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Biological research is in constant need of new methodological developments to assess organization and functions at various scales ranging from whole organisms to interactions between proteins. One of the main ways to evidence and quantify biological phenomena is imaging. Fluorescence microscopy and label-free microscopy are in particular highly active fields of research due to their compatibility with living samples as well as their versatility. The Imabio Young Scientists Network (YSN) is a group of young scientists (PhD students, postdocs and engineers) who are excited about bioimaging and aim to create a proactive network of researchers with the same interest. YSN is endorsed by the bioimaging network GDR Imabio in France, where the initiative was started in 2019. Since then, we aim to organize the Imabio YSN conference every year to expand the network to other European countries, establish new collaborations and ignite new scientific ideas. From 6-8 July 2022, the YSN including researchers from the domains of life sciences, chemistry, physics and computational sciences met at the Third Imabio YSN Conference 2022 in Lyon to discuss the latest bioimaging technologies and biological discoveries. In this Meeting Review, we describe the essence of the scientific debates, highlight remarkable talks, and focus on the Career Development session, which is unique to the YSN conference, providing a career perspective to young scientists and help to answer all their questions at this career stage. This conference was a truly interdisciplinary reunion of scientists who are eager to push the frontiers of bioimaging in order to understand the complexity of biological systems.

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Owing to their unique abilities to manipulate, label, and image individual molecules in vitro and in cellulo, single-molecule techniques provide previously unattainable access to elementary biological processes. In imaging, single-molecule fluorescence resonance energy transfer (smFRET) and protein-induced fluorescence enhancement in vitro can report on conformational changes and molecular interactions, single-molecule pull-down (SiMPull) can capture and analyze the composition and function of native protein complexes, and single-molecule tracking (SMT) in live cells reveals cellular structures and dynamics. In labeling, the abilities to specifically label genomic loci, mRNA, and nascent polypeptides in cells have uncovered chromosome organization and dynamics, transcription and translation dynamics, and gene expression regulation. In manipulation, optical tweezers, integration of single-molecule fluorescence with force measurements, and single-molecule force probes in live cells have transformed our mechanistic understanding of diverse biological processes, ranging from protein folding, nucleic acids-protein interactions to cell surface receptor function.

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Focal modulation microscopy (FMM) has gained significant interest in biological imaging. However, the spatial resolution and penetration depth limit the imaging quality of FMM due to the strong scattering background. Here, we introduce FMM with a Tai Chi aperture (TCFMM) based on diffraction theory to improve the spatial resolution. The results show that the transverse resolution is improved by 61.60% and 41.37% in two orthogonal directions, and the axial resolution is improved by 29.67%, compared with confocal microscopy (CM). The signal background ratio (SBR) of TCFMM is increased by 23.26% compared with CM and remains nearly the same compared with FMM using D-shape apertures (DFMM). These improvements in spatial resolution and SBR indicate that TCFMM has potential in deep tissue imaging.

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STED microscopy is one of several fluorescence microscopy techniques that permit imaging at higher spatial resolution than what the diffraction-limit of light dictates. STED imaging is unique among these super-resolution modalities in being a beam-scanning microscopy technique based on confocal or 2-photon imaging, which provides the advantage of superior optical sectioning in thick samples. Compared to the other super-resolution techniques that are based on widefield microscopy, this makes STED particularly suited for imaging inside live brain tissue, such as in slices or in vivo. Notably, the 50 nm resolution provided by STED microscopy enables analysis of neural morphologies that conventional confocal and 2-photon microscopy approaches cannot resolve, including all-important synaptic structures. Over the course of the last 20 years, STED microscopy has undergone extensive developments towards ever more versatile use, and has facilitated remarkable neurophysiological discoveries. The technique is still not widely adopted for live tissue imaging, even though one of its particular strengths is exactly in resolving the nanoscale dynamics of synaptic structures in brain tissue, as well as in addressing the complex morphologies of glial cells, and revealing the intricate structure of the brain extracellular space. Not least, live tissue STED microscopy has so far hardly been applied in settings of pathophysiology, though also here it shows great promise for providing new insights. This review outlines the technical advantages of STED microscopy for imaging in live brain tissue, and highlights key neurobiological findings brought about by the technique.

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One of the biggest unsolved questions in neuroscience is how molecules and neuronal circuitry create behaviors, and how their misregulation or dysfunction results in neurological disease. Light microscopy is a vital tool for the study of neural molecules and circuits. However, the fundamental optical diffraction limit precludes the use of conventional light microscopy for sufficient characterization of critical signaling compartments and nanoscopic organizations of synapse-associated molecules. We have witnessed rapid development of super-resolution microscopy methods that circumvent the resolution limit by controlling the number of emitting molecules in specific imaging volumes and allow highly resolved imaging in the 10-100 nm range. Most recently, Expansion Microscopy (ExM) emerged as an alternative solution to overcome the diffraction limit by physically magnifying biological specimens, including nervous systems. Here, we discuss how ExM works in general and currently available ExM methods. We then review ExM imaging in a wide range of nervous systems, including Caenorhabditis elegans, Drosophila, zebrafish, mouse, and human, and their applications to synaptic imaging, neuronal tracing, and the study of neurological disease. Finally, we provide our prospects for expansion microscopy as a powerful nanoscale imaging tool in the neurosciences.

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Super-resolution microscopy has revolutionised the way we observe biological systems. These methods are now a staple of fluorescence microscopy. Researchers have used super-resolution methods in myriad systems to extract nanoscale spatial information on multiple interacting parts. These methods are continually being extended and reimagined to further push their resolving power and achieve truly single protein resolution. Here, we explore the most recent advances at the frontier of the 'super-resolution' limit and what opportunities remain for further improvements in the near future.

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Fluorescence lifetime microscopy (FLIM) and Förster's resonance energy transfer (FRET) are advanced optical tools that neuroscientists can employ to interrogate the structure and function of complex biological systems in vitro and in vivo using light. In neurobiology they are primarily used to study protein-protein interactions, to study conformational changes in protein complexes, and to monitor genetically encoded FRET-based biosensors. These methods are ideally suited to optically monitor changes in neurons that are triggered optogenetically. Utilization of this technique by neuroscientists has been limited, since a broad understanding of FLIM and FRET requires familiarity with the interactions of light and matter on a quantum mechanical level, and because the ultra-fast instrumentation used to measure fluorescent lifetimes and resonance energy transfer are more at home in a physics lab than in a biology lab. In this overview, we aim to help neuroscientists overcome these obstacles and thus feel more comfortable with the FLIM-FRET method. Our goal is to aid researchers in the neuroscience community to achieve a better understanding of the fundamentals of FLIM-FRET and encourage them to fully leverage its powerful ability as a research tool. Published 2020. U.S. Government.

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Rationale: Intraoperative visualization in small surgical cavities and hard-to-access areas are essential requirements for modern, minimally invasive surgeries and demand significant miniaturization. However, current optical imagers require multiple hard-to-miniaturize components including lenses, filters and optical fibers. These components restrict both the form-factor and maneuverability of these imagers, and imagers largely remain stand-alone devices with centimeter-scale dimensions. Methods: We have engineered INSITE (Immunotargeted Nanoparticle Single-Chip Imaging Technology), which integrates the unique optical properties of lanthanide-based alloyed upconverting nanoparticles (aUCNPs) with the time-resolved imaging of a 25-micron thin CMOS-based (complementary metal oxide semiconductor) imager. We have synthesized core/shell aUCNPs of different compositions and imaged their visible emission with INSITE under either NIR-I and NIR-II photoexcitation. We characterized aUCNP imaging with INSITE across both varying aUCNP composition and 980 nm and 1550 nm excitation wavelengths. To demonstrate clinical experimental validity, we also conducted an intratumoral injection into LNCaP prostate tumors in a male nude mouse that was subsequently excised and imaged with INSITE. Results: Under the low illumination fluences compatible with live animal imaging, we measure aUCNP radiative lifetimes of 600 µs - 1.3 ms, which provides strong signal for time-resolved INSITE imaging. Core/shell NaEr0.6Yb0.4F4 aUCNPs show the highest INSITE signal when illuminated at either 980 nm or 1550 nm, with signal from NIR-I excitation about an order of magnitude brighter than from NIR-II excitation. The 55 µm spatial resolution achievable with this approach is demonstrated through imaging of aUCNPs in PDMS (polydimethylsiloxane) micro-wells, showing resolution of micrometer-scale targets with single-pixel precision. INSITE imaging of intratumoral NaEr0.8Yb0.2F4 aUCNPs shows a signal-to-background ratio of 9, limited only by photodiode dark current and electronic noise. Conclusion: This work demonstrates INSITE imaging of aUCNPs in tumors, achieving an imaging platform that is thinned to just a 25 µm-thin, planar form-factor, with both NIR-I and NIR-II excitation. Based on a highly paralleled array structure INSITE is scalable, enabling direct coupling with a wide array of surgical and robotic tools for seamless integration with tissue actuation, resection or ablation.

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Recently, fluorescence-based super-resolution techniques such as stimulated emission depletion (STED) and stochastic optical reconstruction microscopy (STORM) have been developed to achieve near molecular-scale resolution. However, such a super-resolution technique for nonlinear label-free microscopy based on second harmonic generation (SHG) is lacking. Since SHG is label-free and does not involve real-energy level transitions, fluorescence-based super-resolution techniques such as STED cannot be applied to improve the resolution. In addition, due to the coherent and non-isotropic emission nature of SHG, single-molecule localization techniques based on isotropic emission of fluorescent molecule such as STORM will not be appropriate. Single molecule SHG microscopy is largely hindered due to the very weak nonlinear optical scattering cross sections of SHG scattering processes. Thus, enhancing SHG using plasmonic nanostructures and nanoantennas has recently gained much attention owing to the potential of various nanoscale geometries to tightly confine electromagnetic fields into small volumes. This confinement provides substantial enhancement of electromagnetic field in nanoscale regions of interest, which can significantly boost the nonlinear signal produced by molecules located in the plasmonic hotspots. However, to date, plasmon-enhanced SHG has been primarily applied for the measurement of bulk properties of the materials/molecules, and single molecule SHG imaging along with its orientation information has not been realized yet. Herein, we achieved simultaneous visualization and three-dimensional (3D) orientation imaging of individual rhodamine 6G (R6G) molecules in the presence of plasmonic silver nanohole arrays. SHG and two-photon fluorescence microscopy experiments together with finite-difference time-domain (FDTD) simulations revealed a ∼106-fold nonlinear enhancement factor at the hot spots on the plasmonic silver nanohole substrate, enabling detection of single molecules using SHG. The position and 3D orientation of R6G molecules were determined using the template matching algorithm by comparing the experimental data with the calculated dipole emission images. These findings could enable SHG-based single molecule detection and orientation imaging of molecules which could lead to a wide range of applications from nanophotonics to super-resolution SHG imaging of biological cells and tissues.

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The ability to visualize and quantitatively measure dynamic biological processes in vivo and at high spatiotemporal resolution is of fundamental importance to experimental investigations in developmental biology. Light-sheet microscopy is particularly well suited to providing such data, since it offers exceptionally high imaging speed and good spatial resolution while minimizing light-induced damage to the specimen. We review core principles and recent advances in light-sheet microscopy, with a focus on concepts and implementations relevant for applications in developmental biology. We discuss how light-sheet microcopy has helped advance our understanding of developmental processes from single-molecule to whole-organism studies, assess the potential for synergies with other state-of-the-art technologies, and introduce methods for computational image and data analysis. Finally, we explore the future trajectory of light-sheet microscopy, discuss key efforts to disseminate new light-sheet technology, and identify exciting opportunities for further advances.

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In this photo essay, we present a sampling of technologies from laboratories at the forefront of whole-brain clearing and imaging for high-resolution analysis of cell populations and neuronal circuits. The data presented here were provided for the eponymous Mini-Symposium presented at the Society for Neuroscience's 2018 annual meeting.

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Superresolution microscopy (SM) techniques are among the revolutionary methods for molecular and cellular observations in the 21st century. SM techniques overcome optical limitations, and several new observations using SM lead us to expect these techniques to have a large impact on neuroscience in the near future. Several types of SM have been developed, including structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), and photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM), each with special features. In this Minisymposium, experts in these different types of SM discuss the new structural and functional information about specific important molecules in neuroscience that has been gained with SM. Using these techniques, we have revealed novel mechanisms of endocytosis in nerve growth, fusion pore dynamics, and described quantitative new properties of excitatory and inhibitory synapses. Additional powerful techniques, including single molecule-guided Bayesian localization SM (SIMBA) and expansion microscopy (ExM), alone or combined with super-resolution observation, are also introduced in this session.

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Advances in fluorescent dye and protein design have led to a wider color palette for microscopy. Nathan Blow explores how this happened and what is still to come.

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Insulin secretory granule (SG) turnover consists of several highly regulated processes allowing for proper ß-cell function and insulin secretion. Besides the spatial distribution of insulin SGs, their age has great impact on the likelihood of their secretion and their behaviour within the ß-cell. While quantitative measurements performed decades ago demonstrated the preferential secretion of young insulin, new experimental approaches aim to investigate insulin ageing at the granular level. Live-cell imaging, automated image analysis and correlative light and electron microscopy have fostered knowledge of age-defined insulin SG dynamics, their interaction with the cytoskeleton and ultrastructural features. Here, we review our recent work in regards to the connection between insulin SG age, SG dynamics, intracellular location and interaction with other proteins.

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Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called 'green revolution', has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called 'super-resolution' fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques.

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Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.

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From bacteria to circulating tumor cells, advances in flow cytometry technology are pushing the boundaries of cell biology research.

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Over the past two decades, single-molecule techniques have evolved into robust tools to study many fundamental biological processes. The combination of optical tweezers with fluorescence microscopy and microfluidics provides a powerful single-molecule manipulation and visualization technique that has found widespread application in biology. In this combined approach, the spatial (~nm) and temporal (~ms) resolution, as well as the force scale (~pN) accessible to optical tweezers is complemented with the power of fluorescence microscopy. Thereby, it provides information on the local presence, identity, spatial dynamics, and conformational dynamics of single biomolecules. Together, these techniques allow comprehensive studies of, among others, molecular motors, protein-protein and protein-DNA interactions, biomolecular conformational changes, and mechanotransduction pathways. In this chapter, recent applications of fluorescence microscopy in combination with optical trapping are discussed. After an introductory section, we provide a description of instrumentation together with the current capabilities and limitations of the approaches. Next we summarize recent studies that applied this combination of techniques in biological systems and highlight some representative biological assays to mark the exquisite opportunities that optical tweezers combined with fluorescence microscopy provide.

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