Nanosized mesoporous silica particles with high colloidal stability attract growing attention as drug delivery systems for targeted cancer treatment and as bioimaging devices. This Perspective describes recent breakthroughs in mesoporous silica nanoparticle design to demonstrate their high potential as multifunctional drug delivery nanocarriers. These types of nanoparticles can feature a well-defined and tunable porosity at the nanometer scale, high loading capacity, and multiple functionality for targeting and entering different types of cells. We focus on the requirements for an efficient stimuli-responsive and thus controllable release of cargo into cancer cells and discuss design principles for smart and autonomous nanocarrier systems. Mesoporous silica nanoparticles are viewed as a promising and flexible platform for numerous biomedical applications.

We describe a method, based on single-molecule imaging, that allows the real-time visualization of the infection pathway of single viruses in living cells, each labeled with only one fluorescent dye molecule. The tracking of single viruses removes ensemble averaging. Diffusion trajectories with high spatial and time resolution show various modes of motion of adeno-associated viruses (AAV) during their infection pathway into living HeLa cells: (i) consecutive virus touching at the cell surface and fast endocytosis; (ii) free and anomalous diffusion of the endosome and the virus in the cytoplasm and the nucleus; and (iii) directed motion by motor proteins in the cytoplasm and in nuclear tubular structures. The real-time visualization of the infection pathway of single AAVs shows a much faster infection than was generally observed so far.

In this article, we demonstrate the new method of pulsed interleaved excitation (PIE), which can be used to extend the capabilities of multiple-color fluorescence imaging, fluorescence cross-correlation spectroscopy (FCCS), and single-pair fluorescence resonance energy transfer (spFRET) measurements. In PIE, multiple excitation sources are interleaved such that the fluorescence emission generated from one pulse is complete before the next excitation pulse arrives. Hence, the excitation source for each detected photon is known. Typical repetition rates used for PIE are between approximately 1 and 50 MHz. PIE has many applications in various fluorescence methods. Using PIE, dual-color measurements can be performed with a single detector. In fluorescence imaging with multicolor detection, spectral cross talk can be removed, improving the contrast of the image. Using PIE with FCCS, we can eliminate spectral cross talk, making the method sensitive to weaker interactions. FCCS measurements with complexes that undergo FRET can be analyzed quantitatively. Under specific conditions, the FRET efficiency can be determined directly from the amplitude of the measured correlation functions without any calibration factors. We also show the application of PIE to spFRET measurements, where complexes that have low FRET efficiency can be distinguished from those that do not have an active acceptor.