Reduced Artifacts in Nanoscale Ratiometric Imaging and Sensing via Point-Spread Function Matching.

Ratiometric analysis of two or more fluorescence signals is a staple of quantitative imaging. However, this analysis becomes complicated at (near-) diffraction limited resolutions due to differences in how the emission colors are imaged by the microscope optics. We investigate this and find that point-spread function (PSF) mismatch between different emission wavelengths readily introduces spurious structuring in ratiometric images.

Celluloepidemiology-A paradigm for quantifying infectious disease dynamics on a population level.

To complement serology as a tool in public health interventions, we introduced the "celluloepidemiology" paradigm where we leveraged pathogen-specific T cell responses at a population level to advance our epidemiological understanding of infectious diseases, using SARS-CoV-2 as a model. Applying flow cytometry and machine learning on data from more than 500 individuals, we showed that the number of T cells with positive expression of functional markers not only could distinguish patients who recovered from COVID-19 from controls and pre-COVID donors but also identify previously unrecognized asymptomatic patients from mild, moderate, and severe recovered patients. The celluloepidemiology approach was uniquely capable to differentiate health care worker groups with different SARS-CoV-2 exposures from each other.

Simple generation of cleavable labels for multiplexed imaging.

The most common methods for multiplexed immunohistochemistry rely on cyclic procedures, whereby cells or tissues are repeatedly stained, imaged, and regenerated. Here, we present a simple and inexpensive approach for amine-targeted labeling of antibodies using a linker that can be easily cleaved by a mild reducing agent. This method requires only inexpensive and readily-available reagents, and can be carried out without synthetic experience in a simple one-pot reaction.

Application of Deep Sequencing in Phage Display.

This chapter describes the workflow to implement deep sequencing into standard phage display experiments on protein libraries. By harvesting the power of high throughput of these techniques, it allows for comprehensive analysis of the naïve library and library evolution in response to selection by ligand binding. The mutagenized target region of the protein variants encoded by the phage pool is analyzed by Illumina paired-end sequencing.

Smart genetically-encoded biosensors for the chemical monitoring of living systems.

Genetically-encoded biosensors provide the all-optical and non-invasive visualization of dynamic biochemical events within living systems, which has allowed the discovery of profound new insights. Twenty-five years of biosensor development has steadily improved their performance and has provided us with an ever increasing biosensor repertoire. In this feature article, we present recent advances made in biosensor development and provide a perspective on the future direction of the field.

Comparative Assessment of NMR Probes for the Experimental Description of Protein Folding Pathways with High-Pressure NMR.

Multidimensional NMR intrinsically provides multiple probes that can be used for deciphering the folding pathways of proteins: NH amide and CαHα groups are strategically located on the backbone of the protein, while CH groups, on the side-chain of methylated residues, are involved in important stabilizing interactions in the hydrophobic core. Combined with high hydrostatic pressure, these observables provide a powerful tool to explore the conformational landscapes of proteins. In the present study, we made a comparative assessment of the NH, CαHα, and CH groups for analyzing the unfolding pathway of ∆+PHS Staphylococcal Nuclease.

High-Throughput Sequencing of Phage Display Libraries Reveals Parasitic Enrichment of Indel Mutants Caused by Amplification Bias.

The combination of phage display technology with high-throughput sequencing enables in-depth analysis of library diversity and selection-driven dynamics. We applied short-read sequencing of the mutagenized region on focused display libraries of two homologous nucleic acid modification eraser proteins-AlkB and FTO-biopanned against methylated DNA. This revealed enriched genotypes with small indels and concomitant doubtful amino acid motifs within the FTO library.