Imaging approaches for monitoring three-dimensional cell and tissue culture systems.

The past decade has seen an increasing demand for more complex, reproducible and physiologically relevant tissue cultures that can mimic the structural and biological features of living tissues. Monitoring the viability, development and responses of such tissues in real-time are challenging due to the complexities of cell culture physical characteristics and the environments in which these cultures need to be maintained in. Significant developments in optics, such as optical manipulation, improved detection and data analysis, have made optical imaging a preferred choice for many three-dimensional (3D) cell culture monitoring applications.

Erratum: Label-free imaging of fibroblast membrane interfaces and protein signatures with vibrational infrared photothermal and phase signals: publisher's note.

[This corrects the article on p. 303 in vol. 12, PMID: 33520386.

Label-free imaging of fibroblast membrane interfaces and protein signatures with vibrational infrared photothermal and phase signals.

Label-free vibrational imaging of biological samples has attracted significant interest due to its integration of structural and chemical information. Vibrational infrared photothermal amplitude and phase signal (VIPPS) imaging provide label-free chemical identification by targeting the characteristic resonances of biological compounds that are present in the mid-infrared fingerprint region (3 µm - 12 µm). High contrast imaging of subcellular features and chemical identification of protein secondary structures in unlabeled and labeled fibroblast cells embedded in a collagen-rich extracellular matrix is demonstrated by combining contrast from absorption signatures (amplitude signals) with sensitive detection of different heat properties (lock-in phase signals).

Conformational States Control Lck Switching between Free and Confined Diffusion Modes in T Cells.

T cell receptor phosphorylation by Lck is an essential step in T cell activation. It is known that the conformational states of Lck control enzymatic activity; however, the underlying principles of how Lck finds its substrate over the plasma membrane remain elusive. Here, single-particle tracking is paired with photoactivatable localization microscopy to observe the diffusive modes of Lck in the plasma membrane.

tagPAINT: covalent labelling of genetically encoded protein tags for DNA-PAINT imaging.

Recently, DNA-PAINT single-molecule localization microscopy (SMLM) has shown great promise for quantitative imaging; however, labelling strategies thus far have relied on multivalent and affinity-based approaches. Here, the covalent labelling of expressed protein tags (SNAP tag and Halo tag) with single DNA-docking strands and application of SMLM via DNA-PAINT is demonstrated. tagPAINT is then used for T-cell receptor signalling proteins at the immune synapse as a proof of principle.

Observing the Reversible Single Molecule Electrochemistry of Alexa Fluor 647 Dyes by Total Internal Reflection Fluorescence Microscopy.

Alexa Fluor 647 is a widely used fluorescent probe for cell bioimaging and super-resolution microscopy. Herein, the reversible fluorescence switching of Alexa Fluor 647 conjugated to bovine serum albumin (BSA) and adsorbed onto indium tin oxide (ITO) electrodes under electrochemical potential control at the level of single protein molecules is reported. The modulation of the fluorescence as a function of potential was observed using total internal reflectance fluorescence (TIRF) microscopy.

Stoichiometric quantification of spatially dense assemblies with qPAINT.

Quantitative PAINT (qPAINT) is a useful method for counting well-separated molecules within nanoscale assemblies. But whether cross-reactivity in densely-packed arrangements perturbs measurements is unknown. Here we establish that qPAINT measurements are robust even when target molecules are separated by as little as 3 nm, sufficiently close that single-stranded DNA binding sites can interact.