Romain F. Laine

University College London | UCL · MRC Laboratory for Molecular Cell Biology

Unwanted sample drift is a common issue that plagues microscopy experiments, preventing accurate temporal quantification of biological processes. While multiple methods and tools exist to correct images post-acquisition, performing drift correction of large 3D videos using open-source solutions remains challenging and time-consuming. Here we presen...

In recent years, the development of analytical approaches to super-resolution microscopy has highlighted the possibility of recovering super-resolution information from short sequences of wide-field images. Our recently developed method, SRRF (Super-Resolution Radial Fluctuations), enables long-term live-cell imaging beyond the resolution limit wit...

Deep Learning (DL) methods are increasingly recognised as powerful analytical tools for microscopy. Their potential to outperform conventional image processing pipelines is now well established. Despite the enthusiasm and innovations fuelled by DL technology, the need to access powerful and compatible resources, install multiple computational tools...

Cellular mechanics play a crucial role in tissue homeostasis and are often misregulated in disease. Traction force microscopy is one of the key methods that has enabled researchers to study fundamental aspects of mechanobiology; however, traction force microscopy is limited by poor resolution. Here, we propose a simplified protocol and imaging stra...

The three-dimensional imaging of mesoscopic samples with Optical Projection Tomography (OPT) has become a powerful tool for biomedical phenotyping studies. OPT uses visible light to visualize the 3D morphology of large transparent samples. To enable a wider application of OPT, we present OptiJ, a low-cost, fully open-source OPT system capable of im...

Cellular mechanics play a crucial role in tissue morphogenesis and homeostasis and are often misregulated in disease. Traction force microscopy (TFM) is one of the key methods that has enabled researchers to study fundamental aspects of mechanobiology; however, the power of TFM is limited by poor resolution and low throughput. Here, we propose a si...

Artificial Intelligence based on Deep Learning (DL) is opening new horizons in biomedical research and promises to revolutionize the microscopy field. It is now transitioning from the hands of experts in computer sciences to biomedical researchers. Here, we introduce recent developments in DL applied to microscopy, in a manner accessible to non-exp...

The nematode worm Caenorhabditis elegans has emerged as an important model organism to study the molecular mechanisms of protein misfolding diseases associated with amyloid formation because of its small size, ease of genetic manipulation and optical transparency. Obtaining a reliable and quantitative read-out of protein aggregation in this system,...

The three-dimensional imaging of mesoscopic samples with Optical Projection Tomography (OPT) has become a powerful tool for biomedical phenotyping studies. OPT uses visible light to visualize the 3D morphology of large transparent samples. To enable a wider application of OPT, we present OptiJ, a low-cost, fully open-source OPT system capable of im...

Reduced protein homeostasis leading to increased protein instability is a common molecular feature of aging, but it remains unclear whether this is a cause or consequence of the aging process. In neurodegenerative diseases and other amyloidoses, specific proteins self-assemble into amyloid fibrils and accumulate as pathological aggregates in differ...

Combining and multiplexing microscopy approaches is crucial to understand cellular events, but requires elaborate workflows. Here, we present a robust, open-source approach for treating, labelling and imaging live or fixed cells in automated sequences. NanoJ-Fluidics is based on low-cost Lego hardware controlled by ImageJ-based software, making hig...

Artificial Intelligence based on Deep Learning is opening new horizons in Biomedical research and promises to revolutionize the Microscopy field. Slowly, it now transitions from the hands of experts in Computer Sciences to researchers in Cell Biology. Here, we introduce recent developments in Deep Learning applied to Microscopy, in a manner accessi...

Artificial Intelligence based on Deep Learning is opening new horizons in Biomedical research and promises to revolutionize the Microscopy field. Slowly, it now transitions from the hands of experts in Computer Sciences to researchers in Cell Biology. Here, we introduce recent developments in Deep Learning applied to Microscopy, in a manner accessi...

Super-resolution microscopy (SRM) has become essential for the study of nanoscale biological processes. This type of imaging often requires the use of specialised image analysis tools to process a large volume of recorded data and extract quantitative information. In recent years, our team has built an open-source image analysis framework for SRM d...

Optical super-resolution microscopy techniques enable high molecular specificity with high spatial resolution and constitute a set of powerful tools in the investigation of the structure of supramolecular assemblies such as viruses. Here, we report on a new methodology which combines Structured Illumination Microscopy (SIM) with machine learning al...

Super-resolution microscopy has become essential for the study of nanoscale biological processes. This type of imaging often requires the use of specialised image analysis tools to process a large volume of recorded data and extract quantitative information. In recent years, our team has built an open-source image analysis framework for super-resol...

Reduced protein homeostasis and increased protein instability is a common feature of aging. Yet it remains unclear whether protein instability is a cause of aging. In neurodegenerative diseases and amyloidoses, specific proteins self-assemble into amyloid fibrils and accumulate as pathological solid aggregates in a variety of tissues. More recently...

Living crystallization-driven self-assembly (CDSA) is a seeded growth method for crystallizable block copolymers (BCPs) and related amphiphiles in solution and has recently emerged as a highly promising and versatile route to uniform core–shell nanoparticles (micelles) with control of dimensions and architecture. However, the factors that influence...

The nematode worm Caenorhabditis elegans has emerged as an important model organism to study the molecular mechanisms of protein misfolding diseases associated with amyloid formation because of its small size, ease of genetic manipulation, and optical transparency. Obtaining a reliable and quantitative read-out of protein aggregation in this system...

Fluorescence microscopy can reveal all aspects of cellular mechanisms, from molecular details to dynamics, thanks to approaches such as super-resolution and live-cell imaging. Each of its modalities requires specific sample preparation and imaging conditions to obtain high-quality, artefact-free images, ultimately providing complementary informatio...

Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated syna...

Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N-terminus which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C-terminus of alpha-synuclein, therewith increasing its lipid binding capacity. Using CEST-NMR we reveal that alpha-synuclein interacts with isolated synapt...

Nascent proteins can be positioned rapidly at precise subcellular locations by local protein synthesis (LPS) to facilitate localized growth responses. Axon arbor architecture, a major determinant of synaptic connectivity, is shaped by localized growth responses but it is unknown whether LPS influences these responses in vivo. Using high resolution...

Nascent proteins can be positioned rapidly at precise subcellular locations by local protein synthesis (LPS) to facilitate localized growth responses. Axon arbor architecture, a major determinant of synaptic connectivity, is shaped by localized growth responses, but it is unknown whether LPS influences these responses in vivo. Using high-resolution...

Movie S5. Related to Figure 2. Acute translation inhibition reduces branching dynamics of somaless RGC axons in vivo The movie was captured at 30 spf and the rate of movie is 8 fps.

Movie S6. Related to Figure 4. β-actin knockdown inhibits branching dynamics of RGC axons in vivo The movie was captured at 30 spf and the rate of movie is 8 fps.

Movie S9. Related to Figure 6. FRAP of Venus control and Venus-β-actin in RGC axon terminals in vivo The movie was created at 60 spf and played at the rate of 2 fps.

Movie S1. Related to Figure 1. RNA granules dock at axonal sites in advance of branch emergence in vivo The movie was captured at 3.3 seconds per frame (spf) and the rate of movie is 40 frames per second (fps).

The movie was captured at 3.8spf and the rate of movie is 100 fps.

Movie S3. Related to Figure 1. Trafficking of RNA granules out of branch before branch retraction in vivo The movie was captured at 5.6 spf and the rate of movie is 30 fps.

Movie S7. Related to Figure 5. Local inhibition of β-actin translation inhibits branching dynamics in somaless RGC axons in vivo The movie was captured at 30 spf and the rate of movie is 8 fps.

Movie S8. Related to Figure 5 and S6. Local inhibition of β-actin translation does not result in observable defects in RGC axon navigation in vivo The movie was captured at 300 spf and the rate of movie is 6 fps.

Movie S10. Related to Figure 7. Focal translation of Venus-B-actin promotes hotspot formation at branches in vivo The movie was captured at 1 spf and the rate of movie is 60 fps.

Movie S11. Related to Figure 8. Focal translation of Venus-B-actin takes place in close proximity to docked RNA granules in vivo The movie was captured at 1 spf and the rate of movie is 30 fps.

Movie S4. Related to Figure 1, S1, S2 and S3. RNA granules and mitochondria dynamics The movie was captured at 5 spf and the rate of movie is 10 fps.

Abstract Local mRNA translation occurs in growing axons enabling precise control of the proteome in response to signals. To measure quantitatively the spatiotemporal dynamics of protein synthesis in growth cones, we further developed a technique for single molecule translation imaging (SMTI). We report that Netrin-1 triggers a burst of β-actin synt...

Local mRNA translation occurs in growing axons enabling precise control of the proteome in response to signals. To measure quantitatively the spatiotemporal dynamics of protein synthesis in growth cones, we further developed a technique for single molecule translation imaging (SMTI). We report that Netrin-1 triggers a burst of β-actin synthesis at...

The aggregation promoter heparin is commonly used to study the aggregation kinetics and biophysical properties of protein amyloids. However, the underlying mechanism for amyloid promotion by heparin remains poorly understood. In the case of the neuropeptide β-endorphin that can reversibly adopt a functional amyloid form in nature, aggregation in th...

Aggregation by design Amyloid aggregation is driven by short sequences within proteins that self-assemble into characteristic amyloid structures. About 30 human proteins are implicated in amyloid-associated diseases, but many more contain short sequences that are potentially amyloidogenic. Gallardo et al. designed a peptide based on an amyloidogeni...

For more than 20 years, single-molecule spectroscopy has been providing invaluable insights into nature at the molecular level. The field has received a powerful boost with the development of the technique into super-resolution imaging methods ca 10 years ago, that overcome the limitations imposed by optical diffraction. Today, single molecule supe...

Significance The self-assembly of normally soluble proteins into fibrillar amyloid structures is associated with a range of neurodegenerative disorders. Here, we monitor the fate of different forms of α-synuclein (AS), a protein implicated in Parkinson’s disease, via optical nanoscopy directly in neuronal cells. We show that exogenously added prefo...

The aggregation promoter heparin is commonly used to study the aggregation kinetics and biophysical properties of protein amyloids. However, the underlying mechanism for amyloid promotion by heparin remains poorly understood. In the case of the neuropeptide β-endorphin that can reversibly adopt a functional amyloid form in nature, aggregation in th...

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Analytical methods that enable visualization of nanomaterials derived from solution self-assembly processes in organic solvents are highly desirable. Here, we demonstrate the use of stimulated emission depletion microscopy (STED) and single molecule localization microscopy (SMLM) to map living crystallization-driven block copolymer (BCP) self-assem...

Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here we show that dynamin-dependent endocytosis plays a major role in this process. Do...

Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here we show that dynamin-dependent endocytosis plays a major role in this process. Do...

FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the compl...

Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-reso-lution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combi-nation...

Background Endoplasmic Reticulum lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases. Lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a ra...

We demonstrate the application of Fluorescence Lifetime Imaging (FLIM) to read out Förster resonant energy transfer (FRET) based biosensors for studying the spatio-temporal dynamics of signalling pathways in cells undergoing chemotaxis.

We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using...

ΔC11CFP decay components (left) and their contributions (right) at different emission wavelength. All decays were fitted with a triple exponential model. (TIF)

Sequence of forward and reverse primers used for the creation of ΔC11CFP. The Ochre STOP codon is shown in red. (PDF)

Temperature dependence of mTFP1 decay components (left) and contributions (right). All decays were fitted with a double exponential model. (TIF)

mTFP1 decay components (left) and their contributions (right) at different emission wavelength. All decays were fitted with a double exponential model. (TIF)

Sequences of the forward and reverse primers for the creation of mTFP-TnC-Cit from TN-L15 and pmTFP-N. (PDF)

Temperature dependence of ΔC11CFP decay components (left) and contributions (right). All decays were fitted with a triple exponential model. (TIF)

Calpains belong to a family of calcium-dependant cysteine proteases that are ubiquitously expressed in mammals and have roles in numerous physiological processes. Inappropriate calpain activation, caused by a loss of calcium homeostasis, has been implicated in various disease states. To date, in vivo studies looking at the activity of calpains have...

We describe a new light transport model, which was applied to three-dimensional lifetime imaging of Förster resonance energy transfer in mice in vivo. The model is an approximation to the radiative transfer equation and combines light diffusion and ray optics. This approximation is well adopted to wide-field time-gated intensity-based data acquisit...