FROM AUTHOR]

Efficient and safe delivery of nanoparticles (NPs) into the cytosol of living cells constitutes a major methodological challenge in bio‐nanotechnology. Electroporation allows direct transfer of NPs into the cytosol by forming transient pores in the cell membrane, but it is criticized for invasiveness, and the applicable particle sizes are not well defined. Here, in order to establish principles for efficient delivery of NPs into the cytosol with minimal cytotoxicity, the influence of the size of NPs on their electroporation and intracellular behavior is investigated. For this study, fluorescent dye‐loaded polymer NPs with core sizes between 10 and 40 nm are prepared. Optimizing the electroporation protocol allows minimizing contributions of endocytosis and to study directly the effect of NP size on electroporation. NPs of <20 nm hydrodynamic size are efficiently delivered into the cytosol, whereas this is not the case for NPs of >30 nm. Moreover, only particles of core size <15 nm diffuse freely throughout the cytosol. While electroporation at excessive electric fields induces cytotoxicity, the use of small NPs <20 nm allows efficient delivery at mild electroporation conditions. These results give clear methodological and design guidelines for the safe delivery of NPs for intracellular applications. [ABSTRACT FROM AUTHOR]

Dye‐loaded polymer nanoparticles (NPs) emerge as a powerful tool for bioimaging applications, owing to their exceptional brightness and controlled small size. However, aggregation‐caused quenching (ACQ) and leakage of dyes at high loading remain important challenges of these nanomaterials. The use of bulky hydrophobic counterions has been recently proposed as an effective approach to minimize ACQ and dye leakage, but the role of counterion structure is still poorly understood. Here, a systematic study based on ten counterions, ranging from small hydrophilic perchlorate up to large hydrophobic tetraphenylborate derivatives, reveals how counterion nature can control encapsulation and emission of a cationic dye (rhodamine B octadecyl ester) in NPs prepared by nanoprecipitation of a biodegradable polymer, poly‐lactide‐co‐glycolide (PLGA). We found that increase in counterion hydrophobicity enhances dye encapsulation efficiency and prevents dye adsorption at the particle surface. Cellular imaging studies revealed that ≥95 % encapsulation efficiency, achieved with most hydrophobic counterions (fluorinated tetraphenylborates), is absolutely required because non‐encapsulated dye species at the surface of NPs are the origin of dye leakage and strong fluorescence background in cells. The size of counterions is found to be essential to prevent ACQ, where the largest species, serving as effective spacer between dyes, provide the highest fluorescence quantum yield. Moreover, we found that the most hydrophobic counterions favor dye–dye coupling inside NPs, leading to ON/OFF fluorescence switching of single particles. By contrast, less hydrophobic counterions tend to disperse dyes in the polymer matrix favoring stable emission of NPs. The obtained structure‐property relationships validate the counterion‐based approach as a mature concept to fight ACQ and dye leakage in the development of advanced polymeric nanomaterials with controlled optical properties. Counterion‐enhanced encapsulation and emission: A systematic study based on ten counterions revealed that their size and hydrophobicity is essential to prevent aggregation‐caused quenching and ensure efficient encapsulation of a cationic rhodamine dye in polymeric nanoparticles. Here, the use of counterions is established as mature concept for preparation of bright and leakage‐free fluorescent nanomaterials for bioimaging applications. [ABSTRACT FROM AUTHOR]