Recognising the importance and impact of Imaging Scientists: Global guidelines for establishing career paths within core facilities.

In the dynamic landscape of scientific research, imaging core facilities are vital hubs propelling collaboration and innovation at the technology development and dissemination frontier. Here, we present a collaborative effort led by Global BioImaging (GBI), introducing international recommendations geared towards elevating the careers of Imaging Scientists in core facilities. Despite the critical role of Imaging Scientists in modern research ecosystems, challenges persist in recognising their value, aligning performance metrics and providing avenues for career progression and job security.

Building a FAIR image data ecosystem for microscopy communities.

Bioimaging has now entered the era of big data with faster-than-ever development of complex microscopy technologies leading to increasingly complex datasets. This enormous increase in data size and informational complexity within those datasets has brought with it several difficulties in terms of common and harmonized data handling, analysis, and management practices, which are currently hampering the full potential of image data being realized. Here, we outline a wide range of efforts and solutions currently being developed by the microscopy community to address these challenges on the path towards FAIR bioimaging data.

QUAREP-LiMi: A community-driven initiative to establish guidelines for quality assessment and reproducibility for instruments and images in light microscopy.

A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted.

Chromophore-assisted laser inactivation of alpha- and gamma-tubulin SNAP-tag fusion proteins inside living cells.

Chromophore-assisted laser inactivation (CALI) can help to unravel localized activities of target proteins at defined times and locations within living cells. Covalent SNAP-tag labeling of fusion proteins with fluorophores such as fluorescein is a fast and highly specific tool to attach the photosensitizer to its target protein in vivo for selective inactivation of the fusion protein. Here, we demonstrate the effectiveness and specificity of SNAP-tag-based CALI by acute inactivation of alpha-tubulin and gamma-tubulin SNAP-tag fusions during live imaging assays of cell division.

Fluorophores for live cell imaging of AGT fusion proteins across the visible spectrum.

O6-alkylguanine-DNA alkyltransferase (AGT) fusion proteins can be specifically and covalently labeled with fluorescent O6-benzylguanine (O6-BG) derivatives for multicolor live cell imaging approaches. Here, we characterize several new BG fluorophores suitable for in vivo AGT labeling that display fluorescence emission maxima covering the visible spectrum from 472 to 673 nm, thereby extending the spectral limits set by fluorescent proteins. We show that the photostability of the cell-permeable dyes BG Rhodamine Green (BG505) and CP tetramethylrhodamine (CP-TMR) is in the range of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP), and that BG diethylaminomethyl coumarin (BGDEAC), a derivative of coumarin, is even more stable than enhanced cyan fluorescent protein (ECFP).

Labeling of fusion proteins with synthetic fluorophores in live cells.

A general approach for the sequential labeling of fusion proteins of O(6)-alkylguanine-DNA alkyltransferase (AGT) with different fluorophores in mammalian cells is presented. AGT fusion proteins with different localizations in the cell can be labeled specifically with different fluorophores, and the fluorescence labeling can be used for applications such as multicolor analysis of dynamic processes and fluorescence resonance energy transfer measurements. The facile access to a variety of different AGT substrates as well as the specificity of the labeling reaction should make the approach an important tool to study protein function in live cells.

Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro.

The in vivo and in vitro labeling of fusion proteins with synthetic molecules capable of probing and controlling protein function has the potential to become an important method in functional genomics and proteomics. We have recently introduced an approach for the specific labeling of fusion proteins, which is based on the generation of fusion proteins with the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) and the irreversible reaction of hAGT with O6-benzylguanine derivatives. Here, we report optimized protocols for the synthesis of O6-benzylguanine derivatives and the use of such derivatives for the labeling of different hAGT fusion proteins in vivo and in vitro.

Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo.

We report here the generation of mutants of the human O(6)-alkylguanine-DNA alkyltransferase (hAGT) for the efficient in vivo labeling of fusion proteins with synthetic reporter molecules. Libraries of hAGT were displayed on phage, and mutants capable of efficiently reacting with the inhibitor O(6)-benzylguanine were selected based on their ability to irreversibly transfer the benzyl group to a reactive cysteine residue. Using synthetic O(6)-benzylguanine derivatives, the selected mutant proteins allow for a highly efficient covalent labeling of hAGT fusion proteins in vivo and in vitro with small molecules and therefore should become important tools for studying protein function in living cells.

A general method for the covalent labeling of fusion proteins with small molecules in vivo.

Characterizing the movement, interactions, and chemical microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin.