Genetically encoded fluorescent calcium indicators allow cellular-resolution recording of physiology. However, bright, genetically targetable indicators that can be multiplexed with existing tools in vivo are needed for simultaneous imaging of multiple signals. Here we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several that efficiently label the brain in animals. When bound to a near-infrared dye-ligand, WHaloCaMP shows a 7× increase in fluorescence intensity and a 2.1-ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a to image Ca responses in vivo in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae and to quantify Ca concentration using fluorescence lifetime imaging microscopy (FLIM).

The structure of axonal arbors controls how signals from individual neurons are routed within the mammalian brain. However, the arbors of very few long-range projection neurons have been reconstructed in their entirety, as axons with diameters as small as 100 nm arborize in target regions dispersed over many millimeters of tissue. We introduce a platform for high-resolution, three-dimensional fluorescence imaging of complete tissue volumes that enables the visualization and reconstruction of long-range axonal arbors. This platform relies on a high-speed two-photon microscope integrated with a tissue vibratome and a suite of computational tools for large-scale image data. We demonstrate the power of this approach by reconstructing the axonal arbors of multiple neurons in the motor cortex across a single mouse brain.

Enzyme kinetics measurements are a standard component of undergraduate biochemistry laboratories. The combination of serine hydrolases and fluorogenic enzyme substrates provides a rapid, sensitive, and general method for measuring enzyme kinetics in an undergraduate biochemistry laboratory. In this method, the kinetic activity of multiple protein variants is determined in parallel using a microplate reader, multichannel pipets, serial dilutions, and fluorogenic ester substrates. The utility of this methodology is illustrated by the measurement of differential enzyme activity in microplate volumes in triplicate with small protein samples and low activity enzyme variants. Enzyme kinetic measurements using fluorogenic substrates are, thus, adaptable for use with student-purified enzyme variants and for comparative enzyme kinetics studies. The rapid setup and analysis of these kinetic experiments not only provides advanced undergraduates with experience in a fundamental biochemical technique, but also provides the adaptability for use in inquiry-based laboratories.

Spontaneously blinking fluorophores permit the detection and localization of individual molecules without reducing buffers or caging groups, thus simplifying single-molecule localization microscopy (SMLM). The intrinsic blinking properties of such dyes are dictated by molecular structure and modulated by environment, which can limit utility. We report a series of tuned spontaneously blinking dyes with duty cycles that span two orders of magnitude, allowing facile SMLM in cells and dense biomolecular structures.

Chemical synapses between axons and dendrites mediate much of the brain’s intercellular communication. Here we describe a new kind of synapse – the axo-ciliary synapse - between axons and primary cilia. By employing enhanced focused ion beam – scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between the serotonergic axons arising from the brainstem, and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, 5-hydroxytryptamine receptor 6 (HTR6), whose mutation is associated with learning and memory defects. Using a newly developed cilia-targeted serotonin sensor, we show that optogenetic stimulation of serotonergic axons results in serotonin release onto cilia. Ciliary HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway. Ablation of this pathway results in nuclear actin and chromatin accessibility changes in CA1 pyramidal neurons. Axo-ciliary synapses serve as a distinct mechanism for neuromodulators to program neuron transcription through privileged access to the nuclear compartment.

Maintenance of the bacterial homeostasis initially emanates from interactions between proteins and the bacterial nucleoid. Investigating their spatial correlation requires high spatial resolution, especially in tiny, highly confined and crowded bacterial cells. Here, we present super-resolution microscopy using a palette of fluorescent labels that bind transiently to either the membrane or the nucleoid of fixed E. coli cells. The presented labels are easily applicable, versatile and allow long-term single-molecule super-resolution imaging independent of photobleaching. The different spectral properties allow for multiplexed imaging in combination with other localisation-based super-resolution imaging techniques. As examples for applications, we demonstrate correlated super-resolution imaging of the bacterial nucleoid with the position of genetic loci, of nascent DNA in correlation to the entire nucleoid, and of the nucleoid of metabolically arrested cells. We furthermore show that DNA- and membrane-targeting labels can be combined with photoactivatable fluorescent proteins and visualise the nano-scale distribution of RNA polymerase relative to the nucleoid in drug-treated E. coli cells.

The fast turnover of membrane components through endocytosis and recycling allows precise control of the composition of the plasma membrane. Endocytic recycling can be rapid with some molecules returning to the plasma membrane with a <5 minutes. Existing methods to study these trafficking pathways utilize chemical, radioactive, or fluorescent labeling of cell surface receptors in pulse-chase experiments, which require tedious washing steps and manual collection of samples. Here, we introduce a live-cell endocytic recycling assay, based on a newly designed cell-impermeable, fluorogenic ligand for HaloTag: 'Janelia Fluor 635i' (JFi; i=impermeant) which allows real-time detection of membrane receptor recycling at steady state. We used this method to study the effect of iron depletion on transferrin receptor (TfR) recycling using the chelator desferrioxamine. We found this perturbation significantly increases the TfR recycling rate. The high temporal resolution and simplicity of this assay provides a clear advantage over extant methods and makes it ideal for large scale cellular imaging studies. This assay can be adapted to examine other cellular kinetic parameters such as protein turnover and biosynthetic trafficking.

Small molecule fluorophores are essential tools for chemical biology. A benefit of synthetic dyes is the ability to employ chemical approaches to control the properties and direct the position of the fluorophore. Applying modern synthetic organic chemistry strategies enables efficient tailoring of the chemical structure to obtain probes for specific biological experiments. Chemistry can also be used to activate fluorophores; new fluorogenic enzyme substrates and photoactivatable compounds with improved properties have been prepared that facilitate advanced imaging experiments with low background fluorescence. Finally, chemical reactions in live cells can be used to direct the spatial distribution of the fluorophore, allowing labeling of defined cellular regions with synthetic dyes.

Differential Scanning Fluorimetry (DSF) is a biophysical assay that is used to estimate protein stability in vitro. In a DSF experiment, the increased fluorescence of a solvatochromatic dye, such as Sypro Orange, is used to detect the unfolding of a protein during heating. However, Sypro Orange is only compatible with a minority of proteins (< 30%), limiting the scope of this method. We recently reported that protein-adaptive DSF (paDSF) can partially solve this problem, wherein the protein is initially pre-screened against ∼300 chemically diverse dyes, termed the Aurora collection. While this approach significantly improves the number of targets amenable to DSF, it still fails to produce protein-dye pairs for some proteins. Here, we report the expansion of the dye collection to Aurora 2.0, which includes a total of 517 structurally diverse molecules and multiple new chemotypes. To assess performance, these dyes were screened against a panel of ∼100 proteins, which were selected, in part, to represent the most challenging targets (e.g. small size). From this effort, Aurora 2.0 achieved an impressive success rate of 94%, including producing dyes for some targets that were not matched in the original collection. These findings support the idea that larger, more chemically diverse libraries improve the likelihood of detecting melting transitions across a wider range of proteins. We propose that Aurora 2.0 makes paDSF an increasingly powerful method for studying protein stability, ligand binding and other biophysical properties in high throughput.

In 2023, the ImaBio consortium (imabio-cnrs.fr), an interdisciplinary life microscopy research group at the Centre National de la Recherche Scientifique, celebrated its 20th anniversary. ImaBio contributes to the biological imaging community through organization of MiFoBio conferences, which are interdisciplinary conferences featuring lectures and hands-on workshops that attract specialists from around the world. MiFoBio conferences provide the community with an opportunity to reflect on the evolution of the field, and the 2023 event offered retrospective talks discussing the past 20 years of topics in microscopy, including imaging of multicellular assemblies, image analysis, quantification of molecular motions and interactions within cells, advancements in fluorescent labels, and laser technology for multiphoton and label-free imaging of thick biological samples. In this Perspective, we compile summaries of these presentations overviewing 20 years of advancements in a specific area of microscopy, each of which concludes with a brief look towards the future. The full presentations are available on the ImaBio YouTube channel (youtube.com/@gdrimabio5724).