† These authors contributed equally.visualize living cells with super-resolution at multiple reconstructed frames per second in real time. We demonstrate the versatility of this new platform by imaging cellular organelle dynamics based on live-cell fluorescent stains as well as with fluorescent protein stained samples.

Super-resolution (SR) fluorescence microscopy is typically carried out on research microscopes equipped with high-NA TIRF objectives and powerful laser light sources. Super-resolution optical fluctuation imaging (SOFI) is a fast SR technique capable of live-cell imaging, that is compatible with many wide-field microscope systems. However, especially when employing fluorescent proteins, a key part of the imaging system is a very sensitive and well calibrated camera sensor. The substantial costs of such systems preclude many research groups from employing SR imaging techniques. Here, we examine to what extent SOFI can be performed using a range of imaging hardware comprising different technologies and costs. In particular, we quantitatively compare the performance of an industry-grade CMOS camera to both state-of-the-art emCCD and sCMOS detectors, with SOFIspecific metrics. We show that SOFI data can be obtained using a cost-efficient industry-grade sensor, both on commercial and home-built microscope systems, though our analysis also readily exposes the merits of the per-pixel corrections performed in scientific cameras.

We investigate the internal structure of smart core–shell microgels by super-resolution fluorescence microscopy by combining of 3D single molecule localization and structured illumination microscopy using freely diffusing fluorescent dyes.

Injection techniques to deliver macromolecules to cells such as microinjection have been around for decades with applications ranging from probing whole organisms to the injection of fluorescent molecules into single cells. A similar technique that has raised recent interest is nanoinjection. The pipettes used here are much smaller and allow for the precise deposition of molecules into single cells via electrokinetics with minimal influence on the cells’ health. Unfortunately, the equipment utilized for nanoinjection originates from scanning ion conductance microscopy (SICM) and is therefore expensive and not portable, but usually fixed to a specific microscope setup. The level of precision that these systems achieve is much higher than what is needed for the more robust nanoinjection process. We present Mobile Nanoinjection (MoNa), a portable, cost-efficient and easy to build system for the injection of single cells. Sacrificing unnecessary sub-nanometer accuracy and low ion current noise levels, we were able to inject single living cells with high accuracy. We determined the noise of the MoNa system and investigated the injection conditions for 16 prominent fluorescent labels and fluorophores. Further, we performed proof of concepts by injection of ATTO655-Phalloidin and MitoTracker Deep Red to living human osteosarcoma (U2OS) cells and of living adult human inferior turbinate stem cells (ITSC’s) following neuronal differentiation with the MoNa system. We achieved significant cost reductions of the nanoinjection technology and gained full portability and compatibility to most optical microscopes.

Digital micromirror devices (DMDs) are spatial light modulators that employ the electro-mechanical movement of miniaturized mirrors to steer and thus modulate the light reflected off a mirror array. Their wide availability, low cost and high speed make them a popular choice both in consumer electronics such as video projectors, and scientific applications such as microscopy. High-end fluorescence microscopy systems typically employ laser light sources, which by their nature provide coherent excitation light. In super-resolution microscopy applications that use light modulation, most notably structured illumination microscopy (SIM), the coherent nature of the excitation light becomes a requirement to achieve optimal interference pattern contrast. The universal combination of DMDs and coherent light sources, especially when working with multiple different wavelengths, is unfortunately not straight forward. The substructure of the tilted micromirror array gives rise to a blazed grating, which has to be understood and which must be taken into account when designing a DMD-based illumination system. Here, we present a set of simulation frameworks that explore the use of DMDs in conjunction with coherent light sources, motivated by their application in SIM, but which are generalizable to other light patterning applications. This framework provides all the tools to explore and compute DMD-based diffraction effects and to simulate possible system alignment configurations computationally, which simplifies the system design process and provides guidance for setting up DMD-based microscopes. This article is part of the Theo Murphy meeting ‘Super-resolution structured illumination microscopy (part 1)’.